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IMMUNOTECHNOLOGY

Dr. K. BALAKRISHNAN.
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Chapter 1
INTRODUCTION TO IMMUNOLOGY
1.1 History and Milestones of Immunology

Human fight against smallpox represents the first ever breakthrough in immunology.
Edward Jenner in 1798 laid the foundation for the process of immunization against this
dreaded disease. Jenner collected pus from cowpox sores on the hands of milkmaid Sarah
Nelmes and inoculated eight-year-old James Phipps. Phipps developed fever but nothing
more. Then Jenner inoculated Phipps with pus from active smallpox. The boy developed
no reaction to the smallpox inoculation. A few enlightened physicians took up the idea.
Their confirmation of Jenner’s observations gradually led to acceptance of “vaccination”.
Cowpox is the result of the “vacca” virus. Hence the term “vaccination” (vacca in Latin
for ‘cow’).

Lois Pasteur was a scientist interested in fermentation of beer wine meat decay, which at
the time was also regarded as fermentation. He was the first to isolate microorganisms
from ferments. He was able to purify them and then introduce them to fresh material to
transfer the fermentation process. He also demonstrated that this transfer could be
stopped by heating (pasteurization). He later became involved in examining silkworm
blight that was seriously affecting France’s silk industry in the 1850s. He was able to
transfer his experiences in fermentation and demonstrate the presence of a microorganism
in affected worms. He could show that transfer of the microbe from affected to
unaffected worms transferred the condition.

Pasteur started studying anthrax in domestic animals, a significant cause of death for
domesticated animals at the time. By 1840 scientists were already aware of rod-shaped
microbes in the blood of anthrax-infected animals. Pasteur was able to recognize the
similarities between these microbes and the ones he had seen at work in fermentation
decay processes. Once again Pasteur isolated the microbe and showed that injection into
unaffected animals transferred the disease. By 1878 Pasteur examined chicken cholera. It
was a devastating disease for the poultry industry. Pasteur from his previous experience
worked to isolate the microbe and demonstrated its presence by culturing the causative
agent (what we now call Pasteurella multocida, a bacterium), transferring it from
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affected to unaffected animals. However, he accidentally took this work a step further.
Pasteur attempted in one experiment to transfer the microbe to unaffected animals as he
had done before. But the culture he used was old, unknown to him; the culture was what
we describe as attenuated, weakened and, therefore, limited in its infective capability.
The chicken got sick but later recovered. Realizing his mistake in using an attenuated
culture he later reinjected the chickens using a fresh culture. However, the chicken did
not die as he expected. He recognized that the old attenuated culture was a form of
vaccine against chicken cholera.

1.2 Koch-Pasteur Germ Theorem

German scientist Robert Koch was the first to isolate the anthrax microbe although it was
Pasteur who demonstrated its ability to transfer disease. Koch, unaware of Pasteur’s
work, also demonstrated the ability of the anthrax microbe to transfer the disease. There
was a stiff competition between Koch and Pasteur. While Pasteur worked from the
applied side of microbial science, Koch was a key theoretician, advancing much of the
germ theory based on analysis of Pasteur’s work. In 1881, following on from his
experiments on chicken cholera, Pasteur produced an attenuated form of anthrax to use as
a vaccine. Then, Pasteur went on to produce attenuated vaccines for swine erysipelas
rabies.

In spite of the fast pace of development of germ theory of disease by Koch and Pasteur,
many were still reluctant to accept it applied to humans. Koch isolated for the first time
the microbe that caused the human disease tuberculosis in 1882. Koch outlined the
parameters required for identification of a disease causing agent, called “Koch’s
Postulate”. These requirements still stay when identifying infective organisms.

Thus the field of immunology was born from the work of just two people in the 1880s.
Most historians define the turning point as the publication of Pasteur’s work on an
attenuated chicken cholera vaccine. In the 1880-90s immunization with attenuated
vaccines was taken up across Europe and America for a number of diseases. However,
examination of many more diseases frustrated scientists. Of course most viral based
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conditions would not reveal any bacterial microorganism that could be cultured used in a
vaccine. Even for some bacteria based conditions such as syphilis, tuberculosis and
salmonellosis, development of vaccines was unsuccessful. Two major questions to be
answered were how infection by bacteria could cause tissue to degrade and how vaccines
worked to defend an individual against death from infection. Pasteur and Koch made
little attempt to explain the mechanisms of disease and the human’s body’s defense
against it.

In 1888 Emile Roux and Alexre Yersin isolated a soluble toxin from cultures of
diphtheria. The bacterium itself is only found in the throat but its destructive effects are
found throughout the body. The bacterium must be sending out an invisible factor, most
likely chemical in nature, to cause the body-wide destruction. This idea was the
hypothesis of Roux and Yersin. They filtered diphtheria cultures to remove the bacteria
and then used the remaining fluid filtrate (we call supernatant) to inject into healthy
animals. As expected the animals showed diphtheria lesions but without any obvious
presence of bacteria.

In the next step Emil von Behring and Shibasaburo Kitasato took serum from animals
infected with diphtheria and injected it into healthy animals. When these animals were
later inoculated with diphtheria they were found to be resistant to infection. We now
know this method of conferring resistance as “passive immunity”. This first
demonstration of defense against infection was revealed and described as mediated by
“antitoxin”. It was clear to Behring and Kitasato that the antitoxin was specific only for
diphtheria; it did not confer any defense against other forms of infection. We now know
this antitoxin to be antibodies produced specifically against the diphtheria microbe.
Rudolf Kraus in 1897 first visualized the reaction of antitoxins to bacteria by simply
adding serum from infected animals to a culture of the bacteria and seeing a cloudy
precipitate develop as the antibodies bound the bacteria together.

Other scientists took different approaches and revealed serum-based responses towards
bacteria and their products. Initially, these serum properties were given a range of
different names such as precipitins, bacteriolysins and agglutinins. Long before
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antibodies were actually isolated and identified in serum, Paul Erlich had put forward his
hypothesis for the formation of antibodies. The words antigen and antibody were first
used in 1900. It was clear to Erlich and others that a specific antigen elicited production
of a specific antibody that apparently did not react to other antigens. Erlich introduced a
number of ideas that later came to be proved correct. He hypothesized that antibodies
were distinct molecular structures with specialized receptor areas. He believed that
specialized cells encountered antigens bound to them via receptors on the cell surface.
This binding of antigen then triggered a response of production of antibodies to be
released from the cell to attack the antigen. He understood that antigen and antibody
would fit together like a “lock and key”. A different key would not fit the same lock and
vice versa. However, he went wrong in two important points. First, he suggested that the
cells that produced antibody could make any type of antibody. He saw the cell as capable
of reading the structure of the antigen bound to its surface and then making an antibody
receptor to it in whatever shape was required to bind the antigen. He also suggested that
the antigen-antibody interaction was by chemical bonding rather than physical. However,
Karl Lsteiner and others quickly clarified these faults in Erlich’s theory. So, by 1900 the
medical world was aware that the body had a comprehensive defense system against
infection based on the production of antibodies. By this time Anton van Leeuwenhoek
was able to describe organisms not visible to the naked eye, with the help of the
microscope he had discovered.
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Table 1. Milestones in immunology and immunotechnology

1798 Edward Jenner, Smallpox vaccination
1862 Ernst Haeckel, Recognition of phagocytosis
1877 Paul Erlich, recognition of mast cells
1879 Louis Pasteur, Attenuated chicken cholera vaccine development
1883 Elie Metchnikoff Cellular theory of vaccination
1885 Louis Pasteur, Rabies vaccination development
1888 Pierre Roux and Alexre Yersin, Bacterial toxins
1888 George Nuttall, Bactericidal action of blood
1891 Robert Koch, Delayed type hypersensitivity
1894 Richard Pfeiffer, Bacteriolysis
1895 Jules Bordet, Complement antibody activity in bacteriolysis
1900 Paul Erlich, Antibody formation theory
1901 Karl Lansteiner, A, B O blood groupings
1901-08 Carl Jensen and Leo Loeb, Transplantable tumors
1902 Paul Portier and Charles Richet, Anaphylaxis
1903 Almroth Wright and Stewart Douglas, Opsonization reactions
1906 Clemens von Pirquet, coined the word allergy
1907 Svante Arrhenius, coined the term immunochemistry
1910 Emil von Dungern, and Ludwik Hirszfeld, Inheritance of ABO blood groups
1910 Peyton Rous, Viral immunology theory
1914 Clarence Little, Genetics theory of tumor transplantation
1915-20 Leonell Strong and Clarence Little, Inbred mouse strains
1917 Karl Lansteiner, Haptens
1921 Carl Prausnitz and Heinz Kustner, Cutaneous reactions
1924 L Aschoff, Reticuloendothelial system
1926 Lloyd Felton and GH Bailey, Isolation of pure antibody preparation
1934-08 John Marrack, Antigen-antibody binding hypothesis
1936 Peter Gorer, Identification of the H-2 antigen in mice
1940 Karl Lansteiner and Alexer Weiner, Identification of the Rh antigens
1941 Albert Coons, Immunofluorescence technique
1942 Jules Freund and Katherine McDermott, Adjuvants
Karl Lansteiner and Merill Chase, Cellular transfer of sensitivity in
1942 guinea pigs (anaphylaxis)
1944 Peter Medwar, Immunological hypothesis of allograft rejection
1948 Astrid Fagraeus, Demonstration of antibody production in plasma B cells
1948 George Snell, Congenic mouse lines
1949 Macfarlane Burnet and Frank Fenner, Immunological tolerance hypothesis
1950 Richard Gershon K Kondo, Discovery of suppressor T cells

1952 Ogden Bruton, discovery of agammagobulinemia (antibody immunodeficiency)

1953 Morton Simonsen WJ Dempster, Graft-versus-host reaction
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1953 James Riley and Geoffrey West, Discovery of histamine in mast cells
Rupert Billingham, Leslie Brent, Peter Medwar, and Milan Hasek,
1953
Immunological tolerance hypothesis
1955-1959 Niels Jerne, David Talmage, Macfarlane Burnet, Clonal selection theory
1957 Ernest Witebsky et al., Induction of autoimmunity in animals
1957 Alick Isaacs and Jean Lindemann, Discovery of interferon (cytokine)
1958-62 Jean Dausset et al., Human leukocyte antigens
1959-62 Rodney Porter et al., Discovery of antibody structure
1959 James Gowans, Lymphocyte circulation
1961-62 Jaques Miller et al., Discovery of thymus involvement in cellular immunity
1961-62 Noel Warner et al., Distinction of cellular humoral immune responses
1963 Jaques Oudin et al., antibody idiotypes
1964-8 Anthony Davis et al., T B cell cooperation in immune response
1965 Thomas Tomasi et al., Secretory immunoglobulin antibodies
1967 Kimishige Ishizaka et al., Identification of IgE as the reaginic antibody
1971 Donald Bailey, Recombinant inbred mouse strains
1974 Rolf Zinkernagel and Peter Doherty, MHC restriction
1975 Kohler Milstein, Monoclonal antibodies used in genetic analysis
1983 Luc Montagnier,Discovery of HIV
1984 Robert Good, Failed treatment of severe combined immunodeficiency
(SCID, David the bubble boy) by bone marrow grafting
1984 (Kendall A. Smith) The first single cell analysis of lymphocyte proliferation
(Doreen Cantrell)
1985-onwards Rapid identification of genes for immune cells, antibodies, cytokines other
immunological structures
1985 Tonegawa, Hood et al., Identification of immunoglobulin genes
1985-7 Leroy Hood et al., Identification of genes for the T cell receptor
1985-1987 Identification of genes for the T cell receptor
1986 Hepatitis B vaccine produced by genetic engineering
1986 Th1 vs Th2 model of T helper cell function (Timothy Mosmann)
1988 Discovery of biochemical initiators of T-cell activation: CD4- CD8-p56lck
complexes (Christopher E. Rudd)
1990 Yamamoto et al., Molecular differences between the genes for
blood groups O A between those for A B
1990 NIH team, Gene therapy for SCID using cultured T cells
1993 NIH team, Treatment of SCID using genetically altered umbilical cord cells
1990 Gene therapy for SCID
1994 'Danger' model of immunological tolerance (Polly Matzinger)
1995 Regulatory T cells (Shimon Sakaguchi)
1996-1998 Identification of Toll-like receptors
2001 Discovery of FOXP3 - the gene directing regulatory T cell development
2005 Development of human papillomavirus vaccine (Ian Frazer)
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Table 2. Nobel laureates in the filed of immunonology and immunotechnology

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1901 Emil Adolf von Behring (1854-1917), "for his serum therapy to treat diphtheria"
(First ever Nobel Prize in Physiology or Medicine)
1908 Eli Metchnikoff (1845-1916) Paul Ehrlich (1854-1915), "for study of the
immune system"
1919 Jules Bordet (1870-1961), "for discovery of the complement system in the
immune system"
1930 Karl Lsteiner (1868-1943), "for discovery of human blood types"
1960 Peter B. Medawar (1915-1987) Frank Macfarlane Burnet (1899-1985), "for the
discovery that the immune system of the fetus learns how to distinguish between
self non
1972 Gerald Maurice Edelman (1929) Rodney Robert Porter (1917-1985)
1980 Baruj Benacerraf (1920), Jean Dausset (1916) George Davis Snell (1903), "for
discovery of the Major histocompatibility complex genes which encode cell
surface molecules important for the immune system's distinction between self –
non-self
1984 Niels Jerne (1911-1994), Georges J. F. Köhler (1946-1995) César Milstein
(19272002) "for work on the immune system the production of monoclonal
antibodies"
1987 Susumu Tonegawa (1939), "for discovering how the large diversity of
antibodies is produced genetically"
1989 J. Michael Bishop (1936) Harold E. Varmus (1939), "for discovering the
cellular origins of retroviral oncogenes"
1996 Peter C. Doherty (1940) Rolf M. Zinkernagel (1944) "for describing how MHC
molecules are used by white blood cells to detect kill virus
1997 S.B. Prusiner (USA) for the discovery of prions as a new biological principle of
infection.
1999 G. Blobel (USA) for discoveries concerning signal transduction.

1.3 Organs of Immune System

The organs of immune system are called lymphoid organs because they are the home for
lymphocytes, the white blood cells that are key role players of the immune system.
Within these organs, the lymphocytes grow, develop and are transported to blood for
immunological surveillance. Bone marrow, the soft tissue in the hollow center of bones,
is the ultimate source of all blood cells, including the immune cells. The thymus is an
organ that lies behind the breastbone; lymphocytes known as T lymphocytes, or just T
cells, mature there. The spleen is a flattened organ at the upper left of the abdomen. Like
the lymph nodes, the spleen contains specialized compartments where immune cells
gather and face the antigens. In addition to these organs, clumps of lymphoid tissue are
found in many parts of the body, especially in the linings of the digestive tract and the
airways and lungs. These tissues include the tonsils, adenoids and appendix (Fig.1).
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1.3.1 Thymus

In human anatomy, the thymus is an organ located in the upper anterior portion of the
chest cavity just behind the sternum (Fig.2). Hormones produced by this organ stimulate
the production of certain infection-fighting cells. It is of central importance in the
maturation of T cells. The thymus was known to the Ancient Greeks. Galen was the first
to note that the size of the organ changed over the duration of a person's life. Due to the
large numbers of dying lymphocytes, the thymus was originally dismissed as a
"lymphocyte graveyard", without functional importance. The importance of the thymus in
the immune system was discovered in 1961 by Jacques Miller, by surgically removing
the thymus from three day old mice, and observing the subsequent deficiency in a
lymphocyte population, subsequently named T cells after the organ of their origin.
Recently, advances in immunology have allowed the function of the thymus in T cell
maturation to be more fully elucidated.

In the two thymic lobes, lymphocyte precursors from the bone-marrow become
thymocytes, and subsequently mature into T cells. Once mature, T cells emigrate from
the thymus and constitute the peripheral T cell repertoire responsible for directing many
facets of the adaptive immune system. Loss of the thymus at an early age through genetic
mutation (as in DiGeorge Syndrome) or its surgical removal results in severe
immunodeficiency and a high susceptibility to infection.

The stock of T-lymphocytes is built up in early life. Therefore, the function of the thymus
is diminished in adults. It is, hence, largely degenerated in elderly adults and is barely
identifiable, consisting mostly of fatty tissue; however it continues to function as an
endocrine gland important in stimulating the immune system.

The ability of T cells to recognize foreign antigens is mediated by the T cell receptor. The
T cell receptor undergoes genetic rearrangement during thymocyte maturation, resulting
in each T cell bearing a unique T cell receptor, specific to a limited set of peptide: MHC
combinations. The random nature of the genetic rearrangement results in a requirement of
central tolerance mechanisms to remove or inactivate those T cells which bear a T cell
receptor with the ability to recognize self-peptides.
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The generation of T cells expressing distinct T cell receptors occurs within the thymus,
and can be conceptually divided into three phases:

1. Rare population of hematopoietic progenitor cells enters the thymus from the
blood, and expands by cell division to generate a large population of immature
thymocytes.
2. Immature thymocytes each make distinct T cell receptors by a process of gene
rearrangement.
3. Immature thymocytes undergo a process of selection, based on the specificity of
their T cell receptors. This involves selection of T cells that are functional
(positive selection), and elimination of T cells that are autoreactive (negative
selection).

1.3.2 Spleen

The spleen is an organ found in all vertebrate animals. In humans, the spleen is located in
the abdomen of the body, where it functions in the destruction of redundant red blood
cells, and holds a reservoir of blood. It is one of the centers of activity of the
reticuloendothelial system (part of the immune system). Its absence leads to a
predisposition to certain infections.

The spleen is an organ found in the left upper abdomen above the stomach and
underneath the rib cage. Spleens in healthy adult humans are approximately 9 to 13
centimetres (Fig.3).

The areas and the functions of spleen:

Area Function
Red pulp Mechanical filtration. Removes
unwanted materials from the blood,
including old red blood cells.
Composition
"sinuses" (or "sinusoids") which are filled
with blood
"splenic cords" of reticular fibers
"marginal zone" bordering on white pulp
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White Helps fight infections. Composed of nodules, called Malpighian

pulp corpuscles. These are composed of:
"lymphoid follicles" (or "follicles"), rich in
B-lymphocytes
"periarteriolar lymphoid sheaths" (PALS),
rich in T-lymphocytes

Other functions of the spleen are less prominent, especially in the healthy adult:

• Production of opsonins, properdin, and tuftsin.

• Creation of red blood cells. While the bone marrow is the primary site of
hematopoeisis in the adult, the spleen has important hematopoietic functions up
until the fifth month of gestation. After birth, erythropoietic functions cease
except in some hematologic disorders. As a major lymphoid organ and a central
player in the reticuloendothelial system the spleen retains the ability to produce
lymphocytes and, as such, remains an hematopoietic organ.

• Storage of red blood cells and other formed elements. This is only valid for
certain mammals, such as dogs and horses. In horses roughly 50% of the red
blood cells are stored there. The red blood cells can be released when needed.
These animals also have large hearts in relation to their body size to accommodate
the higher-viscosity blood that results. In humans, however, the spleen does not
function as a depository of red blood cells, but instead it stores platelets in case of
an emergency. Some athletes have tried doping themselves with their own stored
red blood cells to try to achieve the same effect, but the human heart is not
equipped to handle the higher-viscosity blood.

1.3.3 Lymph nodes

A lymph node is an organized collection of lymphoid tissue, through which the lymph
passes on its way to returning to the blood. Lymph nodes are located at intervals along
the lymphatic system (Fig.4). Several afferent lymph vessels bring in lymph, which
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percolates through the substance of the lymph node, and is drained out by an efferent
lymph vessel.

The substance of a lymph node consists of lymphoid follicles in the outer portion called
the cortex, which contains the lymphoid follicles, and an inner portion called medulla,
which is surrounded by the cortex on all sides except for a portion known as the hilum.
The hilum presents as a depression on the surface of the lymph node, which makes the
otherwise spherical or ovoid lymph node bean-shaped. The efferent lymph vessel directly
emerges from the lymph node here. The arteries and veins supplying the lymph node with
blood enter and exit through the hilum.

Lymph follicles are a dense collection of lymphocytes, the number, size and
configuration of which change in accordance with the functional state of the lymph node,
e.g., the follicles expand significantly upon encountering a foreign antigen. The selection
of B cells occurs in the germinal center of the lymph nodes.

Lymph nodes are particularly numerous in the mediastinum in the chest, neck, pelvis,
axilla (armpit), inguinal (groin) region, and in association with the blood vessels of the
intestines.

1.3.4 Lymphatic system

The organs of the immune system are connected with one another and with other organs
of the body by a network of lymphatic vessels, though lymphocytes can travel throughout
the body using the blood vessels. The cells can also travel through a system of lymphatic
vessels that closely lie parallel to the body's veins and arteries. Cells and fluids are
exchanged between blood and lymphatic vessels, enabling the lymphatic system to
monitor the body for invading microbes. The lymphatic vessels carry lymph, a clear fluid
that bathes the body's tissues.

1.4 Antigens

An antigen is defined as a substance capable of triggering the synthesis of antibodies in a

host. Antigenicity is the ability of the substance to bind an antibody molecule with much
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precision and accuracy. The term immunogen has the same meaning and implications as
antigen and, hence, the two terms are used synonymously. Immunogens which are
capable of eliciting allergic or hypersensitivity reactions are called as allergens. The
antigens can be broadly classified into natural antigens including naturally available cells
such as bacteria, viruses, erythrocytes, cells of any type (particulate antigens), soluble
antigens (bacterial toxins, proteins, carbohydrates, glycoproteins and lipoproteins) and
synthetic antigens, i.e., the antigens that are synthetically made (polymers of amino acids
or sugars). The generalized events associated with inflammation are as shown in Fig.5

1.5 Requirements for Immunogenecity

i) Foreigness: The antigenic substance must be genetically foreign to the host.

Sometimes body constituents are recognized as foreign, leading to
autoimmune disease. Normally the body discriminates self from non-self.

ii) Molecular size: The most potent immunogens are macromolecular proteins
with molecular weight of 10,000 and more. Particles with a molecular weight
less than 10,000 are only weakly immunogenic or not immunogenic at all.

iii) Chemical complexity: An immunogenic molecule needs to possess a certain

degree of chemical complexity. Proteins, lipoproteins, lipo-polysaccharides
and polysaccharides are all good examples for antigens. Only pure lipids are
non-immunogenic. A solution of monomeric proteins may actually induce
tolerance, but is highly immunogenic in the polymeric state. Adjuvants are
used to enhance immunogenecity.
iv) Conformation: Three dimensional conformation is important for the
antigenicity of an antigen. The lysozyme molecule, which has several
aminoacids and so fold into a loop with the aid of a disulphide bond, is a good
antigen.
v) Residues: The amino acid sequence is important to the antigenicity of
antigens.
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1.6 Antibody

Antibodies (also known as immunoglobulins, abbreviated Ig) are gamma globulin

proteins that are found in blood or other bodily fluids of vertebrates. They are used by the
immune system to identify and neutralize foreign objects, such as bacteria and viruses.
They are typically made of basic structural units—each with two large heavy chains and
two small light chains—to form, for example, monomers with one unit, dimers with two
units or pentamers with five units. Antibodies are produced by a kind of white blood cell
called a B cell. There are several different types of antibody heavy chains, and several
different kinds of antibodies, which are grouped into different isotypes based on which
heavy chain they possess. Five different antibody isotypes are known in mammals, which
perform different roles, and help direct the appropriate immune response for each
different type of foreign object they encounter.

Although the general structure of all antibodies is very similar, a small region at the tip of
the protein is extremely variable, allowing millions of antibodies with slightly different
tip structures to exist. This region is known as the hypervariable region. Each of these
variants can bind to a different target, known as an antigen. This huge diversity of
antibodies allows the immune system to recognize an equally wide diversity of antigens.
The unique part of the antigen recognized by an antibody is called an epitope. These
epitopes bind with their antibody in a highly specific interaction, called induced fit. It
allows antibodies to identify and bind only their unique antigen in the midst of the
millions of different molecules that make up an organism. Recognition of an antigen by
an antibody tags it for attack by other parts of the immune system. Antibodies can also
neutralize targets directly by, for example, binding to a part of a pathogen that it needs to
cause an infection.

The large and diverse population of antibodies is generated by random combinations of a

set of gene segments that encode different antigen binding sites (or paratopes), followed
by random mutations in this area of the antibody gene, which create further diversity.
Antibody genes also re-organize in a process called class switching that changes the base
of the heavy chain to another, creating a different isotype of the antibody that retains the
antigen-specific variable region. This allows a single antibody to be used by several
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different parts of the immune system. Production of antibodies is the main function of the
humoral immune system.

1.6.1 Antibody forms

Activated B cells differentiate into either antibody-producing cells called plasma cells
that secrete soluble antibody or memory cells that survive in the body for years afterward
in order to allow the immune system to remember an antigen and respond faster upon
future exposures. Antibodies are, therefore, an essential product of the adaptive immune
system that learns and remembers responses to invading pathogens. Antibodies occur in
two forms: a soluble form secreted into the blood and other fluids in the body, and a
membrane-bound form that is attached to the surface of a B cell.

Soluble antibodies that are secreted from an activated B cell (in its plasma cell form) bind
to foreign substances and signal for their destruction by the rest of the immune system.
They may also be called free antibodies (until they bind an antigen and become part of an
immune complex) or secreted antibodies.

The membrane-bound form of an antibody may be called a surface immunoglobulin (sIg)

or a membrane immunoglobulin (mIg). It is part of the B cell receptor (BCR) which
allows a B cell to detect when a specific antigen is present in the body and triggers B cell
activation. The BCR is composed of surface-bound IgD or IgM antibodies and associated
Ig-α and Ig-β heterodimers, which are capable of signal transduction. A typical human B
cell will have 50,000 to 100,000 antibodies bound to its surface. Upon antigen binding,
they cluster in large patches, which can exceed 1 micrometer in diameter, on lipid rafts
that isolate the BCRs from most other cell signaling receptors. These patches may
improve the efficiency of the cellular immune response. In humans, the cell surface is
bare around the B cell receptors for several thousand Ångstroms which further isolates
the BCRs from competing influences.

1.6.2 Isotypes

Name Types Description

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Found in mucosal areas, such as the gut, respiratory tract and urogenital
IgA 2 tract, and prevents colonization by pathogens. Also found in saliva,
tears, and breast milk.

Functions mainly as an antigen receptor on B cells that have not been

IgD 1
exposed to antigens. Its function is less defined than other isotypes.
Binds to allergens and triggers histamine release from mast cells and
IgE 1 basophils, and is involved in allergy. Also protects against parasitic
worms.
In its four forms, provides the majority of antibody-based immunity
IgG 4 against invading pathogens. The only antibody capable of crossing the
placenta to give passive immunity to fetus.

Expressed on the surface of B cells and in a secreted form with very

IgM 1 high avidity. Eliminates pathogens in the early stages of B cell mediated
(humoral) immunity before there is sufficient IgG.

Antibodies can come in different varieties known as isotypes or classes. In placental

mammals there are five antibody isotypes known as IgA, IgD, IgE,IgG and IgM. They
are each named with an "Ig" prefix that stands for immunoglobulin, another name for
antibody, and differ in their biological properties, functional locations and ability to deal
with different antigens, as depicted in the table. The antibody isotype of a B cell changes
during cell development and activation. Immature B cells, which have never been
exposed to an antigen, are known as naïve B cells and express only the IgM isotype in a
cell surface bound form. B cells begin to express both IgM and IgD when they reach
maturity—the co-expression of both these immunoglobulin isotypes renders the B cell
'mature' and ready to respond to antigen. B cell activation follows engagement of the cell
bound antibody molecule with an antigen, causing the cell to divide and differentiate into
an antibody-producing cell called a plasma cell. In this activated form, the B cell starts to
produce antibody in a secreted form rather than a membrane-bound form. Some daughter
cells of the activated B cells undergo isotype switching, a mechanism that causes the
production of antibodies to change from IgM or IgD to the other antibody isotypes, IgE,
IgA or IgG.

1.6.3 Structure

Antibodies are heavy (~150kDa) globular plasma proteins that are also known as
immunoglobulins. They have sugar chains added to some of their amino acid residues. In
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other words, antibodies are glycoproteins. The basic functional unit of each antibody is
an immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibodies can
also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like teleost
fish IgM, or pentameric with five Ig units, like mammalian IgM. Several immunoglobulin
domains make up the two heavy chains (red and blue) and the two light chains (green and
yellow) of an antibody. The immunoglobulin domains are composed of between 7 (IgC)
and 9 (IgV) β-strands.

1.6.4 Immunoglobulin domains

The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two
identical heavy chains and two identical light chains connected by disulfide bonds. Each
chain is composed of structural domains called Ig domains. These domains contain about
70-110 amino acids and are classified into different categories (for example, variable or
IgV, and constant or IgC) according to their size and function. They have a characteristic
immunoglobulin fold in which two beta sheets create a “sandwich” shape, held together
by interactions between conserved cysteines and other charged amino acids.

1.6.4.1 Heavy chain

There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ,
and μ. The type of heavy chain present defines the class of antibody; these chains are
found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Distinct heavy chains
differ in size and composition; α and γ contain approximately 450 amino acids, while μ
and ε have approximately 550 amino acids.

Each heavy chain has two regions, the constant region and the variable region. The
constant region is identical in all antibodies of the same isotype, but differs in antibodies
of different isotypes. Heavy chains γ, α and δ have a constant region composed of three
tandem (in a line) Ig domains, and a hinge region for added flexibility; heavy chains μ
and ε have a constant region composed of four immunoglobulin domains. The variable
region of the heavy chain differs in antibodies produced by different B cells, but is the
same for all antibodies produced by a single B cell or B cell clone. The variable region of
 18

each heavy chain is approximately 110 amino acids long and is composed of a single Ig
domain.

1.6.4.2 Light chain

In mammals there are two types of light chain, which are called lambda (λ) and kappa
(κ). A light chain has two successive domains: one constant domain and one variable
domain. The approximate length of a light chain is 211 to 217 amino acids. Each
antibody contains two light chains that are always identical; only one type of light chain,
κ or λ, is present per antibody in mammals. Other types of light chains, such as the iota (ι)
chain, are found in lower vertebrates like Chondrichthyes and Teleostei.

1.6.4.3 Fab and Fc Regions

Some parts of an antibody have unique functions. The tips of the Y, for example, contain
the site that bind antigen and, therefore, recognize specific foreign objects. This region of
the antibody is called the Fab (fragment, antigen binding) region. It is composed of one
constant and one variable domain from each heavy and light chain of the antibody. The
paratope is shaped at the amino terminal end of the antibody monomer by the variable
domains from the heavy and light chains (Fig.6).

The base of the Y plays a role in modulating immune cell activity. This region is called
the Fc (Fragment, crystallizable) region, and is composed of two heavy chains that
contribute two or three constant domains depending on the class of the antibody. By
binding to specific proteins the Fc region ensures that each antibody generates an
appropriate immune response for a given antigen. The Fc region also binds to various cell
receptors, such as Fc receptors, and other immune molecules, such as complement
proteins. By doing this, it mediates different physiological effects including opsonization,
cell lysis, and degranulation of mast cells, basophils and eosinophils.

1.7 Antigen- Antibody Interaction

The reaction between an antigen and the homologous antibody is essentially a reaction
between the epitope of the antigen and paratope of the antibody molecules. The antigen-
 19

binding site situated in the V region of the antibody reacts with the antigenic
determinants on the surface of the antigens. The basic principles of antigen-antibody
interaction are those of interactions between any biomolecular chemical reactions.

The strength of antigen - antibody reactions depend upon antibody affinity and avidity.
Although Ag-Ab reactions are highly specific, in some cases antibody elicited by one
antigen can cross-react with an unrelated antigen. Such cross-reactions occur if two
different antigens share an identical epitope or if antibodies specific for one epitope also
bind to an unrelated epitope possessing similar chemical properties. Antigen-antibody
reactions are characterized with following features:

A. Lock and Key Concept: The combining site of an antibody is located in the Fab
portion of the molecule and is constructed from the hyper-variable regions of the heavy
and light chains. X-Ray crystallography studies of interacting antigens and antibodies
show that the antigenic determinant nestles in a cleft formed by the combining site of the
antibody. Thus, the concept of Ag-Ab reactions is like one of a key (i.e., the Ag), which
fits into a lock (i.e., the Ab).

B. Non-covalent Bonds: The bonds that hold the Ag in the antibody-combining site are
all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, van der
Waals forces and hydrophobic bonds.

C. Reversible: Since Ag-Ab reactions occur via non-covalent bonds, they are reversible.

1.8 Affinity and Avidity

Affinity - Antibody affinity is the strength of the reaction between a single antigenic
determinant and a single combining site on the antibody. It is the sum of the attractive
and repulsive forces operating between the antigenic determinant and the combining site
of the antibody.
 20

The strength of the sum total of non-covalent interactions between a single antigen-
binding site on an antibody and a single epitope is the affinity of the antibody for that
epitope. Low affinity antibodies react with the antigen weakly and tend to dissociate
readily whereas high affinity antibodies bind with the antigen more tightly and remain
bound for a longer time.

Avidity - Avidity is a measure of the overall strength of binding of an antigen with many
antigenic determinants and multivalent antibodies. Affinity refers to the strength of
binding between a single antigenic determinant and an individual antibody-combining
site whereas avidity refers to the overall strength of binding between multivalent antigens
and antibodies. Avidity is influenced by both the valence of the antibody and the valence
of the antigen. Avidity is more than the sum of the individual affinities.

1.9 Specificity and Cross Reactivity

A. Specificity - There is a high degree of specificity in Ag-Ab reactions. Antibodies can

distinguish differences in the primary structure of an antigen, isomeric forms of an
antigen, and secondary and tertiary structures of an antigen.

B. Cross reactivity - Cross reactivity refers to the ability of an individual antibody

combining site to react with more than one antigenic determinant or the ability of a
population of antibody molecules to react with more than one antigen. Cross-reactions
arise because the cross-reacting antigen shares an epitope in common with the
immunizing antigen or because it has an epitope, which is structurally similar to the one
on the immunizing antigen. Although Ag-Ab reactions are highly specific, in some cases
antibody elicited by one antigen can cross-react with an unrelated antigen. Such cross-
reactions occur if two different antigens share an identical epitope or if antibodies
specific for one epitope also bind to an unrelated epitope possessing similar chemical
properties.

A number of viruses and bacteria possess antigenic determinants identical to or similar to

normal host cell components. In some cases these microbial antigens have been shown to
 21

elicit an antibody that cross-reacts with the host cell components resulting in a tissue
damaging autoimmune reactions. The bacterium Streptococcus pyogenes, for example,
expresses cell wall proteins called M-antigens. Antibodies produced to streptococcal M-
antigens have been shown to cross react with several myocardial and skeletal muscle
proteins and have been implicated in heart and kidney damage following streptococcal
infections. The role of other cross-reacting antigens is significant in the development of
autoimmune diseases.

Chapter 2
IMMUNOLOGICAL TOOLS AND IMMUNOTECHNIQUES
2.1 Antibody Mediated Assays
2.1.1 Agglutination, Precipitation and Complement Fixation
A) Agglutination Tests
 22

The interaction between antibody and a particulate antigen results in visible clumping
called agglutination. Antibodies that produce such reactions are called agglutinins.
Agglutination reactions are similar in principle to precipitation reactions; they depend on
the cross linking of polyvalent antigens. Just as an excess of antibody inhibits
precipitation reactions, such excess can also inhibit agglutination reactions; this inhibition
is called the prozone effect. Occasionally, one observes that when the concentration of
antibody is high (i.e., lower dilutions), there is no agglutination and then as the sample is
diluted agglutination occurs. The lack of agglutination at high concentrations of
antibodies is called the prozone effect. Lack of agglutination in the prozone is due to
excess of antibody resulting in very small complexes, which do not clump to form visible
agglutination.

Several mechanisms can cause the prozone effect. First, at high antibody concentrations,
the number of antibody binding sites may greatly exceed the number of epitopes. As a
result, most antibodies bind antigen only univalently instead of multivalently. Antibodies
that bind univalently cannot cross link one antigen to another. Prozone effects are readily
diagnosed by performing the assay at a variety of antibody (or antigen) concentrations.
As one dilutes to an optimum antibody concentration, one finds higher levels of
agglutination. When one uses polyclonal antibodies, the prozone effect can also occur for
another reason.

i) Agglutination Assays

When the antigen is particulate the reaction of an antibody with the antigen can be
detected by agglutination (clumping) of the antigen. When the antigen is an erythrocyte
the term hemagglutination is used. The term agglutinin is used to describe antibodies that
agglutinate particulate antigens.

a) Qualitative agglutination test: Agglutination tests can be used in a qualitative manner

to assay for the presence of an antigen or an antibody. The antibody is mixed with the
particulate antigen and a positive test is indicated by the agglutination of the particulate
 23

antigen. For example, a patient’s red blood cells can be mixed with antibody to a blood
group antigen to determine a person’s blood type. In a second example, a patient’s serum
is mixed with red blood cells of a known blood type to look for the presence of antibodies
to that blood type in the patient’s serum. This is called reverse blood grouping.

b) Quantitative agglutination test: Agglutination tests can also be used to quantitate the
level of antibodies to particulate antigens. In this test one makes serial dilutions of a
sample to be tested for antibody and then adds a fixed number of red blood cells or
bacteria or other such particulate antigen and determines the maximum dilution, which
gives agglutination. The maximum dilution that gives visible agglutination is called the
titer. The results are reported as the reciprocal of the maximal dilution that gives visible
agglutination.

c) Applications of agglutination tests:

1) Determination of blood types or antibodies to blood group antigens.

2) To assess bacterial infections, e.g., a rise in titer to a particular bacterium indicates an

infection with that bacterial type (a fourfold rise in titer is generally taken as a significant
rise in antibody titer).

ii) Passive Hemagglutination

The agglutination test works only with particulate antigens. However, it is possible to
coat erythrocytes with a soluble antigen (e.g., viral antigen, a polysaccharide or a hapten)
and use the coated red blood cells in an agglutination test for antibody to the soluble
antigen. This is called passive hemagglutination (Fig.7). The test is performed just like
the agglutination test. Applications include detection of antibodies to soluble antigens and
detection of antibodies to viral antigens.

iii) Coomb’s test (Anti-globulin test)

 24

a) Direct Coomb’s test: When antibodies bind to erythrocytes, it does not always result in
agglutination. This can result from the Ag/Ab ratio being in antigen excess or antibody
excess or in some cases electrical charges on the red blood cells preventing the effective
cross linking of the cells. These antibodies that bind to but do not cause agglutination of
red blood cells are sometimes referred to as incomplete antibodies. In order to detect the
presence of non-agglutinating antibodies on red blood cells, one simply adds a second
antibody directed against the immunoglobulin (Ab; antibody to antibody) coating the red
cells (Fig.8a). This anti-immunoglobulin can now cross-link the red blood cells and result
in agglutination.

b) Indirect Coomb’s test: If it is necessary to find if a serum sample has antibodies

directed against a particular red blood cell (RBC) and if one wants to be sure that he also
detects potentially non-agglutinating antibodies in the sample, an Indirect Coomb’s test is
performed. This test is done by incubating the red blood cells with the serum sample,
washing out any unbound antibodies and then adding a second antiimmunoglobulin
reagent to cross-link the cells. Applications include detection of anti-Rh antibodies.
Antibodies to the Rh factor generally do not agglutinate red blood cells. Thus, red cells
from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with
these antibodies (Fig.8b). To check for this a direct Coombs test is performed. To see if
the mother has anti-Rh antibodies in her serum an indirect Coombs test is performed.

iv) Hemagglutination Inhibition (or) viral neutralization assay

The agglutination test can be modified to be used for the measurement of soluble
antigens. One measures the ability of soluble antigen to inhibit the agglutination of
antigen-coated red blood cells by antibodies. In this test a fixed amount of antibodies to
the antigen in question is mixed with a fixed amount of red blood cells coated with the
antigen (see passive hemagglutination above). Also included in the mixture are different
amounts of the sample to be analyzed for the presence of the antigen. If the sample
contains the antigen, the soluble antigen will compete with the antigen coated on the
RBC for binding to the antibodies, thereby inhibiting the agglutination of the RBC. By
 25

serially diluting the sample, one can quantitate the amount of antigen in unknown sample
by its titer. This test is called hemagglutination inhibition.

B) Precipitation tests

Antibody and soluble antigen interacting in an aqueous solution or agarose gel form a
insoluble complex (lattice) that eventually develops into a visible precipitate. Antibodies
that aggregate soluble antigens are called precipitins. Although formation of the soluble
Ag-Ab complex occurs within minutes, formation of the visible precipitate occurs rather
more slowly and often takes a day or two for complete precipitation. Formation of an Ag-
Ab lattice depends on the valency of both the antibody and the antigen. The following
points are worth monitoring:

 Affinity, Avidity, Association constant.

 The antibody must be bivalent; a precipitate will not form with monovalent Fab
fragments.
 The antigen must be either bivalent or polyvalent; that is, it must have at least two
copies of the same epitope, or have different epitopes that react with different
antibodies present in polyclonal antisera.

i) Precipitation reactions in fluids

A quantitative precipitin reaction can be performed by placing a constant amount of

antibody in a series of tubes and adding increasing amounts of antigen to the tubes. After
the precipitate forms, each tube is centrifuged to pellet the precipitate, the supernatant is
poured off and the amount of precipitate against increasing antigen concentrations yields
a precipitin curve. Excess of either antibody or antigen interferes with maximal
precipitation, which occurs in the so-called equivalence zone when the ratio of antibody
to antigen is optimum.

As a large multimolecular lattice is formed at equivalence, the complex increases in size

and precipitates out of solution. In the region of antibody excess unreacted antibody is
 26

found in the supernatant along with small soluble complexes consisting of multiple
molecules of antibody bound to a single molecule of antigen. In the region of antigen
excess unreacted antigen can be detected and small complexes are again observed, this
time consisting of one or two molecules of antigen bound to a single molecule of
antibody.

The precipitation reaction can also be used as a rapid test for the presence of antibody or
antigen. The interfacial or ring precipitin test is performed by adding antiserum to a small
tube and layering antigen on top. If the antiserum contains antibodies specific for the test
antigen then the antibody and antigen diffuse toward each other and form a visible band
of precipitation at the interface within a few minute.

ii) Precipitation reactions in gels

Immune precipitates can form not only in solution but also in an agar matrix. When
antigen and antibody diffuse toward one another in agar gel or when antibody is
incorporated into agar gel and antigen diffuses into antibody containing matrix, a visible
line of precipitation will form (Fig.9). These immunodiffusion reactions can be used to
determine relative concentrations of antibodies or antigens to compare antigens or to
determine the relative purity of an antigen preparation. Two frequently used
immunodiffusion techniques are the single radial immunodiffusion method (SRID) and
the double immunodiffusion method; both are carried out in a semisolid medium like
agar or agarose gel.

ii. a) Radial Immunodiffusion (Mancini method)

In radial immunodiffusion, the antibody is incorporated into the agar gel as it is poured
and different dilutions of the antigen are placed in wells punched radially / into the agar.
As the antigen / diffuses into the gel it reacts with the antibody and when the equivalence
 27

point is reached a ring of precipitation is formed (fig.10). The diameter of the ring is
proportional to the log of the concentration of antigen since the amount of antibody is
constant. Thus, by running different concentrations of a standard antigen one can
generate a standard curve from which one can quantify the amount of the antigen in an
unknown sample. Therefore, this is a quantitative test. If more than one ring appears in
the test, more than one antigen / antibody reaction has occurred. This could be due to a
mixture of antigens and / or antibodies. This test is commonly used in the clinical
laboratories for determination of immunoglobulin levels in patient samples.

ii. b) Double Immunodiffusion (Ouchterlony method)

In the Ouchterlony method both antigen and antibody diffuse radially from wells toward
each other, thereby establishing a concentration gradient. Where equivalence is reached, a
visible line of precipitation (band / arch) forms (Fig.11). This simple technique is an
effective qualitative tool for determining the relationship between the antigens and the
types of different Ag- Ab systems present. The patterns of the precipitin lines that form
when two different antigen preparations are placed in adjacent wells indicate whether or
not they share common epitopes. For example, when two antigens share identical
epitopes, the antiserum will form a single precipitin line with each antigen that will grow
toward each other and fuse to form a pattern called identity. If two antigens are unrelated,
the antigen and antibody do not precipitate and, therefore, are free to diffuse passing the
precipitin line forming the precipitin line of the unrelated antigen-antibody system.

If two antigens share some epitopes but one or the other has a unique epitope a pattern of
partial identity is obtained. Antibodies to the common epitope form a line of identity but
antibodies to the unique epitope diffuse passing the precipitin line formed with the unique
epitope of the more complex antigen.

One of the drawbacks of Ouchterlony double diffusion assay is that it takes 18-24 hours
before precipitin lines appear. This limitation can be overcome by using electric field. In
this method positively charged antibody and negatively charged antigen are added to
separate wells in a gel. An electric current is used to drive the immunodiffusion forming a
 28

sharp precipitin line within a few hours and with greater sensitivity than in Ouchterlony
double diffusion.

ii. c) Classical Immunoelectrophoresis(CIE)

In immunoelectrophoresis a complex mixture of antigens is placed in a slide centre well

punched out of an agar gel and the antigens are electrophoresed so that the antigens are
separated according to their charge. After electrophoresis a trough is cut in the gel and
antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced
in the equivalence zone when an Ag/Ab reaction occurs. This test is used for the
qualitative analysis of complex mixtures of antigens, although a crude measure of
quantity (thickness of the line) can be obtained. This test is commonly used for the
analysis of components in a patient’s serum. The serum is placed in the well and antibody
to whole serum in the trough. By comparison to normal serum one can determine whether
there are deficiencies on one or more serum components or whether there is an
overabundance of some serum component (thickness of the line). This test can also be
used to evaluate the purity of isolated serum proteins (Fig.12).

Immunoelectrophoresis, also called gamma globulin electrophoresis or immunoglobulin

electrophoresis, is a method of determining the blood levels of three major
immunoglobulins viz., immunoglobulin M (IgM), immunoglobulin G (IgG) and
immunoglobulin A (IgA). Immunoelectrophoresis is a powerful analytical technique with
High resolving power since it combines separation of antigens by electrophoresis with
immunodiffusion against an antiserum. The increased resolution is of benefit in the
immunological examination of serum proteins. Immunoelectrophoresis aids in the
diagnosis and evaluation of the therapeutic response in many disease /stage/. It is called a
serum protein electrophoresis (a rise in the immunoglobulin level can be detected).
Immunoelectrophoresis is also used frequently to diagnose multiple myeloma, a disease
affecting the bone marrow. Elevated level of myeloma proteins in patient’s serum is a
useful diagnostic criterion for B cell malignancy (Fig.13).
 29

Serum proteins separate in agar gels under the influence of an electric field into albumin,
alpha-1, alpha-2, beta- and gamma- globulins. Immunoelectrophoresis is performed by
placing serum on a slide containing a gel designed specifically for the test. An electric
current is then passed through the gel and immunoglobulins, which contain an electric
charge (+ve charge), migrate through the gel according to the difference in their
individual electric charges. Antiserum is placed alongside the slide to identify the specific
type of immunoglobulin present. The results are used to identify different disease entities,
and to aid in monitoring the course of the disease and the therapeutic response of the
patient to such conditions as immune deficiencies, autoimmune disease, chronic
infections, chronic viral infections and intrauterine fetal infections.

ii. d) Rocket immunoelectrophoresis(RIE)

In this technique negatively charged antigen is electrophoresed in a gel containing

antibody. The precipitate formed between antigen and antibody has the shape of a rocket
(Fig.14), the height of which is proportional to the concentration of antigen in the well.
One limitation of rocket electrophoresis is the need for the antigen to be negatively
charged for electrophoretic movement within the agar matrix. Some proteins, such as
immunoglobulins, are not sufficiently charged to be quantitated by rocket
electrophoresis; but it is possible to quantify several antigens in a mixture at the same
time.

ii. e) Two - Dimensional immunoelectrophoresis

Several antigens in a complex mixture can be quantified simultaneously with a

modification of rocket electrophoresis called two - dimensional immunoelectrophoresis.
In this technique antigen is first separated into components by electrophoresis. The gel is
then laid over another agar gel containing antiserum and electrophoresis is repeated at
right angles to the first direction, forming precipitin peaks similar to those obtained with
rocket electrophoresis. Measurement of size of the peaks allows quantification of a
number of proteins in a complex antigen mixture.
 30

ii. f) Countercurrent electrophoresis

In this test the antigen and antibody are placed in wells punched out of an agar gel and
are electrophoresed into each other where they form a precipitation line. This test works
only if conditions can be found where the antigen and antibody have opposite charges.
This test is primarily qualitative, although from the thickness of the band one can get
some measure of quantity. The major advantage is that the band formation is much
quicker.

ii. g) Countercurrent Immuno-Electrophoresis (CCIE)

Most bacterial antigens, in a slightly alkaline environment, are negatively charged

whereas the antibodies are neutral. This principle is exploited by CIE assays, in which
solutions of antibody and sample fluid to be tested are placed in small wells cut into a
slab of agarose (a gelatin-like matrix through which molecules can diffuse readily) on a
glass surface. A paper or fibre wick is used to connect the two opposite sides of the
agarose to troughs of buffer, formulated for each antibody-antigen system. When an
electrical current is applied through the buffer, the negatively charged antigen molecules
migrate towards the positive electrode and, thus, towards he wells filled with the antibody
(Fig.15). The neutrally charged antibodies are carried towards the negative electrode by
the flow of the slightly alkaline buffer. At some point between the wells, a zone of
equivalence occurs, and the antigen-antibody complexes form a visible precipitin band. A
well- visualized set of precipitin bands is seen in a simple immunodiffusion gel. The
entire procedure usually takes about 1 hour. Any antigen for which antisera are available
can be tested by CIE. The sensitivity appears to be less than that of agglutination,
detecting approximately 0.01 to 0.05 mg/ml antigen. Bands are often difficult to see, and
the agarose gel may require overnight washing in distilled water to remove non-specific
precipitin reactions. Testing positive and negative controls is especially critical, since
sera may contain nonspecifically reacting agents that form nonstable complexes in the
gel. CCIE requires the large quantities of antigen and antibody.

iii) Applications of precipitation reactions

 31

In addition to their importance in vivo, the reactions between antigen and antibody
molecules are important in many laboratory procedures, including many used for the in
vitro diagnosis of diseases. Immunological reactions performed in vitro are called
serological reactions. These are reactions based on the ability to observe or otherwise
detect specific antigen-antibody reactions. It is important that only the specified antigen -
antibody reaction is detected. False / positive results can occur if there is cross reactivity
that is if an antigen reacts with an antibody that was made for reaction with a different
antigen.

C) Flocculation

Flocculation is a special kind of precipitation wherein the antigen is neither cellular nor
soluble, but colloidal. Floccules are formed on the top of Ag-Ab mixture tube. Where as
precipitation occur only in the bottom of the Ag-Ab solution. The precipitate is formed
only over a narrow range of antigen - antibody concentrations. These tests have to be
carefully standardized before use. The flocculation test for syphilis employs the alcoholic
extract of beef heart. In the Kahn test, an alcoholic extract of beef heart washed with
ether is used as the antigen. Treating the extract with cholesterol increases the sensitivity
of the test. In the VDRL slide test, cardiolipin is mixed, as antigen, with 0.05 ml of
diluted, complement - inactivated patient’s serum in a slide. The slide is rotated for 4
minutes and the degree of flocculation is determined in a microscope.

D) Complement Fixation

Antigen/Antibody complexes can also be measured by their ability to fix complement

because an Ag/Ab complex will “consume” complement if it is present whereas free
antigens or antibodies do not. Tests for Ag/Ab complexes that rely on the consumption of
complement are termed complement fixation tests and are used to quantify Ag/Ab
reactions. This test will work only with complement fixing antibodies such as IgG and
IgM.
 32

Antigen is mixed with the test serum to be assayed for antibody and antigen-antibody
complexes are allowed to form. A control tube in which no Ag is added is also prepared.
If no Ag/Ab complexes are present in the tube, none of the complement will be fixed.
However, if Ag/Ab complexes are present, they will fix complement and thereby reduce
the amount of complement in the tube. After allowing for complement fixation by any
Ag/Ab complexes, a standard amount of red blood cells, which have been pre-coated
with anti-erythrocyte antibodies, is added (indicator system). The amount of antibody-
coated RBC is predetermined to be just enough to completely use up all the complement
initially added if it was still there.

If all the complement is free (i.e., no Ag/Ab complexes were formed between the Ag and
Ab in question), RBCs will be lysed. If Ag/Ab complexes were formed between the
particular Ag and Ab in question, some of the complement will be consumed and, thus,
when the antibody - coated RBC’s are added, not all of them will lyse. If all complement
fixed by the test system, the complement is unstable to lyse indicator system. So it is
called as complement fixation test. By simply measuring the amount of RBC lysis
(release of hemoglobin into the medium), one can indirectly quantify Ag/Ab complexes
in the tube. Complement fixation tests are most commonly used to assay for antibody in a
test sample but these tests can be modified to measure the antigen (Fig.16).

E) ELISA (Enzyme Linked Immuno-Sorbent Assay)

The ELISA is a fundamental tool of clinical immunology. Enzyme-Linked Immuno-

sorbent Assay (ELISA) is a useful and powerful method in estimating ng to pg levels in
the solution, such as serum, urine and culture supernatant. Enzyme-linked Immuno-
sorbent Assays combine the specificity of antibodies with the sensitivity of simple
enzyme assays, by using antibodies or antigens coupled to an easily assayable enzyme.
ELISAs can provide useful measurement of antigen or antibody concentration (Fig.17).

Popular enzymes are those which convert a colorless substrate to a colored product, e.g.,
p-nitrophenylphosphate (pNPP), which is converted to yellow p-nitrophenol by alkaline
phosphatase. Substrates used with peroxidase include 2,2¢-azo-bis(3-ethylbenzthiazoline-
 33

6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and tetramethylbenzidine base

(TMB), which yield green, orange and blue colors, respectively. Antibodies can be
labeled conveniently with iodine, enzymes, or biotin.

Applications of ELISA:
1. Measure Ab or Ag.
2. Measure enzyme.
3. Measure hormone.
4. Measure food borne infection.
5. Measure food toxins.

1. Indirect ELISA

In case of indirect ELISA the serum or sample containing the primary antibody is added
to the antigen-coated microtiter well or polystyrene tubes or cellulose membrane and
allowed to react with the antigen. The microtiter wells are washed to remove the unbound
antibody. After the removal of unbound antibody, the antibody bound to the antigen is
detected by adding an enzyme-conjugated secondary antibody, which is specific to the
primary antibody. The free secondary antibody is removed by washing with suitable
buffer. The substrate specific to the enzyme is added and allowed to react to develop a
colored product.

The intensity of the color is measured by spectrophotometric plate readers, which can
measure the absorbance of all the 96 wells in seconds. Indirect ELISA is the method of
choice to detect the presence of serum antibodies against human immunodeficiency virus
(HIV, the causative agent of AIDS). In this assay, recombinant envelope and core
proteins of HIV are adsorbed as solid-phase antigens to microtiter plate wells. Individuals
infected with HIV will produce serum antibodies against the epitopes present on the viral
proteins. Serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of
infection.

2. Sandwich ELISA
 34

In this technique a fixed amount of antibody is coated onto a solid support. To this a test
sample-containing antigen is added and is allowed to bind to the specific antibody coated
onto the microtiter wells. Unbound antigen is removed by washing with buffer. After the
well is washed, a second enzyme-linked antibody specific for a different epitope on the
antigen is added and allowed to react with the bound antigen. The amount of bound
enzyme-linked secondary antibody varies depends upon the antigen in the test sample.
After any free secondary antibody is removed by washing, the substrate is added, and the
colored reaction product is measured.

One of the most useful of the immunoassays is the two antibody “sandwich” ELISA
(Fig.18). This assay is used to determine the antigen concentration in unknown samples.
This ELISA is fast and accurate, and if a purified antigen standard is available, the assay
can determine the absolute amounts of antigen in unknown samples. The sandwich
ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen.
This can be accomplished with either two monoclonal antibodies that recognize discrete
sites in the antigen or one batch of affinity-purified polyclonal antibodies.

To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a
solid phase (floor of the polystyrelene ELISA plates or strips). The antigen is then added
and allowed to complex with the bound antibody. Unbound products are then removed
with a wash, and a labeled second antibody (the “detection” antibody) is allowed to bind
to the antigen, thus completing the “sandwich”. The assay is then quantified by
measuring the amount of labeled second antibody bound to the matrix, through the use of
a colored substrate. A major advantage of this technique is that the antigen need not have
to be purified prior to use. Also, these assays are very specific.

The sensitivity of the sandwich ELISA is dependent on four factors:

(i) the number of molecules of the first antibody that are bound to the solid
phase;
(ii) the avidity of the first antibody for the antigen;
 35

(iii) the avidity of the second antibody for the antigen; and
(iv) the specific activity of the second antibody

3. Competitive ELISA

In this technique, the antibody is first incubated in solution with a sample-containing

antigen. The antigen-antibody mixture is then added to an antigen-coated microtiter well.
The more antigens present in the sample, the less free antibody will be available to bind
to the antigen coated on the well. Addition of enzyme conjugated secondary antibody
specific for the isotype of the primary antibody can be used to quantitate the amount of
primary antibody bound to the well as in indirect ELISA. In competitive assay, however,
the higher the concentration of antigen in the original sample, the lower the absorbance.

4. Chemiluminescence

Measurement of light produced by chemiluminescence during certain chemical reactions

provides a convenient and highly sensitive alternative to absorbance measurements in
ELISA assays. In versions of the ELISA using chemiluminescence, a luxogenic (light-
generating) substrate takes the place of the chromogenic substrate in conventional ELISA
reactions. For example, oxidation of the compound luminol by H2O2 and the enzyme
horseradish peroxidase (HRP) produces light:

The intensity of the light is measured by luminometer.

The advantage of chemiluminescence assays over chromogenic ones is enhanced

sensitivity. In general, the detection limit can be increased at least ten-fold by switching
from a chromogenic to a luxogenic substrate, and with the addition of enhancing agents,
more than 200-fold. In fact, under ideal conditions, as little as 5 x 10-18 moles (5
attomoles) of target antigen can be detected.
 36

F) Western Blotting/ or Immunoblotting

Blotting is a technique used to transfer the electrophoretically separated biomolecules

like nucleic acids or proteins from a gel onto a solid support like nitrocellulose or nylon
membrane and a current applied across the gel thus causing the proteins to move out of
the gel and onto the nitrocellulose where they bind firmly by covalent forces.
Identification of a specific protein in a complex mixture of proteins can be accomplished
by a technique known as Western blotting, named for its similarity to Southern blotting,
which detects DNA fragments, and Northern blotting, which detects mRNA fragments.

Complementarity and hybridization is the principle behind blotting techniques. Molecular

researchers use one of the several forms of complementarity to identify the
macromolecules of interest among a large number of other molecules. Complementarity
is sequence-specific or shape-specific molecular recognition that occurs when two
molecules bind together. The two strands of DNA double helix bind because they have
complementary sequences. An antibody binds to a region of a protein molecule because
they have complementary shapes. Complementarity between a probe molecule and a
target molecule (protein of interest also enzyme substrate complexes) can result in the
formation of a probe-target complex. This complex can then be located if the probe
molecules are tagged with radioactive substance or an enzyme or a fluorescent label. The
location of this complex can then be used to get information about the target molecule.
Hybridization reactions are specific: the probes will only bind to targets with
complimentary sequence (or, in the case of antibodies, sites with the correct 3D shape
i.e., CDRs).

The support for DNA immobilization was nitrocellulose, which was used at early stages
as powder and as sheets latter. Nitrocellulose and reinforced PVDF membranes are
generally used in Western blotting.

The whole technique of Western blotting is divided into seven steps:

 37

i) Gel electrophoresis (SDS-PAGE)

ii) Transfer to solid support
iii) Blocking of non-specific binding sites on the membrane
iv) Preparation of antibody
v) Hybridization
vi) Washing
vii) Detection of probe-target hybrids.

i) Gel electrophoresis

This technique is simple, rapid and capable of resolving proteins and nucleic acids that
can not be separated by other methods. This technique helps in separating molecules on
the basis of their size. Gels are usually cast from agarose or poly-acrylamide. SDS-PAGE
(sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is commonly used to
separate proteins. These gels are solid and consist of a matrix of long thin molecules with
submicroscopic pores. The size of the pores can be controlled by varying the chemical
composition of the gel.

The gel cast is soaked with buffer. An appropriate SDS/PAGE loading dye (includes dye
markers) is added to the protein solution. The proteins are loaded and then separated by
electrophoresis by either one or two dimensional electrophoresis on a SDS-PAGE gel.
Pre-stained standards can be used to mark the progression and the efficiency of the
electrophoresis. It is usually more convenient to use a discontinuous SDS-PAGE gel,
using two different polyacrylamide concentrations to create stacking and sequencing
sections of the gel.

Proteins can contain either a net positive or negative charge and this is dependent upon
pH of the solution (TEMED converts protein into negative charged particle). In SDS-
PAGE, the protein is denatured by treating it with a reducing agent to cleave disulphide
bonds and coated with negative charges from the detergent, SDS. This coated protein will
move towards the positive electrode irrespective of its normal physiological net charge.
The gel matrix is polyacrylamide, which forms pores the size of which is regulated by the
 38

acrylamide concentration. Thus, smaller proteins will move quickly through the gel than
larger proteins; therefore, proteins of varying molecular weights can be separated based
on their masses.

ii) Transfer onto a solid support

After the proteins are separated in gel according to their molecular weight, they must be
transferred to a solid support before hybridization. This is because hybridization does not
work well in a gel. This transfer process is called blotting and is this is the reason why
these hybridization techniques are called blots. Usually, the solid support is a sheet of
nitrocellulose paper. The developments in the field of recombinant DNA techniques have
resulted in a variety of new approaches to the analysis of genes and gene products. The
gel, nitro-cellulose paper and Whatman paper are washed with a transfer buffer
containing glycine, Tris, SDS and methanol. A Western transfer apparatus is prepared by
sandwiching two pieces of Whatman paper, the nitro-cellulose membrane, the SDS-
PAGE gel, and two more pieces of Whatman paper, in that order, between two wet
porous pads. This sandwich formation is pressed between the two plastic panes of the
Western transfer apparatus with the nitro-cellulose nearest to the positive electrode of the
apparatus. The apparatus is run overnight with about 30 volts electricity and stirring in a
cold room.

iii) Blocking of non-specific binding sites on the membrane

The surface of the filter has the separated molecules on it, as well as many spaces
between the lanes, etc., where no molecules have yet bound. If the probe is added
directly to the filter now, the probe would stick to these blank pans of the filter, like the
molecules transferred from the gel did. This would result in a filter completely covered
with the probe, which would make it impossible to locate the probe-target hybrids. For
this reason, the filters are soaked in a blocking solution, which contains a high
concentration of (DNA, RNA or) protein. This coats the filter and prevents the probe
from sticking to the filter itself. During hybridization, the probe should bind only to the
target molecule. The non-specific binding sites are filled by incubating the membrane
 39

overnight in a buffer-salt solution, such as TBST, containing dry non-fat milk such as
MarvelTM (about 5%). An alternative blocking solution is TWEEN 20.

iv) Preparation of radio-labeled antibody

The desired protein is purified and crushed with Freund’s complete adjuvant (FCA).
Antibodies are prepared by injecting the target protein into an animal (e.g., goat, rabbit or
mouse) and removing the serum produced from the animal which received the protein
antigen. This serum contains antibody against the antigen administered. These are
primary antibodies, which are proteins. These proteins are labeled by chemically
modifying the side chain of tyrosine residues in antibody with 125I. These labeled primary
antibodies will bind to the specific protein on the nylon membrane and can be visualized
adopting autoradiography.

Antibody-tyrosine + 125I + H2O2————> H2O + 125I-tyrosine-Ab.

The secondary antibody are selected from the catalogue and used against the specific
primary antibody. This secondary antibody is conjugated with an enzyme. The desired
protein can be detected by the colored product formed on adding the substrate specific to
the enzyme conjugate with secondary antibody complex.

v) Hybridization

The labeled probe is added to the blocked filter in buffer and incubated for several hours
to allow the probe molecules to find and fix with the targets.

vi) Washing

After hybrids are formed between the probe and the target, it is necessary to remove any
probe that is on the filter which has not stuck to the target molecules. Because the
nitrocellulose is absorbent, some of the probe soaks into the filter and it must be
removed. To do this, the filter is rinsed repeatedly in several changes of buffer to wash
 40

off any unhybridized probe. If it is not removed, the whole filter will be radioactive and
the specific hybrids will not be detectable.

vii) Detection by autoradiography

The localization and recording of a radiolabelled probe in an X-ray film is known as

autoradiography. It involves the production of an image in a photographic emulsion. Such
emulsions consist of silver halide crystals suspended in a clear phase composed mainly of
gelatin. When a beta particle or X-ray from a radionuclide passes through the emulsion,
the silver ions are converted to silver atoms. This results in a latent image being
produced, which is converted to a visible image when the image is developed.
Development is a system of amplification in which the silver atoms cause the entire silver
halide crystal to be reduced to metallic silver. Unexposed crystals are removed by
dissolution in fixer, giving an autoradiographic image which represents the distribution of
radio-label in the original sample. If the probe is radioactive, the radioactive particles it
emits can expose the X-ray film. If the filter is pressed against the X-ray film and left in
the dark for a few minutes to overnight, the film will be exposed. After development,
there will be dark spots on the film wherever the probe is bound.

viii) Enzymatic development

Detection with enzyme-labeled antibodies is the most popular method, normally using
horseradish peroxidase- or alkaline phosphatase-couple antibodies, and a range of soluble
substrates that yield insoluble colored proteins. The results are easy to obtain, immediate,
visible and do not require special equipment. If an antibody-enzyme conjugate was used
as a probe, this can be detected ~ by soaking the filter in a solution of a substrate for the
enzyme. Usually, the substrate produces an insoluble colored product (a chromogenic
substrate) when acted upon by the enzyme. This produces a deposit of colored product
wherever the probe bound.

G) Radioimmunoassay
 41

One of the most sensitive techniques for detecting antigen or antibody is

radioimmunoassay (RIA). The technique was first developed in 1960 by two
endocrinologists, S. A. Berson and R. Yalow, to determine levels of insulin-anti-insulin
complexes in diabetics. The technique soon proved valuable for measuring hormones,
serum proteins, drugs and vitamins at concentrations 0.001 micrograms per milliliter or
less. In 1977, some years after Berson’s death, the significance of the technique was
acknowledged with a Nobel Prize to Yalow.

The principle of RIA involves competitive binding of radiolabelled antigen and unlabeled
antigen to a high-affinity antibody. The labeled antigen is mixed with the antibody at a
concentration that saturates the antigen-binding sites of the antibody. Then test samples
of unlabeled antigen of unknown concentration are added in progressively larger
amounts. The antibody does not distinguish labeled antigen from unlabeled antigen;
therefore, both labeled and unlabeled antigens compete for available binding sites on the
antibody. As the concentration of unlabeled antigen increases, more labeled antigen will
be displaced from the binding sites. The decrease in the amount of radiolabelled antigen
bound to specific antibody in the presence of the test sample is measured in order to
determine the amount of antigen present in the test sample.

125
The antigen is generally labeled with a gamma emitting isotopes such as I, but beta-
emitting isotopes such as tritium (3H) are also routinely used as labels. The radiolabelled
antigen is part of the assay mixture; the test sample may be a complex mixture, such as
serum or other body fluids, that contains the unlabeled antigen. The first step in setting up
an RIA is to determine the amount of antibody needed to bind 50%-70% of a fixed
quantity of radioactive antigen (Ag*) in the assay mixture. This ratio of antibody to Ag*
is chosen to ensure that the number of epitopes presented by the labeled antigen always
exceeds the total number of antibody binding sites. Consequently, the unlabeled antigen
added to the sample mixture will compete with radiolabelled antigen for the limited
amount of antibody. Even a small amount of unlabeled antigen added to the assay
mixture of labeled antigen and antibody will cause a decrease in the amount of
radioactive antigen bound, and this decrease will be proportional to the amount of
unlabeled antigen added.
 42

To determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to
separate it from free antigen (antigen not bound to Ab), and the radioactivity in the
precipitate is measured. A standard curve can be generated using unlabeled antigen
samples of known concentration (in place of the test sample), and from this plot the
amount of antigen in the test mixture may be precisely determined. Several methods have
been developed for separating the bound antigen from the free antigen in RIA. One
method involves precipitating the Ag-Ab complex with a secondary anti-isotype
antiserum. For example, if the Ag-Ab complex contains rabbit IgG antibody, then goat
anti-rabbit IgG will bind to the rabbit IgG and precipitate the complex. Another method
makes use of the fact that protein a of Staphylococcus aureus has high affinity for IgG. If
the Ag-Ab complex contains an IgG antibody, the complex can be precipitated by mixing
with formalin-killed S. aureus. After removal of the complex by either of these methods,
the amount of free labeled antigen remaining in the supernatant can be measured in a
radiation counter. By subtracting this value from the total amount of labeled antigen
added yields the amount of labeled antigen bound. Various solid-phase RIAs have been
developed that make it easier to separate the Ag-Ab complex from the unbound antigen.

In some cases, the antibody is covalently cross linked to Sepharose beads. The amount of
radiolabelled antigen bound to the beads can be measured after the beads have been
centrifuged and washed. Alternatively, the antibody can be immobilized on polystyrene
or polyvinyl chloride wells and the amount of free labeled antigen in the supernatant can
be determined in a radiation counter. In another approach, the antibody is immobilized on
the walls of microtiter wells and the amount of bound antigen determined. Because the
procedure requires only small amounts of sample and can be conducted in small 96-well
microtiter plates, this procedure is well suited for determining the concentration of a
particular antigen in large numbers of samples. For example, a microtiter RIA has been
widely used to screen for the presence of the hepatitis B virus. RIA screening of donor
blood has sharply reduced the incidence of hepatitis B infections in recipients of blood
transfusions.
 43

A standard curve is obtained by adding increasing concentrations of unlabelled HBsAg to

a fixed quantity of (125I) HBsAg and specific antibody. From the plot of the percentage of
labeled antigen bound vs the concentration of unlabeled antigen, the concentration of
HBsAg in unknown serum samples can be determined.

H) Immunofluorescence

Immuno-fluorescence (IF) is a laboratory technique commonly used to detect antigens of

cells and tissues. In 1944, Albert Coons showed that the antibodies could be labeled with
the molecules that have the property of fluorescence. Fluorescent molecules absorb
velocity of one wavelength (excitation) and emit light of another wavelength (emission).
If antibody molecules are tagged with a fluorescent dye, or fluorochrome, immune
complexes containing these fluorescently labeled antibodies (FA) can be detected by
colored light emission when excited by light of the appropriate wavelength. Antibody
molecules bound to antigens in cells or tissue sections can similarly be visualized. The
emitted light can be viewed in a fluorescent microscope with an UV light source.

In immunofluorescence, fluorescent compounds such as fluorescein and rhodamine are

commonly used, but other highly fluorescent substances such as phycoerythrin, an
intensely colored and highly fluorescent pigment obtained from algae, are also routinely
used. These molecules can be conjugated to the Fc region of an antibody molecule
without affecting the specificity of the antibody. Fluorescent - antibody staining of cell
membrane molecules or tissue sections can be done in two ways i.e., direct or indirect. In
direct staining, the specific antibody (the primary antibody) is directly conjugated with
fluorescein. In indirect staining, the primary antibody is unlabeled and is detected with an
additional fluorochrome-labeled reagent. A number of reagents have been developed for
indirect staining. The most common is a fluorochrome-labeled secondary antibody raised
in one species against antibodies of another species, such as fluorescein-labeled goat anti-
mouse immunoglobulin. Indirect immunofluorescent staining has two advantages over
direct staining. First, the primary antibody does not need to be conjugated with a
fluorochrome. Because the supply of primary antibody is often a limiting factor, indirect
methods avoid the loss of antibody that usually occurs during the conjugation reaction.
 44

Second, indirect methods increase the sensitivity of staining because multiple molecules
of the fluorochrome reagent bind to each primary antibody molecule, increasing the
amount of light emitted at the location of each primary antibody molecule.

The technique is also suitable for identifying bacterial species, detecting Ag-Ab
complexes in autoimmune diseases, detecting complement components in tissues and
localizing hormones and other cellular products stained in situ. Indeed, a major
application of the fluorescent antibody technique is the localization of antigens in tissue
sections or in subcellular compartments. Because it can be used to map the actual
location of target antigens, fluorescent microscopy is a powerful tool for relating the
molecular architecture of tissues and organs to their overall gross anatomy.

Fluorescein, an organic dye that is the most widely used label for immunofluorescence
procedures, absorbs blue light (490 nm) and emits an intense yellow-green fluorescence
(517 nm).

Rhodamine, another organic dye, absorbs in the yellow-green range (515 nm) and emits
a deep red fluorescence (546 nm). Because it emits fluorescence at a longer wavelength
than fluorescein, it can be used in two-color immunofluorescence assays. An antibody
specific to one determinant is labeled with fluorescein, and an antibody recognizing a
different antigen is labeled with rhodamine. The location of the fluorescein-tagged
antibody will be visible by its yellow-green color, easy to distinguish from the red color
emitted where the rhodamine tagged antibody has bound. By conjugating fluorescein to
one antibody and rhodamine to another antibody, one can, for example, visualize
simultaneously two different cell-membrane antigens on the same cell.

Phycoerythrin is an efficient absorber of light (~30- fold greater than fluorescein) and a
brilliant emitter of red fluorescence, favoring its wide use as a label for
immunofluorescence.

2. 2 Cell-Mediated Assays/ Cellular assays

 45

1. Plaque forming cell assay, 2. Rosette forming assay, 3. NBT assay, (Ros assay)
RNi assay.

A) ELISPOT Assay

The Enzyme-Linked Immuno-SPOT (ELISPOT) assay is a common method for

monitoring immune responses in humans and animals. It was developed by Cecil
Czerkinsky in 1983.

The ELISPOT assay is a modified version of the ELISA immunoassay. ELISPOT assays
were originally developed to enumerate B cells secreting antigen-specific antibodies, and
have subsequently been adapted for various tasks, especially the identification and
enumeration of cytokine-producing cells at the single cell level. At appropriate conditions
the ELISPOT assay allows visualization of the secretory product of individual activated
or responding cells. Each spot that develops in the assay represents a single reactive cell.
Thus, the ELISPOT assay provides both qualitative (type of immune protein) and
quantitative (number of responding cells) information.

By virtue of precise sensitivity of the ELISPOT assay, frequency analysis of rare cell
populations (e.g., antigen-specific responses) which were not possible before are now
relatively easy. This exceptional sensitivity is in part due to the fact that the product is
rapidly captured around the secreting cell: before it is either diluted in the supernatant,
captured by receptors of adjacent cells, or degraded. This makes ELISPOT assays much
more sensitive than conventional ELISA measurements. Limits of detection are below
1/100,000 rendering the assay uniquely useful for monitoring antigen-specific responses,
applicable to a wide range of areas of immunology research, including cancer,
transplantation, infectious disease, and vaccine development. The assay has gained a
recent increase in popularity, especially as a surrogate measure for CTL responses, in
large part because it is both reliable and highly sensitive.

While ELISPOT assay techniques have existed for more than two decades now
advancements are still being made in the assay. Modern ELISPOT analysis is typically
performed using ELISPOT readers, which employ computer vision techniques to
enumerate the actively producing cells. This allows much of the analysis process to be
 46

automated, and permits a greater level of accuracy than what can be achieved using
manual inspection.

Procedure

As noted above, the ELISPOT assays employ a technique very similar to the sandwich
enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or
polyclonal capture antibody is coated aseptically onto a PVDF (polyvinylidene fluoride)
-backed microplate. These antibodies are chosen for their specificity for the analyte in
question. The plate is blocked, usually with a serum protein that is non-reactive with any
of the antibodies in the assay. After this, cells of interest are plated out at varying
densities, along with antigen or mitogen, and then placed in a humidified 37°C CO2
incubator for a specified period of time.

Cytokine (or other cell product of interest) secreted by activated cells is captured locally
by the coated antibody on the high surface area PVDF membrane. After washing the
wells to remove cells, debris and media components, a biotinylated polyclonal antibody
specific for the chosen analyte is added to the wells. This antibody is reactive with a
distinct epitope of the target cytokine and thus is employed to detect the captured
cytokine. Following a wash to remove any unbound biotinylated antibody, the detected
cytokine is then visualized using an avidin-HRP, and a precipitating substrate (e.g., AEC,
BCIP/NBT). The colored end product (a spot, usually a blackish blue) typically
represents an individual cytokine-producing cell. The spots can be counted manually
(e.g., with a dissecting microscope) or using an automated reader to capture the
microwell images and to analyze spot number and size.

B) HLA Tissue Typing (Histocompatibility Testing)

The histocompatibility antigens of the recipient (patients) and the potential donor are
detected by serological typing (using predefined anti-sera for different HLA alleles)
and/or PCR (polymerase chain reaction) based SSP (sequence-specific primers) and
SSOP (sequence-specific oligonucleotide probe) methods.

i) Methods of tissue typing

 47

HLA Typing is done for finding out the differences in HLA alleles of donor and recipient
cells. Routine HLA typing focuses only on HLA-A, HLA-B and HLA-DR since they are
involved in graft versus host reactions or rejections. This is done through serological
tests. Standard set of sera from multiple donors previously sensitized to different HLA
molecules (by multiple pregnancies or transfusions). Each sample of sera is mixed with
patient’s lymphocytes in separate wells of sixty or seventy two well Terasaki plates. A
source of rabbit complement is added to the wells, along with fluorescent dye, which
enters only dead cells. After incubation the wells are observed under phase contrast
microscope for the presence of dead cells. B lymphocytes are used as target cells since
they express both class I and class II MHC molecules. HLA type and the haplotype is
determined by visualizing dead cells.

ii) Cross matching

This is done after identifying a potential donor normally blood relative i.e. siblings, son
or daughter. It determines whether the patient has antibodies that specifically react with
donor cells. Patient’s serum in different dilutions in Terasaki plate is tested with donor
lymphocytes viability through cyototoxic and flow cytometric assays called MLR (mixed
leukocyte reaction) Micro Lymphocytotoxicity Assay.

iii) Immunosuppression

Cyclosporin, mycophenolate mofetils and steroids are being used for the non-specific
suppression of receipient’s/patient’s immune response against graft or transplanted organ.
These drugs, along with other treatment modalities, prolong the life of the graft and avoid
the rejection episode immediately after the transplantation.

D) Lymphocyte Transformation Test (LTT)

There is an important need to determine metal allergies in large numbers of individuals

who may be exposed either occupationally or environmentally. An in vitro test has the
significant advantage over skin tests of avoiding exposing people to the metal during
 48

testing, which may exacerbate, or even cause, sensitization. Berellium is one such metal.
One of the most widely used test systems for this purpose is the lymphocyte
transformation test (LTT). Its development was based on the observation that incubation
of lymphocytes with phytohemagglutinin, a mitogen, leads to cell activation and
proliferation. Therefore, it is also called the “lymphocyte proliferation test” (LPT). The
test has been applied by different research groups for the evaluation of various cell-
mediated immune reactions. The principle of the LTT is based on the fact that
lymphocytes, which have been sensitized by a certain antigen (“memory cells”),
transform into blasts and proliferate when they are again exposed to this antigen.
According to the different biological mechanisms occurring in a transforming cell, there
are several chemical and physical methods to measure this transformation into blasts.
Some such methods are determination of metabolic processes, biochemical alterations
such as protein biosynthesis, or the synthesis of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA).

E) Mantoux Test/ Skin sensitivity Test

The Mantoux test (or Mantoux screening test, Tuberculin Sensitivity Test, Pirquet test, or
PPD test for Purified Protein Derivative) is a diagnostic tool for tuberculosis. It is one of
the two major tuberculin skin tests for tuberculosis used in the world. Until 2005, the
Heaf test was used in the United Kingdom instead of the Mantoux.

Tuberculin is a glycerine extract of the tubercule bacilli. Purified protein derivative

(PPD) tuberculin is a precipitate of non-species-specific molecule obtained from filtrates
of sterilized, concentrated cultures. It was first described by Robert Koch in 1891. The
test is named after Charles Mantoux, a French physician who developed on the work of
Koch and Clemens von Pirquet to create this test in 1907.

A standard dose of 5 Tuberculin units (0.1 mL) (The standard Mantoux test in the UK
consists of an intradermal injection of 2TU of Statens Serum Institute (SSI) tuberculin
RT23 in 0.1ml solution for injection is injected intradermally (into the skin) and read 48
to 72 hours later. A person who has been exposed to the Mycobacterium is expected to
 49

mount an immune response in the skin containing the bacterial proteins.The reaction is
read by measuring the diameter of induration (palpable raised hardened area) across the
forearm (perpendicular to the long axis) in millimeters. No induration should be recorded
as “0 mm”. Erythema (redness) cannot be measured; however, it indicates the presence of
the infectious agent (Mycobacterium tuberculosis) in the individual.

2.3 Immunofluorescent Techniques

a) Flow cytometry (FACS)

Flow cytometry is a powerful tool in modern biomedical investigations. Flow cytometry

is a technique for measuring both scattered light and fluorescent light from single cells at
very rapid rates. Typically up to 5000 cells can be analyzed per second. Using various
fluorochromes this allows a cell population to be analyzed for cells showing certain
characteristics such as the presence of a particular enzyme, cellular constituent or other
gene product. The information it can provide is invaluable in helping to diagnose certain
cancers as well as aiding basic research in many aspects of cell biology such as the cell
cycle and gene expression.

The most numerous flow cytometers are those used for complete blood cell counts in
clinical laboratories - these do not employ fluorescence. More versatile research
instruments employ fluorescence, hence, may be distinguished as flow cytofluorometers.
They are present in medical centers and research institutions, where they are used for
diagnosis as well as research. Ploidy and cell cycle analysis of cancers are the major
diagnostic use. Lymphomas and leukemias are intensively studied for surface markers of
diagnostic and prognostic value. It is commonly used to exclude dead cells, cell
aggregates, and cell debris from the fluorescence data. It is sufficient to distinguish
lymphocytes and monocytes from granulocytes in blood leukocyte samples. Although
less expensive alternative technologies are under development, at the present, flow
cytometry has been the method of choice for monitoring CD4 lymphocyte levels in the
blood of AIDS patients.
 50

The term “FACS” is Becton-Dickinson’s registered trademark and is an acronym for

Fluorescence-Activated Cell Sorter. Another major brand of flow cytometers is Coulter
Electronics.

i) Principles of working

The cells may be alive or fixed at the time of measurement, but must be in monodisperse
(single cell) suspension. They are passed single-file through a LASER beam by
continuous flow of a fine stream of the suspension. Each cell scatters some of the laser
light, and also emits fluorescent light excited by the laser. Fluorescence intensities are
typically measured at several different wavelengths simultaneously for each cell.
Fluorescent probes are used to report the quantities of specific components of the cells
(Fig.19).

Fluorescent antibodies are often used to report the densities of specific surface receptors
and, thus, to distinguish subpopulations of differentiated cell types, including cells
expressing a trans-gene. By making them fluorescent, the binding of viruses or hormones
to surface receptors can be measured. Intracellular components can also be reported by
fluorescent probes, including total DNA/cell (allowing cell cycle analysis), newly
synthesized DNA, specific nucleotide sequences in DNA or mRNA, filamentous actin,
and any structure for which an antibody is available. Flow cytometry can also monitor
rapid changes in intracellular free calcium, membrane potential, pH, or free fatty acids.

Flow cytometers involve sophisticated fluidics, laser optics, electronic detectors, analog
to digital converters, and computers. The optics deliver laser light focused to a beam a
few cell diameters across. The fluidics hydrodynamically focus the cell stream to within
an uncertainty of a small fraction of a cell diameter, and in sorters, break the stream into
uniform-sized droplets to separate individual cells. The electronics quantify the faint
flashes of scattered and fluorescent light and, under computer control, electrically charge
droplets containing cells of interest so that they can be deflected into a separate test tube
or culture wells. The computer records data for thousands of cells per sample, and
displays the data graphically.
 51

ii) Detection

The cells flow single-file past a laser beam (or in some more complicated instruments,
two laser beams). The momentary pulse of fluorescence emitted as the cell crosses the
beam is measured by photomultipliers at a 90o angle from the beam. Typically, 2-3
detectors are used with different wavelength bandpass filters, allowing the simultaneous
detection of emissions at different wavelengths from different fluorochromes in a single
cell. In addition to fluorescence, two types of light scatter are measured. Low-angle
forward scatter (often called “forward scatter”) is roughly proportional to the diameter
of the cell. Orthogonal, 90o or “side scatter” is proportional to the granularity: neutrophil
granulocytes have higher side scatter than do lymphocytes, which are agranular. Thus, in
the FACScan, each cell can provide up to five numbers: size, granularity, plus green, red,
and far-red fluorescence intensities.

iii) Applications

The major applications in the clinical laboratory are as follows:

1. Monitoring of solid tumors and AIDS patients (CD4/CD8 ratio).
2. Immunophenotyping leukemia and lymphomas and monitoring residual disease.
3. Determining CD34 counts for hematopoietic reconstitution.
4. Monitoring organ transplant patients for rejection.
5. Counting reticulocyte.
6. DNA analysis of S-phase fraction of solid tumors.

iv) Research applications

Flow cytometry in the recent times is in extensive use in the field of drug discovery.
Because of the multi-parameter capability, antibodies can be used to identify cells and
cytokines or cytokine receptors to identify specific populations of functional cells. This
approach leads to increased understanding of cellular communication. It is also possible
to use fluorescent probes to many physiological functions such as metabolic processes,
 52

ion channels, organelles and intracellular pH. These assays are extremely important in
understanding the effects of drugs on cell physiology. Finally, molecular phenotyping
using in situ PCR, in situ hybridization and the use of fluorescent markers for isolation of
transfected cells are significantly increasing.

C) Immuno-electron Microscopy

The fine specificity of antibodies has made them powerful tools for visualizing specific
intracellular tissue components by immuno-electron microscopy. In this technique, an
electron dense label is either conjugated to the Fc portion of a specific antibody or
conjugated to an anti-immunoglobulin reagent for indirect staining. A number of electron
dense labels have been employed, including ferritin and colloidal gold. Because electron
dense label absorbs electrons, it can be visualized with the electron microscope as small
black dots. In case of immuno-gold labeling, different antibodies can be conjugated with
gold particles of different sizes, allowing identification of several antigens within a cell
by the different sizes of the electron dense gold particles attached to the antibodies. This
technique allows the investigator to identify antibody / antigen complexes that localize to
a particular subcellular organelle or compartment by using the Protein A gold technique.
The tissue is first fixed in 4.0% para-formaldehyde and processed for embedding in a low
temperature embedding resin (Lowicryl).

Protein A gold labeling of a prostatic endocrine-paracrine cell demonstrating localization

of calcitonin (inset) to the neuroscretory granules Paraffin-embedded tissue (same tissue)
sections are cut and screened by light microscopy using a streptavidin-biotin technique to
determine the appropriate antibody concentration to be used for the EM immuno-gold
procedure. Ultra-thin sections from the same resin-embedded block(s) are cut, incubated
with primary antibody and then incubated with Protein A gold particles (size range is 5
nm to 20 nm). The gold particles bind to the Fc portion of the antibody and are detected
by EM. A variation of the large block “pop-off” technique for immuno-electron
microscopy is also available.

Chapter 3
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HYBRIDOMA TECHNOLOGY

Most antigens offer multiple epitopes and, therefore, induce proliferation and
differentiation of a variety of B-cell clones, each derived from a B cell that recognizes a
particular epitope. The resulting serum antibodies are heterogeneous, comprising a
mixture of antibodies, each specific for one epitope. Such a polyclonal antibody response
facilitates the localization, phagocytosis, and complement-mediated lysis of antigen. It,
thus, has clear advantages for the organism in vivo. Unfortunately, the antibody
heterogeneity that increases immune protection in vivo often reduces the efficacy of an
antiserum for various in vitro uses. For most research, diagnostic, and therapeutic
purposes, monoclonal antibodies derived from a single clone and, thus, specific for a
single epitope, are preferable. Direct biochemical purification of a monoclonal antibody
from a polyclonal antibody preparation is not feasible. In 1975, Georges Kohler and
Cesar Milstein devised a method for preparing monoclonal antibody, which quickly
became one of immunology’s key technologies. By fusing a normal activated, antibody-
producing B cell with a myeloma cell (a cancerous plasma cell), they were able to
generate a hybrid cell, called a hybridoma. This cell possessed the immortal growth
properties of the myeloma cell and secreted the antibody produced by the B cell. The
resulting clones of hybridoma cells, which secrete large quantities of monoclonal
antibody, can be cultured indefinitely. Two features of the antibody-epitope relationship
are key to the use of monoclonal antibodies as molecular tools are i) specificity (the
antibody binds only to its particular epitope) and ii) sufficiency (the epitope can bind to
the antibody on its own, i.e., the presence of the whole antigen molecule is not
necessary). Kohler and Milstein were awarded Nobel Prize for discovering the
hybridoma technology.

3.1 Production of Monoclonal Antibodies

In order to isolate a B lymphocyte producing a certain antibody, one has first to induce
the production of such a B cell in an organism. For example, if we need an antibody for
avian SERCA2 protein, one would inject the protein into a mouse. This is typically done
in two doses, an initial “priming” dose and a second “booster” dose 10 days later. Since
 54

the protein is of foreign origin, the mouse immune system recognizes it as such and soon
some of the B cells in the mouse would begin the production of the antibody to avian
SERCA2.

A sample of B cells is extracted from the spleen of the mouse and added to a culture of
myeloma cells (cancer cells). The intended result is the formation of hybridomas, cells
formed by the fusion of a B cell and a myeloma cell. The fusion is brought about by
using polyethylene glycol, a virus or by electroporation. The next step is to select for the
hybridomas. The myeloma cells are HGPRT- and the B cells are HGPRT+. HGPRT is
hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in the synthesis of
nucleotides from hypoxanthine, an amino acid. The culture is grown in HAT
(hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+ cells.
The myeloma cells that fuse with another myeloma cell or do not fuse at all die in the
HAT medium since they are HGPRT-. The B cells that fuse with another B cell or do not
fuse at all die because they do not have the capacity to divide indefinitely. Only
hybridomas between B cells and myeloma cells survive, both being HGPRT+ and
cancerous (Fig.20).

The initial collection of B cells used is heterogenous, i.e., they do not all produce the
same antibody or may not produce an antibody at all. Therefore, the hybridoma
population would produce antibodies against more than one epitope or none. A
hybridoma cell is initially tetraploid, having been formed by the fusion of two diploid
cells. However the extra chromosomes are somehow lost in subsequent divisions in a
random manner. This means that one cannot be certain that the hybridomas will all
produce the desired antibody or even any antibody at all. Screening is required to decide
which hybridoma cells are actually producing the desired antibody. Each hybridoma is
cultured and screened after doing SDS-PAGE (sodium dodecyl sulfate - polyacrylamide
gel electrophoresis) and Western blots. The probe used is the epitope of the antibody that
is desired, which may be labeled by radioactivity or immunofluorescence.

3. 2 Applications of Monoclonal Antibodies

 55

1. Identification of substances produced by the body in special circumstances, e.g.,

hormones produced in pregnancy, or abnormal metabolites, which enable screening for
genetic abnormalities, drugs, etc., and other health problems.

2. Identification and quantification of circulating microorganisms, possibly in small

numbers or too small to see under the microscope, e.g., infecting viruses.

3. Industrial processing of blood products such as factor 8, which is needed by

haemophiliacs, could be processed using monoclonal antibodies in such a way as to avoid
viruses which may be present in only a small number of cases but which pose a major
problem when blood products are produced from a pooled source.

4. If a particular type of cancer with cells widespread in the body can be shown to have
different antigens than ordinary cells, then it may be possible to treat the cancer by
monoclonal antibody-based targeting. This could be done by attaching a toxin or poison
to antibodies, which will build up only in the cancer cells and kill them, but have little
effect on ordinary cells which have different antigens. The potent plant poison ricin, in
combination with monoclonal antibody, has been thus investigated.

5. Reducing the possibility of rejection in transplants by more accurate tissue typing

between recipient and possible donors.

6. Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins
or hormones; e.g., monoclonal antibodies to human chorionic gonadotropin (HCG) are
used in pregnancy test kits. Another diagnostic use of monoclonal antibodies is the
diagnosis of AIDS by the ELISA test.

7. Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer,

and some new methods can even target only the cell membrane of cancerous cells.

8. A new cancer drug based on monoclonal antibody technology is Ritoxin (Rituximab)

approved by the FDA.
 56

9. M
 onoclonal antibodies can be used to treat viral diseases, traditionally considered
“untreatable”. In fact, there is some evidence to suggest that antibodies may lead to a cure
for AIDS.

10. M
 onoclonal antibodies can be used to classify strains of a single pathogen; e.g.,
Neisseria gonorrhoeae can be typed using monoclonal antibodies.

11. Monoclonal antibodies are used in research to identify and trace specific cells or
molecules in an organism; e.g., use of monoclonal antibodies to find which proteins are
responsible for cell differentiation in the respiratory system.

Chapter 4
ANTIBODY ENGINEERING

4.1 Engineered Antibodies

Engineered antibodies now represent over 30% of biopharmaceuticals in clinical trials, as

highlighted by recent approvals from the US Food and Drug Administration (FDA).
Recombinant antibodies have been reduced in size, rebuilt into multivalent molecules and
fused with, for example radionuclides, toxins, enzymes, liposomes and viruses. The
emergence of recombinant technologies has revolutionized the selection, humanization
and production of antibodies, superseding hybridoma technology and allowing the design
of antibody-based reagents of any specificity and for very diverse purposes.

The discovery of hybridoma technology by Kohler and Milstein in 1975 heralded a new
era in antibody research and clinical development. Mouse hybridomas were the first
reliable source of monoclonal antibodies and were developed for a number of in vivo
therapeutic applications.

The list of approved antibody therapeutics against cancer and against viral and
inflammatory diseases is growing rapidly, with more than 30 antibodies in late-phase
clinical trials. More recently, innovative structural designs have improved in vivo
 57

pharmacokinetics of the engineered antibodies with expanded immune repertoires. Latest

technologies are being used for attaching additional therapeutic domains, radionuclides,
drugs, enzymes and vaccine-inducing epitopes by recombinant fragments.

4.2 Intact Antibodies, Humanization and De-immunization

Intact antibodies provide high-specificity, high-affinity targeting reagents and are usually
multivalent. Their simultaneous binding to two adjacent antigens increases functional
affinity and confers high retention times, for example, on cell surfaces. In addition, intact
antibodies comprise Fc domains, which can be important for cancer immunotherapy
through their abilities both to recruit cytotoxic effector functions and to extend the serum
half-life, mediated by the neonatal Fc receptor. Unmodified mouse monoclonal
antibodies formed the first wave of FDA-approved immunotherapeutic reagents, although
their in vivo applications were limited because repeated administrations provoked an anti-
mouse immune response (Fig.21).
 58

Simple strategies have been developed to avoid, mask or redirect this human immune
surveillance; these strategies include fusion of mouse variable regions to human constant
regions as 'chimeric antibodies’, 'de-immunization' by removal of T-cell epitopes and
'humanization' by grafting mouse surface residues onto human acceptor antibody
frameworks. Modern alternative strategies now allow selection of fully human antibodies
directly from natural or synthetic repertoires, including live transgenic mice producing
purely human antibodies. Human antibody-display libraries were used to transform a
mouse antibody in vitro into a fully human derivative (D2E7), which is the first FDA-
approved fully human anti-inflammatory antibody. Many other fully human antibodies,
including Efalizumab for the treatment of psoriasis, are in clinical evaluation.

4.3 Design of 'Antibody Fragments' for Unique Clinical Applications

For cytokine inactivation, receptor blockade or viral neutralization, the Fc-induced

effector functions are often unwanted and can be simply removed by proteolysis of intact
antibodies to yield monovalent Fab fragments (ReoPro, Remicade). Proteolysis, however,
does not easily yield molecules smaller than a Fab fragment, and microbial expression of
single-chain Fv (scFv) is currently the favored method of production. In scFvs, the
variable (VH and VL) domains are stably tethered together with a flexible polypeptide
linker. In comparison with whole antibodies, small antibody fragments such as Fab or
scFv exhibit better pharmacokinetics for tissue penetration and also provide full binding
specificity because the antigen-binding surface is unaltered. However, Fab and scFv are
monovalent and often exhibit fast off-rates and poor retention time on the target.
Therefore, Fab and scFv fragments have been engineered into dimeric, trimeric or
tetrameric conjugates to increase functional affinity through the use of either chemical or
genetic cross-links. Various methods have been devised to genetically encode multimeric
scFvs, of which the most successful design was the simple reduction of scFv linker length
to direct the formation of bivalent dimers (diabodies, 60 kDa), triabodies (90 kDa) or
tetrabodies (120 kDa). Indeed, the first clinical trials of scFv fragments are likely to be as
multivalent reagents, because they exhibit high functional affinity and have been very
successful in preclinical studies
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4.4 Pharmacokinetics of Intact Antibodies Versus Fragments

The efficiency of antibodies in vivo, for example in cancer therapy, lies in their capacity
to discriminate among tumor-associated antigens at low levels. Immunotherapy has been
more successful against circulating cancer cells than solid tumors because of better cell
accessibility. This is illustrated by the FDA approval of intact antibodies, Rituxan, for the
treatment of non-Hodgkin lymphoma and Campath and Mylotarg for the treatment of
leukemia. Only two monoclonal antibodies have been approved for the treatment of solid
tumors: Herceptin for the treatment of breast carcinoma and PanoRex for colon cancer (in
Germany). Although the mechanisms of action are still under investigation, Herceptin
appears to utilize Fc receptors and angiogenesis, whereas Rituxan activates apoptosis
through receptor dimerization.

Radiolabeled antibodies are important clinical reagents for both tumor imaging and
therapy and also provide an effective evaluation of pharmacokinetics. The choice of
radionuclide dictates the application. For example, Zevalin is approved for lymphoma
therapy as a rapidly cleared, intact mouse antibody to match the clearance rates of
yttrium-90. Therapeutic administration requires a balance between long dissociation rates
at the target site and slow blood clearance, which can lead to accumulation in the liver
and high radiation exposure of other tissues. Biodistribution studies in solid tumors have
also revealed that whole IgG molecules are too large (150 kDa) for rapid tumor
penetration. The best tumor-targeting reagents comprise an intermediate-sized
multivalent molecule, providing rapid tissue penetration, high target retention and rapid
blood clearance. For example, diabodies (60 kDa) are efficacious with short-lived
radioisotopes for clinical imaging as a result of the fast clearance rates. Larger molecules,
such as minibodies (90 kDa), are used with long-lived radioisotopes and are suitable for
tumor therapy because they achieve a higher total tumor 'load'. Fab dimers (110 kDa)
have also been effective in preclinical studies.

The short half-life of antibody fragments can also be extended by 'pegylation', that is, a
fusion to polyethylene glycol (PEG). Renal and hepatic localization of intact radiolabeled
antibody fragments constitutes a major problem. An important study demonstrated that a
 60

previously undescribed radio- iodination reagent could liberate radionuclide from the
antibody fragment before incorporation into renal cells. The radionuclide is excreted
rapidly, thus decreasing the total renal radiation dose. The development of new
metabolizable chelates will further improve the pharmacokinetics of recombinant
antibodies for cancer targeting. Modifications to surface charge designed to alter the
isoelectric point (pI), such as glycolation, can also reduce the tissue (kidney) uptake. The
improved functional affinity, tumor penetration and biodistribution of these engineered
antibody fragments will stimulate the development of a new generation of reagents for
imaging and therapy.

4.5 Engineering Multiple Specificity in Antibody Fragments

Bispecific antibodies contain two different binding specificities fused together and, in the
most simple example, bind to two adjacent epitopes on a single target antigen, thereby
increasing the avidity. Alternatively, bispecific antibodies can cross-link two different
antigens and are powerful therapeutic reagents, particularly for recruitment of cytotoxic T
cells for cancer treatment. Bispecific antibodies can be produced by fusion of two
hybridoma cell lines into a single 'quadroma' cell line; however, this technique is
complex and time-consuming, and it produces unwanted pairing of the heavy and light
chains. Far more effective methods to couple two different Fab modules incorporate
either chemical or genetic conjugation or fusion to adhesive heterodimeric domains,
including designed CH3 domains. Bispecific diabodies provide an innovative alternative
therapeutic.

4.6 Bifunctional Antibodies

The original 'magic bullet' concept is still alive: antibodies have been fused to a vast
range of molecules that provide important ancillary functions after target binding. These
include radionuclides and also cytotoxic drugs, toxins, peptides, proteins, enzymes and
viruses, the latter for targeted gene therapy. For cancer therapy, bifunctional antibodies
are engineered to effectively target tumor-associated antigens at low levels and then
deliver a cytotoxic payload to tumor cells. The latest antibody-toxin conjugates are stable
 61

in vivo and minimally immunogenic. Antibodies have also been fused to lipids and PEGs,
both to enhance in vivo delivery and pharmacokinetics and to direct drug-loaded
liposomes. As immunoliposomes, anti-transferrin receptor antibodies have been used to
deliver drugs to the brain, passing through the blood-brain barrier. Antibody-enzyme
fusions have also been developed for prodrug activation, primarily for cancer therapy.

4.7 Antibody Libraries: Construction, Display and Selection

Library display has superseded hybridoma technology for the selection of human
antibodies through the creation of large natural and synthetic immune repertoires in vitro.
From these libraries, specific high-affinity antibodies can be selected by linking
phenotype (binding affinity) to genotype, thereby allowing simultaneous recovery of the
gene encoding the selected antibody. Antibodies are usually displayed as monovalent Fab
or scFv fragments and then, as required, reassembled into intact Ig or multivalent variants
after selection. If the repertoire is sufficiently large, a high-affinity Fab or scFv can be
selected directly or, more frequently, the recovered gene can be subjected to cycles of
mutation and further selection to enhance affinity. Furthermore, new methods of selection
and screening have been designed to specifically isolate antibodies with desired
characteristics, such as enhanced stability, high expression or capacity to activate
receptors.

i) Bacteriophage display. Fd phage and Fd phagemid technologies are currently the

most widely used in vitro methods for the display of large repertoires and for the
selection of high-affinity recombinant antibodies against a range of clinically important
target molecules. Innovative selection methods have proved powerful for isolating
antibodies against previously refractory antigens, such as new tumor-associated antigens,
cell surface receptors and HLA-A1-presented peptides. Important improvements in
selection technology have included array screening for high-avidity antibodies and
recovery of internalized phage from live cells to select against internalizing (human)
receptors. Phage technology has been applied to complete proteome analysis using
membrane-based screening.
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ii) Libraries of mRNA-protein complexes. Ribosome display relies on stabilized

complexes of antibody, ribosome and mRNA to replace bacteriophage as the display
platform. Ribosome complexes are constructed totally in vitro, thereby eliminating the
need for cell transformation and allowing the production of large libraries, 1014
members. The system is limited only by the requirement of a ribonuclease-free
environment for selection and buffer compositions suitable for antibody folding. Indeed,
picomolar affinity antibodies have been selected and rapid affinity maturation cycles
carried out using this innovative in vitro method. Covalent display using puromycin-
stabilized mRNA-protein complexes is an alternative strategy to ribosome display.

iii) Cell surface libraries. Before the advent of bacteriophage systems, antibodies had
been displayed on or in bacterial cells, although replica plating had limited screening to
libraries of <108. The recent development of high-speed flow cytometers has re-activated
the efforts in cell surface display, and several high-affinity antibodies have been isolated
by this method.

iv) Transgenic mice. Transgenic mice have been produced that lack the native mouse
immune repertoire and instead harbor most of the human antibody repertoire in the
germline. Injection of antigens into these mice leads to the development of human
antibodies that have undergone mouse somatic hypermutation and selection to relatively
high affinity. Antibodies can be recovered by classic hybridoma technology or, for more
efficient affinity enhancement, by in vitro display and selection technologies.

v) Production, stability and expression levels

Production of antibodies for preclinical and clinical trials has been evaluated in numerous
expression systems, including bacteria, yeast, plant, insect and mammalian cells. Bacteria
are favored for expression of small, non-glycosylated Fab and scFv fragments, usually
with terminal polypeptides such as c-Myc, His or FLAG, for affinity purification.
Mammalian or plant cells are favored for intact antibodies and, occasionally, also for
expression of scFvs, diabodies and minibodies. There is still hope that eukaryotic cell
cultures, such as those of the yeast Pichia pastoris, will allow efficient production of
fully processed scFvs, albeit with high-mannose oligosaccharides.
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v) Affinity maturation
Both transgenic mice and display libraries typically produce human antibodies with
binding affinities (KD) ranging from 10-7 to 10-9 M. Obtaining higher-affinity antibodies is
important for efficient binding to the antigenic target for in vitro diagnosis, viral
neutralization, cell targeting and in vivo imaging. To improve antibody affinity, various
in vitro strategies have recently been optimized to mimic the mammalian in vivo process
of somatic hypermutation and selection. These include site-specific mutagenesis based on
structural information, combinatorial mutagenesis of complementarity-determining
regions (CDRs), random mutagenesis of the entire gene or chain shuffling.

After a decade of developing library display strategies, it is now obvious that the most
successful methods rely on several cycles of mutation, display, selection (recovery) and
gene amplification. These cycles of mutation and selection can be carried out using either
in vitro or in vivo strategies and have been far more effective than precisely designed
alterations for affinity enhancement. Even with the most detailed structural information,
the techniques for design of precisely complementary surfaces through interface
mutations remain in their infancy.

Affinity enhancement can be restricted to mutations in the antigen-binding surface (CDR

loops). Importantly, mutations in the underlying framework regions have frequently
provided large increases in affinity, stability and expression. Random mutations over the
entire V-domain genes can be derived from Escherichia coli mutator cells, homologous
gene rearrangements or error-prone PCR. Sequential 'chain shuffling' of the two V genes
in the Fv module is also 'random' but offers the advantage that only one V domain is
altered at a time, while the other domain is kept constant to provide a defined specificity.
Recent advances include the incorporation of highly mutagenic enzymes such as mRNA
reverse transcriptase and DNA polymerase with no proofreading activity to achieve a
high gene mutation rate. The integration of such polymerases into the ribosome display
and selection process could rapidly generate large libraries of mutants.

vi) Alternative scaffolds

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Intact antibodies, Fab and scFv fragments provide an antigen-binding surface comprising
six CDR loops; these can be mutated, sequentially or collectively, to bind to a vast array
of target molecules. Some target molecules are refractory to the immune repertoire,
however, particularly those with cavities or clefts that require a small penetrating loop for
tight binding. The natural mammalian antibody repertoire simply does not encode
penetrating loops, and only rarely has this type of antibody been selected. Unexpectedly,
both camelids (camels, llamas and related species) and sharks produce natural, single V-
like domain repertoires displaying cavity-penetrating CDR loops that complement the
repertoire of conventional antibodies. This theory has led to a number of attempts to
design single-domain display libraries in vitro, based on V domains and other Ig-like
scaffolds. These small molecules complement both antibody and peptide libraries and are
expected to have improved pharmacokinetics for several clinical applications, including
those that require access to buried (immunosilent) sites or clefts in enzymes, receptors
and viruses.

vii) Clinical applications

Aside from radioimmunotherapy, there are a variety of other clinical applications of

engineered antibodies for viral infection, cancer, autoimmune disease, allograft rejection,
asthma, stroke and glaucoma surgery. Specific clinical applications are discussed below.

a) Pathogen neutralization and antiviral therapy

Antibody binding can directly and effectively block the activity of many pathogens, often
without requiring Fc-mediated cytotoxicity. Indeed, this has always been the promise of
antibody-mediated viral neutralization. The first monoclonal antibody for the treatment of
viral disease, Synagis, was approved by the FDA in 1998. Synagis is a humanized
antibody used for the prevention of severe respiratory syncytial virus (RSV) disease.
Despite this success, and the wide range of antibodies available against human
immunodeficiency type 1 (HIV) and herpes simplex virus (HSV), the use of recombinant
antibodies as therapeutics for viral infection has been limited. Only a few rare antibodies
 65

have exhibited potent neutralization in vitro and antiviral efficacy in animal models. This
is probably due to viral efficiency both in producing escape mutants and in evolving
immunosilent receptor-binding surfaces. For neutralization of other pathogenic
molecules, monomeric Fab molecules have recently been approved by the FDA as
antivenenes (CroFab), and both scFv fragments and oligoclonal antibody mixtures have
been effective against bacterial toxins.

b) Intracellular antibodies

Antibody fragments can be expressed as intracellular proteins, typically as scFvs termed

intrabodies, and equipped with targeting signals either to neutralize intracellular gene
products or to target cellular pathways. For example, expression of p21ras, erbB2,
huntingtin and MHC have all been individually downregulated using antibodies.
Intrabodies also have important antiviral potential, particularly through their targeting of
intracellular action to mandatory viral proteins such as the Vif, Tat or Rev components of
HIV. Antibody frameworks have been adapted that substantially improve expression
levels and solubility in the intracellular reducing environment. Direct in vivo selection
from large libraries will greatly facilitate the isolation of many previously unknown
intracellular antibodies or 'intrabodies'. Obviously, the expression of intrabodies in vivo
can be encoded into gene therapy vectors, and this could ultimately be their most
powerful clinical application.

c) Cancer therapy and cell recruitment strategies

The promise of engineered antibodies for effective cancer therapy, especially

radioconjugates, has been described earlier. Blocking angiogenesis to prevent the
establishment and growth of tumors is becoming an important strategy. Cancer cells can
be destroyed by cell recruitment of cytotoxic T cells, natural-killer (NK) cells or
macrophages that can be targeted by encoding cell surface antibodies (usually scFv).
Alternative cell recruitment strategies include bifunctional antibodies, fused to cytokines
for T-cell stimulation and proliferation at the tumor site.
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d) Innovative vaccine applications

Troybodies are engineered vaccine antibodies containing cryptic T-cell epitopes to

enhance antigen presentation. Troybodies effectively target antigen-presenting cells
(APCs) and, after processing, expose cryptic T-cell epitopes to direct T-cell activation. In
the preferred format, the Fv domain provides APC specificity and the C domains encode
the cryptic T-cell epitopes. These new vaccines can be redesigned to target many
different APCs and enhance immunity to many different T-cell epitopes. Alternative
vaccine strategies include the use of engineered APC-targeted antibodies that direct
adenoviruses to deliver vaccine-inducing epitopes as a gene therapy capsule and B7-
targeted scaffolds (scFv and VL domains) that enable antigen-loading of dendritic cells.

e) Biosensors and microarrays: the future of diagnosis

Antibodies currently provide high-sensitivity reagents for a huge range of diagnostic kits,
accounting for approximating 30% of the $20 billion per year diagnostic industry. It is
therefore not surprising that antibodies are the paradigm for proof-in-principle of new
biosensing devices, focused initially on glass-surface microarrays. More protein-friendly
surfaces being developed as array platforms for antibody-based diagnosis. These
platforms will become increasingly available over the next few years, driven by the
demand for new reagents to diagnose the vast array of biomarkers stemming from
proteomics discovery programs. These platforms will also be developed for robust ex
vivo applications, including the detection of microbial contaminants, pesticides and
biological (warfare) pathogens.

By providing a highly stable, protease-resistant scaffold, engineered recombinant

antibody fragments will continue to be the model for selection of high-affinity clinical
targeting reagents.
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Chapter 5
VACCINOLOGY
5.1 History

The earliest vaccines were based on the concept of variolation originating in China, in
which a person is deliberately infected with a weak form of smallpox as a form of
inoculation. Jenner realized that milkmaids who had contact with cowpox did not get
smallpox. The process of distributing and administrating vaccines is thus referred to as
"vaccination". Jenner's work was continued by Louis Pasteur and others in the 19th
century. Since vaccination against smallpox was much safer than smallpox inoculation,
the latter fell into disuse and was eventually banned in England in 1849.

The 19th and 20th centuries saw the introduction of several successful vaccines against a
number of infectious diseases. These included bacterial and viral diseases, but not (to
date) any parasitic diseases. Vaccines may be dead or inactivated organisms or purified
products derived from them.

5.2 Types

There are four types of traditional vaccines:

• Vaccines containing killed microorganisms - these are previously virulent micro-

organisms which have been killed with chemicals or heat. Examples are vaccines
against flu, cholera, bubonic plague, and hepatitis A.
• Vaccines containing live, attenuated virus microorganisms - these are live micro-
organisms that have been cultivated under conditions that disable their virulent
properties or which use closely-related but less dangerous organisms to produce a
broad immune response. They typically provoke more durable immunological
responses and are the preferred type for healthy adults. Examples include yellow
 68

fever, measles, rubella, and mumps. The live tuberculosis vaccine is not the
contagious strain, but a related strain called "BCG"; it is used in the United States
very infrequently.
• Toxoids - these are inactivated toxic compounds in cases where these (rather than
the micro-organism itself) cause illness. Examples of toxoid-based vaccines
include tetanus and diphtheria. Not all toxoids are for micro-organisms; for
example, Crotalis atrox toxoid is used to vaccinate dogs against rattlesnake bites.
• Subunit - rather than introducing an inactivated or attenuated micro-organism to
an immune system (which would constitute a "whole-agent" vaccine), a fragment
of it can create an immune response. Characteristic examples include the subunit
vaccine against HBV that is composed of only the surface proteins of the virus
(produced in yeast) and the virus-like particle (VLP) vaccine against human
papillomavirus (HPV) that is composed of the viral major capsid protein.

A number of innovative vaccines are also in development and in use:

• Conjugate - certain bacteria have polysaccharide outer coats that are poorly
immunogenic. By linking these outer coats to proteins (e.g. toxins), the immune
system can be led to recognize the polysaccharide as if it were a protein antigen.
This approach is used in the Haemophilus influenzae type B vaccine
• Recombinant Vector - by combining the physiology of one micro-organism and
the DNA of the other, immunity can be created against diseases that have
complex infection processes
• DNA vaccination - in recent years a new type of vaccine, created from an
infectious agent's DNA called DNA vaccination, has been developed. It works by
insertion (and expression, triggering immune system recognition) into human or
animal cells, of viral or bacterial DNA. Some cells of the immune system that
recognize the proteins expressed will mount an attack against these proteins and
cells expressing them. Because these cells live for a very long time, if the
pathogen that normally expresses these proteins is encountered at a later time,
they will be attacked instantly by the immune system. One advantage of DNA
vaccines is that they are very easy to produce and store. As of 2006, DNA
vaccination is still experimental.
 69

While most vaccines are created using inactivated or attenuated compounds from micro-
organisms, synthetic vaccines are composed mainly or wholly of synthetic peptides,
carbohydrates or antigens. Vaccines may be monovalent (also called univalent) or
multivalent (also called polyvalent). A monovalent vaccine is designed to immunize
against a single antigen or single microorganism. A multivalent or polyvalent vaccine is
designed to immunize against two or more strains of the same microorganism, or against
two or more microorganisms.

5.3 Developing immunity

The immune system recognizes vaccine agents as foreign, destroys them, and
'remembers' them. When the virulent version of an agent comes along the body
recognises the protein coat on the virus, and thus is prepared to respond, by (1)
neutralizing the target agent before it can enter cells, and (2) by recognizing and
destroying infected cells before that agent can multiply to vast numbers.

Vaccines have contributed to the eradication of smallpox, one of the most contagious and
deadly diseases known to man. Other diseases such as rubella, polio, measles, mumps,
chickenpox and typhoid are nowhere near as common as they were a hundred years ago.
As long as the vast majority of people are vaccinated, it is much more difficult for an
outbreak of disease to occur, let alone spread. This effect is called herd immunity. Polio,
which is transmitted only between humans, is targeted by an extensive eradication
campaign that has seen endemic polio restricted to only parts of four countries. The
difficulty of reaching all children as well as cultural misunderstandings, however, have
caused the anticipated eradication date to be missed several times.

a) Schedule

In order to provide best protection, children are recommended to receive vaccinations as

soon as their immune systems are sufficiently developed to respond to particular
vaccines, with additional 'booster' shots often required to achieve 'full immunity'. This
has led to the development of complex vaccination schedules.
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Adjuvants are typically used to boost immune response. Adjuvants are sometimes called
the dirty little secret of vaccines in the scientific community, as not much is known about
how adjuvants work. Most often aluminium adjuvants are used, but adjuvants like
squalene are also used in some vaccines and more vaccines with squalene and phosphate
adjuvants are being tested. The efficacy or performance of the vaccine is dependent on a
number of factors:

• the disease itself (for some diseases vaccination performs better than for other
diseases)
• the strain of vaccine (some vaccinations are for different strains of the disease)
• whether one kept to the timetable for the vaccinations
• some individuals are 'non-responders' to certain vaccines, meaning that they do
not generate antibodies even after being vaccinated correctly
• other factors such as ethnicity or genetic predisposition

When a vaccinated individual does develop the disease vaccinated against, the disease is
likely to be milder than without vaccination.

The following are important considerations in the effectiveness of a vaccination program:

1. Careful modelling to anticipate the impact that an immunisation campaign will

have on the epidemiology of the disease in the medium to long term
2. Ongoing surveillance for the relevant disease following introduction of a new
vaccine and
3. Maintaining high immunization rates, even when a disease has become rare.

In 1958 there were 763,094 cases of measles and 552 deaths in the United States. With
the help of new vaccines, the number of cases dropped to fewer than 150 per year
(median of 56). In early 2008, there were 64 suspected cases of measles. 54 out of 64
infections were associated with importation from another country, although only 13%
were actually acquired outside of the United States; 63 of these 64 individuals either had
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never been vaccinated against measles, or were uncertain whether they had been
vaccinated.

b) Other developments in vaccine production

• Rational attenuation. Specific modifications or deletions of genes that confer

virulence reduce or remove the pathogenicity of the microbe while still allowing
an immune response to be generated. This type of rational attenuation can be
viewed as creating a "live" attenuated vaccine.

• Vector-mediated subunit delivery. Introducing a non-infectious, non-pathogenic

subunit into a live vector can prompt an immune response without presence of the
pathogen. This is called vector-mediated subunit delivery. For example, rabies
surface protein gene has been inserted into vaccinia virus.

• Virus-like particles. Capsid proteins of icosahedral viruses assemble without the

presence of a genome. These virus-like particles are antigenically authentic, but
non-infectious. This has been used for HPV-16 and HPV-18 vaccines.

c) Preservatives

Many vaccines need preservatives to prevent serious adverse effects such as the
Staphylococcus infection that, in one 1928 incident, killed 12 of 21 children inoculated
with a diphtheria vaccine that lacked a preservative. Several preservatives are available,
including thiomersal, 2-phenoxyethanol and formaldehyde. Thiomersal is more effective
against bacteria, has better shelf life, and improves vaccine stability, potency, and safety,
but in the U.S., the European Union, and a few other affluent countries, it is no longer
used as a preservative in childhood vaccines, as a precautionary measure due to its
mercury content. Controversial claims have been made that thiomersal contributes to
autism; no convincing scientific evidence supports these claims.

d) Delivery systems
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There are several new delivery systems in development, which will hopefully make
vaccines more efficient to deliver. Possible methods include liposomes and ISCOM
(immune stimulating complex).

e) Plasmids

The use of plasmids has been validated in preclinical studies as a protective vaccine
strategy for cancer and infectious diseases. However, the crossover application into
human studies has been met with poor results based on the inability to provide clinically
relevant benefit. The overall efficacy of plasmid DNA immunization depends on
increasing the plasmid's immunogenicity while also correcting for factors involved in the
specific activation of immune effector cells.

f) Use in nonhumans

Vaccinations of animals are used both to prevent their contracting diseases and to prevent
transmission of disease to humans. Both animals kept as pets and animals raised as
livestock are routinely vaccinated. In some instances, wild populations may be
vaccinated. This is sometimes accomplished with vaccine-laced food spread in a disease-
prone area and has been used to attempt to control rabies in raccoons.

Where rabies occurs, rabies vaccination of dogs may be required by law. Other canine
vaccines include canine distemper, parvovirus, canine adenovirus-2, leptospirosis,
bordatella, canine parainfluenza virus, and Lyme disease among others.

Vaccine development has several trends:

• Vaccines against cancers, autoimmune, and other noninfectious diseases are being
developed.
• Until now, most vaccines have been aimed at infants and children, but adolescents
and adults are increasingly being targeted.
• Combinations of vaccines are becoming more common; vaccines containing five
or more components are used in many parts of the world.
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• New methods of administering vaccines are being developed, such as skin

patches, aerosols via inhalation devices, and eating genetically engineered plants.
• Vaccines are being designed to stimulate innate immune responses, as well as
adaptive.
• Attempts are being made to develop vaccines to help cure chronic infections, as
opposed to preventing disease.
• Vaccines are being developed to defend against bioterrorist attacks such as
anthrax, plague and smallpox.

5.4 Different types of vaccine

Whole virus vaccines. either live or killed, constitute the vast majority of vaccines in use
at present. However, recent advances in molecular biology had provided alternative
methods for producing vaccines. Listed below are the possibilities;-

1. Live whole virus vaccines

2. Killed whole virus vaccines
3. Subunit vaccines: purified or recombinant viral antigen
4. Recombinant virus vaccines
5. Anti-idiotype antibodies
6. DNA vaccines

1. Live vaccines

Live virus vaccines are prepared from attenuated strains that are almost or completely
devoid of pathogenicity but are capable of inducing a protective immune response. They
multiply in the human host and provide continuous antigenic stimulation over a period of
time, Primary vaccine failures are uncommon and are usually the result of inadequate
storage or administration. Another possibility is interference by related viruses as is
suspected in the case of oral polio vaccine in developing countries. Several methods have
been used to attenuate viruses for vaccine production.
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a) Use of a related virus from another animal - the earliest example was the use of
cowpox to prevent smallpox. The origin of the vaccinia viruses used for production is
uncertain.

b) Administration of pathogenic or partially attenuated virus by an unnatural route

- the virulence of the virus is often reduced when administered by an unnatural route.
This principle is used in the immunization of military recruits against adult respiratory
distress syndrome using enterically coated live adenovirus type 4, 7 and (21).

c) Passage of the virus in an "unnatural host" or host cell - the major vaccines used in
man and animals have all been derived this way. After repeated passages, the virus is
administered to the natural host. The initial passages are made in healthy animals or in
primary cell cultures. There are several examples of this approach: the 17D strain of
yellow fever was developed by passage in mice and then in chick embryos. Polioviruses
were passaged in monkey kidney cells and measles in chick embryo fibroblasts. Human
diploid cells are now widely used such as the WI-38 and MRC-5. The molecular basis for
host range mutation is now beginning to be understood.

d) Development of temperature sensitive mutants - this method may be used in

conjunction with the above method.

2. Inactivated whole virus vaccines

These were the easiest preparations to use. The preparation was simply inactivated. The
outer virion coat should be left intact but the replicative function should be destroyed. To
be effective, non-replicating virus vaccines must contain much more antigen than live
vaccines that are able to replicate in the host. Preparation of killed vaccines may take the
route of heat or chemicals. The chemicals used include formaldehyde or beta-
propiolactone. The traditional agent for inactivation of the virus is formalin. Excessive
treatment can destroy immunogenicity whereas insufficient treatment can leave infectious
virus capable of causing disease. Soon after the introduction of inactivated polio vaccine,
there was an outbreak of paralytic poliomyelitis in the USA use to the distribution of
inadequately inactivated polio vaccine. This incident led to a review of the formalin
inactivation procedure and other inactivating agents are now available, such as Beta-
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propiolactone. Another problem was that SV40 was occasionally found as a contaminant
and there were fears of the potential oncogenic nature of the virus.

i) Features of Live and Dead vaccines

__________________________________________________________________

Feature Live Dead

__________________________________________________________________

Dose Low High

No. of doses Single Multiple

Need for adjuvant No Yes

Duration of immunity Many years Less

Antibody response IgG, IgA IgG

CMI Good Poor

Reversion to virulence Possible Not possible

___________________________________________________________________

Because live vaccines replicate inside host cells, remnants of viral antigens are presented
to the cell surface and recognized by cytotoxic cells.

ii) Potential safety problems

a) Live vaccines

1. Under attenuation.
2. Mutation leading to reversion to virulence.
3. Preparation instability.
4. Contaminating viruses in cultured cells.
5. Heat liability.
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6. Should not be given to immunocompromized or pregnant patients.

b) Killed vaccines

1. Incomplete inactivation.
2. Increased risk of allergic reactions due to large amounts of antigen involved.

Potential problems associated with vaccine development:

1. Failure to grow large amounts of organisms in laboratory.

2. Crude antigen preparations often give poor protection. eg. key antigen is not
identified, ignorance of the nature of the protective or the protective immune
response.
3. Live vaccines of certain viruses can induce reactivation (reversion) and/or be
oncogenic in nature.

3. Subunit Vaccines

Originally, non-replicating vaccines were derived from crude preparations of virus from
animal tissues. As the technology for growing viruses to high titres in cell cultures
advanced, it became practicable to purify virus and viral antigens. It is now possible to
identify the peptide sites encompassing the major antigenic sites of viral antigens, from
which highly purified subunit vaccines can be produced. Increasing purification may lead
to loss of immunogenicity, and this may necessitate coupling to an immunogenic carrier
protein or adjuvant, such as an aluminum salt. Examples of purified subunit vaccines
include the HA vaccines for influenza A and B, and HBsAg derived from the plasma of
carriers.

4. Recombinant Viral Proteins

Virus proteins have been expressed in bacteria, yeast, mammalian cells and viruses. E.
coli cells were first to be used for this purpose but the expressed proteins were not
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glycosylated, which was a major drawback since many of the immunogenic proteins of
viruses such as the envelope glycoproteins, were glycosylated. Nevertheless, in many
instances, it was demonstrated that the non-glycosylated protein backbone was just as
immunogenic. Recombinant hepatitis B vaccine is the only recombinant vaccine licensed
at present.

An alternative application of recombinant DNA technology is the production of hybrid

virus vaccines. The best known example is vaccinia; the DNA sequence coding for the
foreign gene is inserted into the plasmid vector along with a vaccinia virus promoter and
vaccinia thymidine kinase sequences. The resultant recombination vector is then
introduced into cells infected with vaccinia virus to generate a virus that expresses the
foreign gene. The recombinant virus vaccine can then multiply in infected cells and
produce the antigens of a wide range of viruses. The genes of several viruses can be
inserted, so the potential exists for producing polyvalent live vaccines. HBsAg, rabies,
HSV and other viruses have been expressed in vaccinia.

Hybrid virus vaccines are stable and stimulate both cellular and humoral immunity. They
are relatively cheap and simple to produce. Being live vaccines, smaller quantities are
required for immunization. As yet, there are no accepted laboratory markers of
attenuation or virulence of vaccinia virus for man. Alterations in the genome of vaccinia
virus during the selection of recombinant may alter the virulence of the virus. The use of
vaccinia also carries the risk of adverse reactions associated with the vaccine and the
virus may spread to susceptible contacts. At present, efforts are being made to attenuate
vaccinia virus further and the possibility of using other recombinant vectors is being
explored, such as attenuated poliovirus and adenovirus.

5. Synthetic Peptides

The development of synthetic peptides that might be useful as vaccines depends on the
identification of immunogenic sites. Several methods have been used. The best known
example is foot and mouth disease, where protection was achieved by immunizing
animals with a linear sequence of 20 aminoacids. Synthetic peptide vaccines would have
many advantages. Their antigens are precisely defined and free from unnecessary
components which may be associated with side effects. They are stable and relatively
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cheap to manufacture. Furthermore, less quality assurance is required. Changes due to

natural variation of the virus can be readily accommodated, which would be a great
advantage for unstable viruses such as influenza.

Synthetic peptides do not readily stimulate T cells. It was generally assumed that,
because of their small size, peptides would behave like haptens and would therefore
require coupling to a protein carrier which is recognized by T-cells. It is now known that
synthetic peptides can be highly immunogenic in their free form provided they contain, in
addition to the B cell epitope, T- cell epitopes recognized by T-helper cells. Such T-cell
epitopes can be provided by carrier protein molecules, foreign antigens. or within the
synthetic peptide molecule itself.

Synthetic peptides are not applicable to all viruses. This approach did not work in the
case of polioviruses because the important antigenic sites were made up of 2 or more
different viral capsid proteins so that it was in a concise 3-D conformation.

a) Advantages of defined viral antigens or peptides include

1. Production and quality control simpler

2. Safer in cases where viruses are oncogenic or establish a persistent infection
3. Feasible even if virus cannot be cultivated

b) Disadvantages

1. May be less immunogenic than conventional inactivated whole-virus vaccines

2. Requires adjuvant
3. Requires primary course of injections followed by boosters
4. Fails to elicit CMI

6. Anti-idiotype Antibodies

The ability of anti-idiotype antibodies to mimic foreign antigens has led to their
development as vaccines to induce immunity against viruses, bacteria and protozoa in
experimental animals. Anti-idiotypes have many potential uses as viral vaccines,
particularly when the antigen is difficult to grow or hazardous. They have been used to
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induce immunity against a wide range of viruses, including HBV, rabies, Newcastle
disease virus and FeLV, reoviruses and polioviruses.

Chapter 6
DNA VACCINES

6.1 Introduction

Encouraging results were reported for DNA vaccines whereby DNA coding for the
foreign antigen is directly injected into the animal so that the foreign antigen is directly
produced by the host cells. In theory these vaccines would be extremely safe and devoid
of side effects since the foreign antigens would be directly produced by the host animal.
In addition, DNA is relatively inexpensive and easier to produce than conventional
vaccines and thus this technology may one day increase the availability of vaccines to
developing countries. Moreover, the time for development is relatively short which may
enable timely immunization against emerging infectious diseases. The major reason is the
DNA vaccines can be generated adopting simple procedures which do not require making
and purification of recombinant proteins and organisms. In addition, DNA vaccines can
theoretically result in more long-term production of an antigenic protein when introduced
into a relatively nondividing tissue, such as muscle. The clinical trials using injections of
DNA to stimulate an immune response against a foreign protein began for HIV,
influenza, herpes simplex virus and, T-cell lymphoma.

6.2 Methods of gene delivery

Several routes of plasmid DNA inoculation have been undertaken in animal models.
These include intra-muscular, subcutaneous, intra-peritoneal, intra-dermal, subcutaneous,
intravenous, oral, rectal, intra-bursal, intra-orbital, intra-tracheal, intra-nasal, and vaginal
routes. In the case of a plasmid DNA vaccine for a tumor, it can be injected into the
tumor site. The most common routes of administration are by injecting the plasmid DNA
dissolved in saline intra-muscularly or intradermally using a hypodermic needle or by
bombarding plasmid DNA coated onto colloidal gold micro-particles in the dermis or
muscle using a gene gun. The gene gun accelerates the entry of particles into the target
tissue by a controlled discharge through a shock wave created by a chemical propellant,
 80

expansion of a compressed gas or an electric spark. The dose used in mice depends on the
method of administration. Usually an immune response is generated when 10-100 μg of
plasmid DNA is injected and 0.1-1 μg when it is administered with a gene gun. The
immune response is increased when 1 or 2 boosters are given. It has been shown that an
increase in the time interval between immunizing doses resulted in an increased immune
response.

The currently used DNA vaccines are constructed based on bacterial plasmids to allow
their propagation in E. coli, and contain the gene encoding the antigen located
downstream of a promoter that is operational in human cells. The promoter of choice is
the intermediate-early promoter of human cytomegalovirus (CMV). Many vaccines also
employ an intron between the promoter and the antigen coding sequence, which is
reported to give better levels of gene expression. DNA can be administered to
experimental animals through several different routes, but intramuscular and intradermal
routes are most commonly.

A device called gene gun has been developed for intradermal immunization. It can propel
DNA-coated, 1-3 picomoles of gold particles into the skin. This procedure can transfect
cells more efficiently than intramuscular injection because only smaller amounts of DNA
are required to achieve immunization. Better TH1 type responses are elicited by
intramuscular injection, whereas the gene gun preferentially induces TH 2-type
responses. An important and useful property of DNA vaccines is their ability to elicit
both cytotoxic and humoral immune responses. The delivery of the nucleoprotein (NP)
gene of influenza virus into mice by intramuscular injection elicited NP-specific
antibodies and Tc cells. Moreover, these animals were protected against a challenge of
live influenza virus. In intramuscular administration, muscle cells become transfected and
become the site in which the antigen is produced. However, muscle cells do not normally
possess the co-stimulatory molecules required for the activation of naive T lymphocytes.
Therefore, the antigen is probably acquired in some way by professional APCs within the
muscle, which then migrate to draining lymph nodes.

6.3 Molecular aspects of DNA Vaccine Production

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DNA vaccines are bacterial plasmids that are designed to express a reporter gene in the
cells of a host. The reporter gene encodes a protein, which could be among others an
antigen of a disease-causing organism, a tumor antigen, an allergen, a cytokine or a co-
stimulatory molecule. One or more sequences known to encode reporter genes in the host
can be inserted in the plasmid. The strategies include the following components:

a) A strong eukaryotic promoter to drive transcription of the reporter gene. Most studies
use viral promoters, in particular the human cytomegalovirus immediate-early promoter.
b) A LacZ gene which encodes the first 146 amino acids of the enzyme beta
galactosidase.
c) A cloning site within the LacZ gene to insert the reporter gene (one or more genes can
be inserted).
(d) A 3’ polyadenylation termination sequence (3’ polyA tail) derived from either the
simian virus 40 or the bovine growth hormone (this provides stability to transcribed
messenger RNA).
(e) A prokaryotic “origin of replication” for plasmid vector amplification in bacteria (E.
coli competent cells are mainly used).
(f) A selectable marker such as an antibiotic resistant gene (usually ampicillin or
kanamycin is used) to select the tranformed E. coli. A plasmid-reporter gene construct is
prepared by treating the plasmid and the DNA containing the reporter gene with the same
restriction endonuclease. E. coli possessing the chromosomal gene that codes for a
segment of beta-galactosidase is cultured on medium containing the recombinant
preparation, isopropylthiogalactoside (IPTG, inducer of the lactose operon), 5-bromo-4-
chloroindolyl-beta-galactoside (X-gal) and an antibiotic (ampicillin or kanamycin). Non-
transformed cells will not grow in the presence of the antibiotic.

6.4 Duration and Strength of the Immune Response

This involves the direct injection of plasmids with loops of DNA that contain genes for
proteins of the pathogenic organism. Once injected into the host's muscle tissue, the DNA
is taken up by host cells, which then start expressing the foreign protein. The protein
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serves as an antigen that stimulates immune responses and provides protective

immunological memory. The efficient delivery of transgenes into cells in vitro is best
accomplished using virus vectors such as adenoviruses or retroviruses. Naked DNA is
taken up by cells much less efficiently; yet it is a very effective means of eliciting
immune responses to the encoded antigens.

A long-lasting immunity is attained when a DNA vaccine is used. Antigen-specific CD4+

T-lymphocytes remained elevated for up to about ten months following immunization
with a DNA vaccine. The CTL responses and antibody levels were observed for up to
about seventeen months in mice immunized with a DNA vaccine containing a reporter
gene coding for an influenza virus protein and a DNA vaccine containing a reporter gene
coding for hepatitis B protein, respectively. The observed prolonged duration of the
immune response in the above-mentioned studies was probably due to the persistence of
the antigen produced in the host. An immunization regimen that may result in an optimal
immune response is to prime the host using the DNA vaccine and subsequently boosting
with the antigen. In one study rhesus monkeys were primed with plasmid DNA
containing HIV env gene. Subsequent boosters were done using a combination of the
plasmid DNA and HIV env protein. Strong CTL and neutralizing antibody activity were
also noticed.

6.5 Th1 and Th2 Lymphocyte Responses

Plasmid DNA vaccines, when injected intramuscularly or intradermally, would induce a

Th1 response because the CpG motifs stimulate the production of IL-12 that favors the
activation of Th1 lymphocytes. On the other hand, if the plasmid DNA is administered
with a gene gun, a Th2 response is generated. Reasons for a biased Th response based on
the method of vaccine administration are yet unknown, but a number of factors need
further investigation. These include, the amount of DNA administered (less when using a
gene gun), host MHC haplotype, nature of the processed antigen and route taken by
administered DNA to arrive at its destination (injection delivers DNA to extracellular
space where it is taken up by cells; gene gun introduces DNA directly into cells).
Injection of BALB/c mice with a plasmid DNA vaccine containing gp63 reporter gene
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from Leishmania major, induced a dominant Th1 response that was protective. In the
case of influenza virus, intra-muscular injection of mice with a plasmid DNA vaccine
encoding influenza NP induced a protective Th1 response.

6.6 Advantages of DNA Vaccines

Plasmid DNA is non-infectious. It does not replicate. It encodes only the antigen of
interest. It does not contain heterologous protein components to which the host may
respond. It induces long lasting cell-mediated (Th1 and CTL) and humoral immunities.
DNA vaccines induce in vivo expression of immunogens and, thus, conserve the native
conformation of epitopes. Conserving an appropriate tertiary structure of proteins is
important for the induction of conformationally specific antibodies and cellular
responses. Also, plasmid DNA can be constructed to include more than one immunogen
gene (bispecific). This would potentially decrease the number of vaccinations required in
children. DNA vaccines offer the possibility of generating effective immune responses
against diseases such as malaria and HIV where other types of vaccines have failed.
Moreover, they may be safer to use than live attenuated vaccines, especially in
immunocompromised hosts. They are stable, easy to freeze dry and reconstitute and can
be manufactured inexpensively in large quantities at high levels of purity.

6.7 Potential Limitations and Demerits

In spite of their multiple advantages, a plasmid DNA vaccine poses some limitations and
potential dangers. It is yet to be established if DNA plasmid vaccines operate in the same
manner in human and mice. Some differences between mice and humans have been
observed. Higher doses of plasmid DNA are needed to induce an immune response in
humans. Immune responses are usually elicited in mice using 0.1-1 μg of plasmid DNA
administered by a gene gun and 10-100 μg (micro gram) administered by injection. In a
clinical trial of a malaria plasmid DNA vaccine, 500-2500 μg of plasmid DNA was
needed to elicit a CTL response. The 6 pair motif (AACGTT) in plasmid DNA that
induces optimal stimulation in mice may not work equally well in humans. This would be
because different CpG motifs might operate in humans and mice. The injection of
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plasmid DNA may induce the production of anti-DNA antibodies and SLE. Systemic
Lupus Erythematosus (SLE) is an autoimmune disease characterized by the presence of
anti-double stranded DNA antibodies in patient’s serum.

Possible harmful effects of bacterial ISS (CpG motifs) include inflammation in the lower
respiratory tract. Therefore, though DNA vaccines are now being tested in Phase I and
Phase II clinical trials, it is felt that further safety studies should be undertaken. The
manner by which they are to be administered, the amount of plasmid DNA to be
administered, the number of boosters to be given and time interval between boosters need
to be optimized.

DNA vaccination in humans is tampered by the fact that delivery of the DNA to cells is
still not optimal, particularly in larger animals. Another concern is the possibility, which
exists with all gene therapy, that the vaccine's DNA will be integrated into host
chromosomes and will turn on oncogenes or turn off tumor suppressor genes. Another
potential downside is that extended immunostimulation by the foreign antigen could in
theory provoke chronic inflammation or autoantibody production

There are also safety issues that need to be addressed before DNA vaccines can be used
safely in humans. The main is the slight chance of random integration of DNA into the
genome of the cells transfected in vivo. This may lead to the interruption of important
cellular genes (insertional mutagenesis), which may predispose the cell from becoming a
cancer cell. Another concern arises from the persistence of DNA. The administered DNA
may remain active for months after immunization. This may lead to a chronic exposure of
antigen to the immune system, leading either to tolerance of the antigen or autoimmunity.