*- Immune respons : its characterized by the produuction of

proteins ( Igs) and specificially reactive

lymphocytes (T-cells ) when an animal
encounters a foreign macromolecules or cells.
The inducing substances are called antigens
i.e. antibody generators or immunogens.

*- Immunogenicity : it the inherent ability of the immunogen

(complete antigen ) to induce a specific immune
response and to react with the products of this
response (i.e. antibodies or the immune
reactive lymphocytes) .
*- Antigenicity : It is the ability of the foreign substance to react
with the products of that response .

Therefore, Antigens are the ligands that react with the products of an immune response
Also, the immunogenicity & antigenicity are two interchangeable terms which will be
used during discussion of the immune reponse during the period of this course.
In addition, HAPTEN HAS AN ANTIGENICITY but HAPTEN PLUS
PROTIEN CARRIER IS IMMUNOGEN
 Overview of the Immune System
Immune System

Adaptive
Innate
(Specific)
(Nonspecific) o
2 line of defense
1o line of defense Protects/re-exposure

Cellular Components Humoral Components Cellular Components Humoral Components

Interactions between the two systems

 Comparison of Innate and Adaptive
Immunity

Innate Immunity Adaptive Immunity

* No time lag * A lag period

* Not antigen specific * Antigen specific

* No memory developed Memory developed

 Functions of the Immune System
(Self/Non-self Discrimination)

• To protect from pathogens

• Intracellular (e.g. viruses and some bacteria
and parasites)
• Extracellular (e.g. most bacteria, fungi and
parasites)

• To eliminate modified or altered self

Infection and Immunity Balance

infection immunity

Bolus of infection x virulence

Disease =
immunity
 Effects of the Immune System

• Beneficial:
• Protection from Invaders
• Elimination of Altered Self

• Detrimental:
• Discomfort and collateral damage (inflammation)
• Damage to self (hypersensitivity or autoimmunity)
Innate (Nonspecific) Immunity
Innate Host Defenses Against Infection
• Anatomical barriers
– Mechanical factors
– Chemical factors
– Biological factors
• Humoral components
– Complement
– Coagulation system
– Cytokines
• Cellular components
– Neutrophils
– Monocytes and macrophages
– NK cells
– Eosinophils
Anatomical Barriers - Mechanical Factors

System or Organ Cell type Mechanism

Skin Squamous epithelium Physical barrier

Desquamation
Mucous Membranes Non-ciliated epithelium Peristalsis
(e.g. GI tract)
Ciliated epithelium (e.g. Mucociliary elevator
respiratory tract)
Epithelium (e.g. Flushing action of
nasopharynx) tears, saliva,
mucus, urine
 Anatomical Barriers - Chemical Factors

System or Organ Component Mechanism

Skin Sweat Anti-microbial fatty

acids
Mucous Membranes HCl (parietal cells) Low pH
Tears and saliva Lysozymes and
Phospholipase A
Defensins (respiratory & GI Antimicrobial
tract)
Sufactants (lung) Opsonin
 Anatomical Barriers - Biological Factors

System or Organ Component Mechanism

Skin and mucous Normal flora ☻Antimicrobial substances

membranes ☻Competition for nutrients
and colonization
Natural immune response (i-Humoral Components)
Component Mechanism

Complement ☻- Lysis of bacteria and some viruses

☻- Opsonin
☻- Increase in vascular permeability
☻- Recruitment and activation of phagocytic cells
☻- With the help of antibodies, they can destroy the
pathogens

Coagulation ☻- Increase vascular permeability

system ☻- Recruitment of phagocytic cells
☻- Β-lysine from platelets (a cationic detergent)
Lactoferrin and ☻- Compete with bacteria for iron, therefore,
transferrin cause bacterial death.
Lysozymes ☻- Breaks down bacterial cell walls causing their lysis.
Cytokines (Interleukins ☻- They had various immunological effects
, Interferon's)
 Interferons (IFNs )
They are natural proteins produced by the cells of the immune system of most
vertebrates in response to challenges by foreign agents such as viruses, bacteria,
parasites and tumor cells. Interferons belong to the large
class of glycoproteins known as cytokines.

;The discovery of interferon

While aiming to develop an improved vaccine for smallpox, two Japanese virologists,
working at the then Institute for Infection Disease at the University of Tokyo, noticed that
rabbit-skin or testis previously inoculated with UV-inactivated virus exhibited inhibited
viral growth inhibitory factor, and began to characterise it by fractionation of the UV-
.irradiated viral homogenates using an ultracentrifuge
:Functions of inteferons
.Interferons in general have several effects in common
They are antiviral and possess antioncogenic properties ►
Macrophage and natural killer lymphocyte activation, and enhancement of major ►
histocompatibility complex glycoprotein classes I and II, and thus presentation of
.foreign (microbial) peptides to T cells

In a majority of cases, the production of interferons is induced in response to ►

microbes such as viruses and bacteria and their products (viral glycoproteins, viral
RNA, bacterial endotoxin, bacterial flagella, CpG DNA), as well as mitogens and
, tumor12interleukin- ,2interleukin ,1other cytokines, for example interleukin
necrosis factor and colony-stimulating factor, that are synthesised in the response
.to the appearance of various antigens in the body
Their metabolism and excretion take place mainly in the liver and kidneys. They rarely
pass the placenta and the blood-brain barrier

Type I interferons
IFN-α and IFN-β are secreted by many cell types including lymphocytes )NK cells, B-
cells and T-cells), macrophages, fibroblasts, endothelial cells, osteoblasts and others.
They stimulate both macrophages and NK cells to elicit and anti-viral response, and
are also active against tumors. IFN-ω is released by leukocytes at the site of viral
.infection or tumors
Type II interferons
IFN-γ is involved in the regulation of the immune and inflammatory responses; in
humans, there is only one type of interferon-gamma. It is produced in activated T-cells
and natural killer cells. IFN-γ has some anti-viral and anti-tumor effects, but these are
.generally weak
1 However, this cytokine potentiates the effects of the type I IFNs. IFN-γ released by Th
cells recruits leukocytes to a site of infection, resulting in increased inflammation. It
also stimulates macrophages to kill bacteria that have been engulfed. IFN-γ released
cells is also important in regulating the1 by Th
response. As IFN-γ is vitally implicated in the regulation of immune response, its 2 Th
production can lead to autoimmune disorders
 Autoimmune diseases
i-What are autoimmune diseases?
Our bodies have an immune system that protects us from disease and infection. But if
you have an autoimmune disease, your immune system attacks itself by mistake, and
you can get sick. Autoimmune diseases can affect connective tissue in your body (the
tissue which binds together body tissues and organs). Autoimmune disease can affect
many parts of your body, like your nerves, muscles, endocrine system (system that
directs your body’s hormones and other chemicals), and digestive system.

Autoimmunity is the failure of an organism to recognize its own constituent

parts (down to the sub-molecular levels) as "self", which results in an immune
response against its own cells and tissues. Any disease that results from such an
aberrant immune response is termed an autoimmune disease. Autoimmune diseases,
therefore are a large group of diseases characterized by abnormal functioning of the
immune system that causes your immune system to produce antibodies against your
own tissues - the prominent examples being Crohn's disease, Diabetes Type 1,
Coeliac disease, Systemic Lupus Erythematosus (SLE), Sjögren's syndrome and
Rheumatoid arthritis (RA).
Prognosis of Autoimmune diseases
Although autoimmune diseases are chronic, the course they take is unpredictable.
A doctor cannot foresee what will happen to the patient based on how the disease
starts. Patients should be monitored closely by their doctors so environmental factors
or triggers that may worsen the disease can be discussed and avoided and new medical
therapy can be started as soon as possible. Frequent visits to a doctor are important in
order for the physician to manage complex treatment regimens and watch for medication
side effects.

Who is at risk for getting autoimmune diseases?

Most autoimmune diseases occur in women, and most often during their childbearing years.
Some of these diseases also affect African American, American Indian, and Latina women more
than white women. These diseases tend to run in families, so your genes, along with the way
your immune system responds to certain triggers or things in the environment, affect your
chances of getting one of these diseases. If you think you may have an autoimmune disease,
ask your family members if they have had symptoms like yours. The good news is that if you
have an autoimmune disease, there ARE things you can do to feel better!
What are the most common symptoms of autoimmune diseases?
There are more than 80 types of autoimmune diseases. Learning the symptoms of some of the
more common autoimmune diseases can help you recognize the signs if you get one. But some
autoimmune diseases share similar symptoms. This makes it hard for doctors to find out if you
really have one of these diseases, and which one it might be. This can make your trip to doctors
long and stressful. The most common symptoms of the autoimmune diseases include
tiredness, depression , sensitivity to cold, weight gain, muscle weakness and cramps, dry hair
tough skin, constipation and sometimes there are no symptoms
 Natural immune response
ii-Cellular Components
Cell Functions

Neutrophils ☻-Phagocytosis and intracellular killing

☻- Inflammation and tissue damage
Macrophages ☻- Phagocytosis and intracellular killing
☻- Extracellular killing of infected or altered self
targets
☻- Tissue repair
☻- Antigen presentation for specific immune
response
NK and LAK cells ☻- Killing of virus-infected cells and altered self
targets
Eosinophils ☻- Killing of certain parasites
 Phagocytosis
and
Intracellular Killing
Phagocyte Response to Infection

• The Signals
–N-formyl methionine-containing
peptides
–Clotting system peptides
–Complement products
–Cytokines released by tissue
macrophages

• Phagocyte response
–Vascular adherence
–Diapedesis
–Chemotaxis
–Activation
–Phagocytosis and killing
Phagocytosis

Steps of Phagocytosis
•Attachment
•Pseudopod extension
•Phagosome formation
•Granule fusion
•Phagolysosome formation
Initiation of Phagocytosis

Attachment via Receptors:

IgG FcR
Complement R
ScavengerR
Toll-like R
Phagocytes - Neutrophils (PNMs)

☻- Characteristic nucleus and cytoplasm

☻- specific granules

☻- CD 66 membrane marker
Phagocytes - Macrophages

• Characteristic nucleus
• Lysosomes
• CD14 membrane marker
Natural Killer (NK) cells

 Also known as large

granular lymphocytes (LGL)
 Kill virus-infected or
malignant cells
 Identified by the presence of
CD56 & CD16 and absence
of CD3
 Activated by IL2 and IFN-γ
to become LAK cells
What are Natural Killer Cells?
Natural killer (NK) cells are an important first line of defense against newly arising malignant cells and
cells infected with viruses, bacteria, and protozoa. They form a distinct group of lymphocytes with no
immunological memory and are independent of the adaptive immune system. Natural killer cells
constitute 5 to 16 percent of the total lymphocyte population. Their specific function is to kill infected and
Most of us have enough natural killer cells (cell .).cancerous cells (AAA Reference Laboratories, Inc
counts) in our body, however many of us don't have enough natural killer cells that are active. These
inactive natural killer cells are present in great numbers in our blood, lymph nodes, organs, and tissue,
but they are not killing foreign invaders such as infectious organisms and malignant cells that constantly
affect all of us
.
What You Should Know about Natural Killer Cells Activity?
It is known that:
Almost all cancer patients have very low levels of natural killer cell activity: usually 0 to 20
Many patients with chronic diseases including Fibromyalgia and Chronic Fatigue Syndrome have low levels in
the range of 10 to 30.
A wide variety of Auto Immune Disorders including Rheumatoid Arthritis, Lupus, Multiple Sclerosis and
others have low levels in the 10 to 30 range
Most patients with chronic and/or recurrent infections (such as Staph, Sinusitis, Bronchitis, Tonsillitis,
Pneumonia, and ear infections, etc.) have low levels in the 10 to 50 range
Many patients with symptomatic EBV, CMV, HPV and other chronic viral infections are in the 0-20 range
It is also known that, there is a direct age related decrease in natural killer cell activity from 20 to 80
years
of age which may partially explain why the risk of cancer increases with each decade of life
Low natural killer cell activity is a significant independent risk factor for the future development of cancer
,as well as other chronic diseases and illnesses.
Also, low natural killer cell activity is a strong predictor of poor prognosis of survival for cancer patients.
Therefore, the higher the natural killer cell activity in patients with cancer the better their prognosis is for
survival
 Non-specific Killer Cells

NK and LAK cells

They all kill foreign
ADCC (K) cell
and altered self
Activated macrophages
targets
Eosinophils
 Intracellular Killing Pathways
a-Respiratory Burst
a-1- Oxygen-Dependent Myeloperoxidase-Independent Reactions

+
G-6-P-dehydrogenase
Glucose +NADP Pentose-P + NADPH

NADPH oxidase -
NADPH + O2 NADP++ O2
Cytochrome b558

- Superoxide dismutase
2O2 + 2H+ H2O2 + 1O2
- -
2O2 + H2O2 OH* + OH + 1O2
Toxic compounds are : Superoxide anion (O2 -), Hydrogen peroxide
(H2O2), Singlet oxygen (1O2) and Hydroxyl
radical (OH*)
 Respiratory Burst (continued)
a-2- Oxygen-Dependent Myeloperoxidase-Dependent Reactions

- myeloperoxidase -
H2O2 + Cl OCl + H2O

- 1O -
2OCl + H2O 2 + Cl + H2O

Toxic compounds: Hypochlorous acid (OCl-), and Singlet oxygen (1O2)

 Respiratory Burst (continued)

Detoxification Reactions

Superoxide dismutase
-
2O2 + 2H+ H2O2 + O2

Catalase
2 H2O2 H2O + O2
 b-Oxygen-Independent Killing in
the Phagolysosome
Effector Molecule
Cationic proteins (cathepsin)
Lysozyme
Lactoferrin
Hydrolytic enzymes (proteases)
Function
Damage to microbial
membranes
Hydrolyses mucopeptides
in the cell wall
Deprives pathogens of iron
Digests killed organisms
Summary of Intracellular Killing
Pathways

Intracellular Killing

Oxygen Oxygen
Dependent Independent

Myleoperoxidase Myleoperoxidase
Dependent Independent
Nitric Oxide Dependent Killing

TNF

TNF

Nitric Oxide
Nitric Oxide
Lymphokine Activated Killer (LAK)
cell

kills
kills
transformed
malignant
and malignant
cells
cells
Regulation of NK Cell Function

•MHC I •KIR •KAR •KAL

•No Killing •Killing

Their metabolism and excretion take place mainly in the liver and kidneys. They rarely
pass the placenta and the blood-brain barrier

Type I interferons
IFN-α and IFN-β are secreted by many cell types including lymphocytes )NK cells, B-
cells and T-cells), macrophages, fibroblasts, endothelial cells, osteoblasts and others.
They stimulate both macrophages and NK cells to elicit and anti-viral response, and
are also active against tumors. IFN-ω is released by leukocytes at the site of viral
.infection or tumors
Type II interferons
IFN-γ is involved in the regulation of the immune and inflammatory responses; in
humans, there is only one type of interferon-gamma. It is produced in activated T-cells
and natural killer cells. IFN-γ has some anti-viral and anti-tumor effects, but these are
.generally weak
1 However, this cytokine potentiates the effects of the type I IFNs. IFN-γ released by Th
cells recruits leukocytes to a site of infection, resulting in increased inflammation. It
also stimulates macrophages to kill bacteria that have been engulfed. IFN-γ released
cells is also important in regulating the1 by Th
response. As IFN-γ is vitally implicated in the regulation of immune response, its 2 Th
production can lead to autoimmune disorders
 Autoimmune diseases
Autoimmunity is the failure of an organism to recognize its own constituent parts
(down to the sub-molecular levels) as "self", which results in an immune response
against its own cells and tissues. Any disease that results from such an aberrant
immune response is termed an autoimmune disease. Autoimmune diseases, therefore
are a large group of diseases characterized by abnormal functioning of the immune
system that causes your immune system to produce antibodies against your own
tissues - the prominent examples being Crohn's disease, Diabetes Type 1, Coeliac
disease, Systemic Lupus Erythematosus (SLE), Sjögren's syndrome and Rheumatoid
arthritis (RA).
Prognosis of Autoimmune diseases
Although autoimmune diseases are chronic, the course they take is unpredictable. A
doctor cannot foresee what will happen to the patient based on how the disease
starts. Patients should be monitored closely by their doctors so environmental factors
or triggers that may worsen the disease can be discussed and avoided and new
medical therapy can be started as soon as possible. Frequent visits to a doctor are
important in order for the physician to manage complex treatment regimens and
watch for medication side effects.
What are autoimmune diseases?
Our bodies have an immune system that protects us from disease and infection. But if
you have an autoimmune disease, your immune system attacks itself by mistake, and you
can get sick. Autoimmune diseases can affect connective tissue in your body (the tissue
which binds together body tissues and organs). Autoimmune disease can affect many
parts of your body, like your nerves, muscles, endocrine system (system that directs your
body’s hormones and other chemicals), and digestive system.
Who is at risk for getting autoimmune diseases?
Most autoimmune diseases occur in women, and most often during their childbearing
years. Some of these diseases also affect African American, American Indian, and Latina
women more than white women. These diseases tend to run in families, so your genes,
along with the way your immune system responds to certain triggers or things in the
environment, affect your chances of getting one of these diseases. If you think you may
have an autoimmune disease, ask your family members if they have had symptoms like
yours. The good news is that if you have an autoimmune disease, there ARE things you
can do to feel better!
What are the most common symptoms of autoimmune diseases?
There are more than 80 types of autoimmune diseases. Learning the symptoms of some
of the more common autoimmune diseases can help you recognize the signs if you get
one. But some autoimmune diseases share similar symptoms. This makes it hard for
doctors to find out if you really have one of these diseases, and which one it might be.
This can make your trip to doctors long and stressful. The most common common
symptoms of the autoimmune diseases include tiredness depression
sensitivity to cold weight gain muscle weakness and cramps dry hair tough skin
constipation sometimes there are no symptoms
What are Natural Killer Cells?
Natural killer (NK) cells are an important first line of defense against newly arising malignant cells and
cells infected with viruses, bacteria, and protozoa. They form a distinct group of lymphocytes with no
immunological memory and are independent of the adaptive immune system. Natural killer cells
constitute 5 to 16 percent of the total lymphocyte population. Their specific function is to kill infected and
Most of us have enough natural killer cells (cell .).cancerous cells (AAA Reference Laboratories, Inc
counts) in our body, however many of us don't have enough natural killer cells that are active. These
inactive natural killer cells are present in great numbers in our blood, lymph nodes, organs, and tissue,
but they are not killing foreign invaders such as infectious organisms and malignant cells that constantly
affect all of us
.
What You Should Know about Natural Killer Cells Activity?
It is known that:
Almost all cancer patients have very low levels of natural killer cell activity: usually 0 to 20
Many patients with chronic diseases including Fibromyalgia and Chronic Fatigue Syndrome have low levels in
the range of 10 to 30.
A wide variety of Auto Immune Disorders including Rheumatoid Arthritis, Lupus, Multiple Sclerosis and
others have low levels in the 10 to 30 range
Most patients with chronic and/or recurrent infections (such as Staph, Sinusitis, Bronchitis, Tonsillitis,
Pneumonia, and ear infections, etc.) have low levels in the 10 to 50 range
Many patients with symptomatic EBV, CMV, HPV and other chronic viral infections are in the 0-20 range
It is also known that, there is a direct age related decrease in natural killer cell activity from 20 to 80
years
of age which may partially explain why the risk of cancer increases with each decade of life
Low natural killer cell activity is a significant independent risk factor for the future development of cancer
,as well as other chronic diseases and illnesses.
Also, low natural killer cell activity is a strong predictor of poor prognosis of survival for cancer patients.
Therefore, the higher the natural killer cell activity in patients with cancer the better their prognosis is for
survival
 Complement: History

Discovered in 1894 by
Bordet
It represents lytic activity
of fresh serum

Its lytic activity destroyed

when heated at 56C
for 30 min
 Complement functions
• Host benefits:
– Opsonization to enhance phagocytosis
– Phagocyte attraction and activation
– Lysis of bacteria and infected cells
– Regulation of antibody responses
– Clearance of immune complexes
– Clearance of apoptotic cells

• Host detriments:
– Inflammation, anaphylaxis
 Proteins of the complement
system (nomenclature)
• C1(qrs), C2, C3, C4, C5, C6, C7, C8, C9
• factors B, D, H and I, properdin (P)
• mannose binding lectin (MBL), MBL associated
serine proteases (MASP-1 MASP-2)
• C1 inhibitor (C1-INH, serpin), C4-binding
protein (C4-BP), decay accelerating factor
(DAF), Complement receptor 1 (CR1), protein-
S (vitronectin)
 Definitions

• C-activation: alteration of C proteins such that they

interact with the next component

• C-fixation: utilization of C by Ag-Ab complexes

• Hemolytic units (CH50): dilution of serum which
lyses 50% of a standardized suspension of Ab-coated
r.b.c

• C-inactivation: denaturation (usually by heat) of an

early C-component resulting in loss of hemolytic activity

• Convertase/esterase: altered C-protein which acts

as a proteolytic enzyme for another C-component
 Activation product of complement
proteins (nomenclature)
Activated component are usually over-lined: e.g.
C1qrs
When enzymatically cleaved, the larger moiety,
binds to the activation complex or membrane
and the smaller peptide is released in the
microenvironment
Letter “b” is usually added to the larger,
membrane-binding, peptide and “a” to the
smaller peptide (e.g., C3b/C3a, C4b/C4a,
C5b/C5a), EXCEPT C2 (the larger, membrane-
binding moiety is C2a; the smaller one is C2b)
 Pathways of complement
activation
CLASSICAL LECTIN ALTERNATIVE
PATHWAY PATHWAY PATHWAY

antibody antibody
dependent independent

Activation of C3 and
generation of C5 convertase

activation
of C5

LYTIC ATTACK
PATHWAY
 Components of the Classical
Pathway

C3 C4

C1 complex
 Classical Pathway
Generation of C3-convertase
 Classical Pathway
Generation of C3-convertase

_____
C4b2a is C3 convertase

C4b
 Classical Pathway
Generation of C5-convertase

________
C4b2a3b is C5 convertase;
it leads into the Membrane
Attack Pathway

C3b
C4b
 Biological Activities of Classical
Pathway Components
Component Biological Activity

C2b Prokinin; cleaved by plasmin to yield kinin, which

results in edema
C3a Anaphylotoxin; can activate basophils and mast
cells to degranulate resulting in increased vascular
permeability and contraction of smooth muscle cells,
which may lead to anaphylaxis
C3b Opsonin
Activation of phagocytic cells
C4a Anaphylaotoxin

C4b Opsonin
54
C1-inhibitor deficiency:
hereditary angioedema
Components of mannose-binding
lectin pathway

MBL MASP1
Mannose-binding lectin pathway

_____
C4b2a is C3 convertase; it
will lead to the generation of
C5 convertase
MASP1

MBL
 Components of the
alternative pathway

C3
Spontaneous C3 activation

Generation of C3 convertase

C3 i b
C3b

C3iBb complex has a very short half life

 C3-activation
the amplification loop

If spontaneously-generated
C3b is not degraded

C3b b C3 b
 C3-activation
the amplification loop

C3 b b C3b

C3b
 General Introduction
The immune system is a set of mechanisms that protect an organism from◙
.infection by identifying and killing pathogens
This task is extremely difficult, since pathogens range from viruses to parasitic worms ◙
. and these diverse threats must be detected with absolute specificity amongst normal cells and tissues
Pathogens are also constantly evolving new ways to avoid detection by the immune ◙
.system and successfully infect their hosts
.To meet this challenge, multiple mechanisms have evolved to recognize and neutralize pathogens ◙
These mechanisms include antimicrobial peptides called defensins, pattern ◙
.recognition receptors, and the complement system
More sophisticated mechanisms, however, developed relatively recently, with the ◙
evolution of vertebrates. The immune systems of vertebrates such as humans
consist of many types of proteins, cells, organs, and tissues, which interact in an
.elaborate and dynamic network
As part of this more complex immune response, the vertebrate system adapts over ◙
.time to recognize particular pathogens more efficiently
The adaptation process creates immunological memories and allows even ◙
more effective protection during future encounters with these pathogens.This
.process of acquired immunity is the basis of vaccination
 General Introduction (continued)
◙Disorders in the immune system can cause diseases.
◙Immunodeficiency diseases occur when the immune system is less active
than normal, resulting in recurring and life-threatening infections.
◙Immunodeficiency can either be the result of a genetic disease, such as severe
combined immunodeficiency, or be produced by pharmaceuticals or an infection,
such as the acquired immune deficiency syndrome (AIDS) that is caused by the
retrovirus HIV .
◙In contrast, autoimmune diseases result from a hyperactive immune system
attacking normal tissues as if they were foreign organisms. Common
autoimmune diseases include rheumatoid arthritis, diabetes mellitus type 1 and lupus

erythematosus.
◙ Therefore, immunity or the resistance is the sum of all naturally occurring and
acquired defense mechanisms that protect the organism from infectious
diseases.and the study of this mechanisms that a host has evolved to get rid itself
of pathogens and other foreign substances.
◙The immune system so, has at least three major functional properties that
distinguish it
from all the body's other defenses:
Immunity (resistance):
It the sum of all naturally occurring defense mechanisms
that protect the organism (or host) from infectious
diseases. In addition, it include the study of the
mechanisms that a host has evolved to get rid itself from
the invading pathogens and other foreign substances.
.
The immune system so, has at least three major
functional properties that distinguish it from all the body's
other defenses:

The first: Is its extreme specificity, the ability to

recognize and distinguish among a large or
vast number of different target molecules, and
to respond (or not respond) to each of these
individually
Second: The immune system discriminates between self
(body ingredients ) and non self ( foreign bodies),
so that it normally coexists peacefully with all
of the immunerable proteins and other organic
materials that make up the host but responds
vigorously against foreign substances,
including cells or tissues from other people .

Third: The immune system has memory, that is, the ability
to be molded by its experiences so that subsequent
encounters with a particular foreign pathogen
provoke more rapid and more vigorous responses
than occurred at the initial encounter.
A- Non a specific or innate immune response:
This consists of the pre-existing defenses of an
animal, such as barrier layers and secretions.
It has the following properties:
i- It does not exhibit high specificity.
ii- It does not depend on a complete (specific)
recognition of the antigen.
iii- A single mechanism protect against many
pathogen.
B- Specific or adaptive immune response:
This response involves the cells of the
immune system and frequently leads to a state
of immune memory, and finally destroying the
invading organisms.
 Comparison between the non-specific and the specific
immunity

Non-specific Immunity Specific Immunity

Response is antigen- Response is antigen-

independent (Not antigen- dependent (antigen-specific)
specific)
There is immediate maximal There is a lag time between
response exposure and maximal
response
Exposure to the Pathogen did Exposure to the Pathogen
not produce immunological produce immunological
memory memory
2- The Non-Specific (Innate Immune)
Response
1- First defense line:
a-Anatomical barriers:
- Skin which physically preventing the interaction
between the host and the pathogen.
- Intestinal movement and mucus coating their walls.
- Oscillation of broncho-pulmonary cilia.
b-secretory molecules:
-These secretions include organic acids in skin secretions,
thiocyanate in saliva, low molecular weight fatty acids, bile acids
in lower gastric intestinal tract, transferring, lactoferrin,
lyzozyme, interferons, fibronectin, complement, etc. in serum,
interferons and tumor necrosis factor at the site of inflammation.
2. Second defense line:
They represent the Cellular components, and they include
phagocytic cells either polynuclear phagocytes or mononuclear
phagocytes and NK cells.
Polynuclear phagocytes:
Neutrophils (Polymorph nuclear cells PMNs) are the most important
cellular components in bacterial destruction. They are relatively large
and most abundant white blood cells (65% of leucocytes) with lobed
nucleus and cytoplasmic granules (lysosomes

All phagocytic cells have receptors for a variety of molecules. Most

pertinent to non-specific immune response are receptors for IgG-Fc,
complement, interferon, TNF and certain bacterial components.
Receptor interactions with these ligands promote phagocytosis and
activation for efficient killing of pathogens
The figure shows a Neutrophil in a blood film

Example of Phagocytosis: A macrophage

attacking E.coli
 Chemotactic response to inflammatory stimulus
And the steps of this type of response
1- Adherence 2- Diapedesis 3- going to the inflammatory site 4- Re-activation of
adherence via histamine and thrombin secretions.
Histopathology of bladder shows eggs of
Schistosoma haematobium surrounded by intense
infiltrates of eosinophils
 3-Front defense line:
The major physiologic roles of natural killer cell (NK cells) appear to be in
the early host defense against microbial agents. Nk cells, therefore, help to
protect against a range of infections before the T-cell and B-cell response
have developed. NK cells may thus function as a bridge between the innate
and the acquired immune systems, acting as a front line of defense , while
producing cytokines to promote the development of a specific immune
response.

NK cells and their activation

1- Derived from bone marrow.
2- Lack most markers for T and B cells
(no TCR or CD3).
3- Don’t undergo thymic maturation.
4- Express CD56, a specific NKs marker
5- Express a low affinity receptor for Fc
portion of IgG called FcR (CD16),
also expressed on granulocytes and
macrophages.
6-Cytokines especially IL -2 promotes further
differentiation in to lymphokine – activated
killer cells (LAK).
Acute-phase Response
Most soluble mediators of innate immune response are
found in relatively small amounts, with the exception of C3,
in the serum under normal conditions.
The concentrations of several of these proteins, however,
can increase as much as 1000-fold during serious
infections, as part of accordinated protective reaction called
the hepatic acute-phase-response. In this response, the
liver temporarily increases its synthesis of more than
adozen different serum proteins that participate in anti-
microbial defense, including complement factors C3 and B,
the mannose binding proteins, C-reactive protein, serum
amyloid protein P, and others.
The response occurs when hepatocytes are exposed to
certain cytokines
3.1 Cells of the immune system
 Immunity (resistance): It the sum of all naturally occurring
defense mechanisms that protect human from infectious disease

Non – specific Specific

Naturally acquired
( Innate ) ( Acquired )
- Mucous membranes
- Phagocytic cells - Placental transfer of antibodies( Passive )

- Enzymes in secretion - Recovery from disease ( Active )

-Interferons ( α,β,γ) - - Administration of antitoxin ( Passive )

- NKCs- - Vaccinations ( Active )
-Skin-
. Macrophages-
Artificially acquired
 Natural ( Innate )
Specific ( Adaptive )
-Less specific .
or
- Skin & mucous membrane .
(Acquired)
-NK cells .
- Complement cascade .
- Phagocytosis .
- C- reactive protein .

Active Passive
-Induced by contact with foreign antigens .- - Induced by antibody performed in -

- Consist of clinical infection , immunization with live or - another host-

killed infectious agents or their toxins . - - Ab injected in the incubation period -
- Long term. - - Short term .
 Naturally acquired Artificially acquired

active passive active passive

First: Non – specific Immunity ( Innate)

:-
This is a physiologic mechanism which is inherent or innate with the -
following properties

A single
mechanisms
Do not depends on specific Protect
It does not exhibit specificity recognition of a foreign Against
material many
paths
Definition :- the body forms his OWN IMMUNITY when
stimulated (sensitized ) by introduction of
immunogenic agent.
Natural Infection
Types *living attenuated vaccine * killed vaccine .
Artificial bacterial products
*Endotoxins.
* Exotoxins.

Others .
Characters :- * slowly developed .
*longer duration
(and leave a potential immunity , so there is A rapid response
in the future to the Same antigen ) leads to ??
*-Homogenous antibodies
*- Cellular defense mechanism play a role
Mechanism of Acquired immunity :-
Humeral Ab
Cellular T_Cells
classification of acquired Immunity:-
- passive Acquired Immunity :-
Definition: acquired Immunity by given already form antibodies or antitoxic serum or gamma
globulins from normal or convalescent individuals or Trans placental or lactation .
Trans placental .
Natural
Types Lactation (Colostrum).
Antitoxin serum tetanus. (Anti_ cobra venom)
Artificial
Gamma globulins.

- characters :- * -Rapidly developed .

* -Short duration .
[ Rapidly eliminated in 2-4 WKS due to the formation of anti – antibodies (a disadvantage )].
*-Heterogeneous antibodies .
* -Cellular mechanism not stimulated .
(No memory ).

* - Side effects:-
*- Hyper sensitivity reactions against the
foreign serum
*-Neurological affection in some cases
( Encephalitis ).
*-Superadded in infections
e.g. (AIDS & HEPAT) .
- Humoral immunity - Cellular immunity .
Antibody mediated immunity. - Cell mediated immunity .
( B- lymphocyte) - (T- lymphocytes-Mediated)

help help

CD4 CD8
B- lymphocyte Helper T- LYMPH . Cytotoxic T-lymphocyte
(Protection is mediated by
the produced antibodies)

TH1 TH2
 Haematopoietic stem cell

Lymphoid stem cell Myeloerythroid progenitor

NK cell
B-lymphocyte T-lymphocyte

monocyte neutrophil eosinophil macrophage basophile RBC platelets

 * B-
Lymphocytes
This cell type consists ( 20 – 25%) of the total peripheral lymphocytes
n mammals , they mature in bone marrow , then, migrate to secondary
lymphoid organs ( e.g. spleen & Lymph nodes ) .

* Upon exposure to antigen , B-Lymphocytes are stimulated to proliferate ,

(large lymphocytes) differentiate and mature into LARGE PLASMA CELLS

* The large mature B-Lymphocytes have

short life span ( days to weeks ) .

Secreted
Immunoglobulins or Humoral antibodies

L-CHAIN

Antibody
H-CHAIN
* Some large mature B-Lymphocytes (B- cells ) can be converted
into small B- cells which have long life span

This type of cells is

Secondary involved in the
Immune Memory cells And serve as
Response

Activation & differentiation of B-Lymphocytes , in certain instances ,

needs a Helper T- Lymphocytes activity to enhance the above to
processes in that B-Lymphocytes.
 T-Lymphocytes :-

*- THEY CONSTITUTE 65 -80 10 of total peripheral lymphocytes .

*- They have long life span ( months to years ).
*- They mature in thymus gland before migrating to lymphoid organs
*- Upon exposure to antigen , T -cell proliferate .

How ever , their specific effectors molecules are not secreted

and remains firmly Attached to their cellular membranes

Giving what is called

cell-mediated immune response

*- They are involved in a variety of cell-mediated immunological responses

defense against
malignant cells
graft rejection
hyper sensitivity
reactions
bacteria & protozoa

Fungi viruses
 T-CELLS

T-HELPER (TH) T-SUPRESSOR(TS) T-CYTOTOXIC (TCs) T-DELAYED

T- Helper :Their Surface Antigen : is T4 (CD4) .
Helper-
*They Promote Maturation Of Antigen .
*Stimulated B and T cells. Sensitivity
And
Cells ( TdH)
Enhance their response
T – suppressor cells: * Their Surface antigen is T8(CD8). and
* they suppress the effect of T – helper cells .
T-CELL MEDIATED
i.e.
*Suppress T &B – response . IMMUNITY
T –cytotoxrc: * their Surface antigen T8(CD).
(Tcmi )
* they specifically destroy target cells.

virus infected cells unacceptable grafted cells

tumor cells
T – delayed hypersensitivity & T cell mediated immunity.CD4 (T4)
*they are responsible for delayed hypersensitivity reactions to different
antigens , particularly those of intra cellular parasites & contact allergen .
In general : * some of the stimulated T-cells release soluble substances
lymphokines that modulate the behavior of other cells.
*- most antigens which have a small number of epitopes and require carrier need
T – cell cooperation with B- cells
for antibodies production .

* Deficiency of B – cells (and\or) T-helper cells

leads to defective synthesis of antibodies.

* its over activity lead to Autoimmune disorders

the majority of B-lymphocytes express both surface IgM & IgD, very few express
surface IgG & IgA or IgE in the circulation.

*the majority of B-cells also carry class 2 major histocompatibility complex )class П
MHC) products which are functionally important in

Regulation of immune response

2- T cell Activation
When a T cell encounters an antigen presenting cell (APC), the specificity of
its TCR determines the outcome.

Only if the TCR recognizes its particular antigen MHC combination does
activation occur.

The recognition of appropriately presented antigen activates T cells to

proliferate, differentiate and perform their effector functions.

Activation of helper T-cells leads to the production of lymphokines that

promote cellular and humoral immune responses, whereas activation of
cytotoxic T cells results in killing of the antigen bearing cells.
 Co -operation of innate & specific Immunity
in
Host defense against infection

*Antibodies promote Phagocytosis or activate complement to kill microbes

*T-lymphocytes enhance phogocytic and microbial functions of macrophages

INNATE IMMUNITY SPECIFIC IMMUNITY complement

In direct lyses by C.

+ +

BACTERIA PHOGOCYTE PHOGOCYTE Opsonization

B-Lymph
INEFFECTIVE BACTERIA And Phagocytosis
Direct lyses
+ SERM
Bacterial lyses +
COMPLEMENT BACTERIA B-Lymph
a
Ab b LYSIS
BACTERIA
s
Cell
+ Mediated
BACTERIA PHOGOCYTE bacteria T-Lymph response
 Embryo Liver stem cell In Bone marrow

central or
primary
lymphoid
organs
(tissues)

Secondary
Lymphoid
Organs
Spleen or +
Bone marrow
+ A9
A9 T_Cells B_Cells

Effector PLASMA
Killer
cells
memory cells CELLS

B T HUMORAL ANTIBODIES
 Specific memory and self-limitation of Immune response

Ag A Secondary anti A
infection response
Serum Primary Anti A
AB RESPONSE

weeks 12 weeks

*- Antigen enhance THE production of specific Antibody A.

*- the secondary response to Ag A is more rapid and larger
then the primary response ( memory cells ) .
*- Antibodies Titer decline ( with time ) after each immunization .
 Specific immune response :
It is developed as a result of exposure to a variety of agents capable of
inducing an immune response
( i.e. immunogens )
vaccines microbes that colonize Macro molecules
in the diet
in the body
A special case Antigen in the form of hapten
Hapten is a micomolecule may be conjugate with a carrier protein in the
blood to be immunogen (antigen)
Specific immune response

Humoral cellular
B. Cells T-CELLS
*- They are two interrelated & interdependent mechanisms .
 Specific immune response can be further
Classified according to its components into

primary secondary
Initial exposure to a particular on farther or
Infectious agent or immunogen repeated exposure
Induction phase of lymphocytes to antigen ( same )
proliferation T-CELLS
B-CELLS PLASMA CELLS increased
resistance
develops
Sensitized T-CELLS Antibodies
Cellular Immune response humoral through

Humoral Cellular
response response
Acquired immune response
Has both good ( desirable ) and
Bad ( undesirable ) consequence

undesirable
Desirable

Protection Allergies (hypersensitivity)

From infections
agents
Autoimmune diseases
Immune
response
Control of
Pre-cancerous Graft rejection
growths
 Interactions & functions of the major
components of the immune system

ANTIBODY – MEDIATED CELL MEDIATED REPONSE

IMMUNE RESPONSE Two major components
Main defense against
T-HLPER & MACROPHAGES Cytotoxic
* exteracellular, encapsulated
pathogenic bacteria Intracellular bacteria T-CELLS
e.g. streptococci & staphylococci * ( mycobacterium &tuberculosis) Viruses
*Neutralizations of toxins e.g.( • * Fungi Acts by
tetanus)
Destroying
* viruses ) Hepatitis C,A,B…….(•
Virus- infected
cells
 T-CELLS
B-CELLS
LYMPHOKINES
HELPER CYTOTOXIC
IL-2,IL-4.IL-5
CD4 CD8
PLASMA CELL

IL_2 IL_2

ANTIBODIES
Neutralize ACTIVATED HELPER Activated
Toxins
+ AND MACRO PHAGES Cytotoxic cells
COMPLEMENT
+
INHIBIT Kill
NEUTROPHILS
INTRACELLULAR Virus – infected
Bacteria cells
KILLING OF
&
BACTERIA
fungi
 VIRUS infection
cell

MHC Class I

T-Cell
receptor
virus
MHC
Killing Class II
CD8
IgM
CYTOTOXIC T-Cell
RECEPTOR
B-CELLS
CD4
( T_HELPER )
INTERLEUKIN-4
INTERLEUKIN-5
VIRUS ANTIGEN
Defense mechanism against viral infection
VIRUS
*- Recognition of phases :- antigen recognition ( binding of Ag to specific
receptor on mature lymphocyte ( exist prior to ag exposure )
*- activation proliferation & differentiation of lymphocytes is the sequence
of events induced in lymphocytes as result of Ag recognition .
*- Effectors phases elimination of antigen [ is the stage of the response
At which the sensitized cells perform the function that (eliminate of Ag)
Some antigen – stimulated lymphocytes die by process called programmed cell
death ( apaptosis ).
Elimination
OF Ag
T
OR + Ag
B Phagocytosis
NATIVE
complement
LYMPHOCYTES

Recognition ACTIVATION Effector

programmed cell phase
phase PHASE
Death
Immunogenicety ability to induce immune response

Antigenicity ability of the substance to react specifically with

immune system

must be
Antigenic Immunogenic
are not necessary to be
Happen is incomplete antigen ( di nitro phenol or penicillin)
It cannot stimulate humoral or cellular reactions but can react with these
products specifically so it is Antigenic not immunogenic
If they reacted with larger carrier protein (e.g., albumin , globulin or
synthetic poly peptide ) . It will be Immunogenic
Animals injected with this hapten – protein
Complex will make antibodies to this hapten ,
Only if it is ( hapten ) covalently linked to
the carrier (chemically bonded)
 CARRIER HAPTEN ANTIBODY
PRTOCAL
PROTEIN

i NO YES NO

II
YES NO Anti carrier only
III YES YES Anti carrier only
( not chemically linked)
IV YES YES Anti carrier
(CHEMICALLY LINKED ) &
Anti hapten
Immune response :- its characterized by the production of proteins
called immunoglobulins( Igs) and specificially reactive lymphocytes (T-
cells ), which carry their own effector molecules on their surfaces, when
an animal encounters a foreign macromolecules or cells .
The inducing substances are called antigens i.e ( antibody generators )
or immunogens
*- Immunogenicity & antigenicity : Interchangeable terms used during
discussion of the immune reponse.
*- Immunogenicity : it the inherent ability of asubstance ( Immunogen (
complete antigen ) to induce a specific immune response and to react with
the product of this response .

*- Antigenicity : the ability of the inducing substance (Antigen) to react

with the products of the immune response (i.e. the antibodies and|or the
effector molecules of the T-lymphocytes). .
HAPTEN HAS AN ANTIGENIC Properties but the HAPTEN PLUS
PROTIEN CARRIER IS IMMUNOGEN
Antigens are the a ligands that react with the products of an
immune response .
Hapten-carrier conjugates have native
antigenic determinants of the carrier as
well as new determinants of the hapten
Epitope ( - antigenic determinants ) :-

are the sites either (on or) within the antigen with which antibodies or T-cells
receptor reacts
paratope :- the sites on antibodies which react with the antigen .

epitope size ( small )

conformational linear
conformational
site are on antigen surface
or internal that expressed only when the
antigen has been partially degraded in
vivo
valency of antigen :- e.g multivalent
i.e the antigen molecule carry a number of different epitopes
( some times 2 or>)

some of which specify antibody A others specify antibody B .

valency = total no . of epitopes the antigen pocesses .

 Antigenic determinants are usually
limited to those portions of the antigen
that are accessible to antibodies shown
in black for this iron-containing protein
 EPITOPE (ANTIGENIC DETEREMNANT):-
The portion of Ag that binds specifically with
the binding site of Ab (paratope) or a receptor(s) on T_lymphocyte
SIZE CONFORMATIONAL STRUCTURE
The size and the structure of epitope are complementary to that of paratope
.i.e. they must have approximately the same dimensions
WITH RESPECT TO THEIR STRUCTURE ,A g MAY HAVE THE FOLLOWING
CHRACTES :-
Ag may have only a single epitope of a given specificity on its surface which is
capable to bind with antibodies , such Ag is called UNIVALENT AND UNIDETRMINANT
(one kind of specificity ) for example hapten
Ag may have two or more epitopes (which determine the specificity ), the A g in
this case is called MULTIVALENT (which determine the number).
If the epitopes are of the same type, the Ag is called also UNIDETERMINANT
(UNIDETERMINANT MULTIVALENT and if they are of different types called
MULTIDETERMINANT (specificity, MULTIDETERMINANT- MULTIVALENT ).

UNIVALENT MULTIVALENT MULTIVALENT

UNIDETREMNANT UNIDETREMINANT MULTIDETRMENANT
In an antigen, the same antigenic determinant
repeated many times
T-dependent antigens are characterized by a few copies
of many different antigenic determinants
 .factors Affecting Immunogenicity

Foreigness chemical complexity molecular

size
A – foreigness :-
the immunogenic substance must be forign to prduce immune response .
The greater the foreignness, the more will be the reponse
*- identical twins smaller or no response
*- brothers with the higher immune response
same tissues compatibility
the same blood groups .etc ………….
B.CHEMICAL COMPLEXITY :-
*- MOST of organic molecules are immunogenic expert lipids
*- proteins are the strongest immunogenic substance .
*-Polysaccharides most of them are haptens but they become complete Ag in
cases of
* peneumococcal polysaccharide .
* Lip polysaccharides in cell membrane of gram (– ve ) bacteria.
*- Glycoprotein's :-
Are immunogenic ex blood group Ags ( A,B,AB,O,RH )
*- POLYPEPTIDES & nucleic acids :-
Are weak immunogens
*- lipids :- are not antigenic or immunogenic
C.molecular size :-
usually the larger the molecule the stronger the Immunogenicety .

M.Wt below 5000 DA ARE NOT IMMUNOGENIC

MACRO MOLECULES are the most potent immunogens .
( e.g. albumin m.wt 40.000 Da
Globulin m.wt 160 kDa
Macrocyanin m.wt 1000 kDa
 The valence of A.g increases proportionally with molecular size .

Macro molecules are easily to induce phagocytic ( as example ) and easier to

be phagocytosed
Quaternary structure are the most Immunogenic
The more complexity , the more Immunogenicety
Superantigens activate a large fraction of
T cells in contrast to conventional T-
dependent antigens
 MULTIVALENT since it has only one kind of determinant but many of such
determinant on each molecule

Ex. Many poly saccharides & homo polymer (e-g peptide chain of the
some .A. Acids .)

*- some antigens are multi determinant & valent such molecules have many
epitopes of different kinds (multi specificity ) but only one of each kind ( mono
valent )
Ex. Most proteins .

*- High M.WT , chemically complexed compounds or polymerized proteins

(quaternary structure or heteropolymerized proteins are usually .

*-Multi determinant Ag ( multi specific) , multivalent Ag (more than one

epitope of each kind)
)What kind &How many of such kind )
Antibody binding site ( Paratope ).

Binding of Ag & Ab

Affinity :- the strength of attraction and binding between an epitope( mono

valent ) of an Ag and the antigen combining site of Ab molecule ( Paratope
).
Avidity :- The strength with which ( multivalcnt ) Ag bind to its
antibodies ( Abs). ( chemical complexity )

This depends on the affinities of the individual

combining sites of the determinants
on the antigen
 ANTIBODIES and their STRUCTURES

Electrophoretic separation of serum proteins

 ANTIBODY STRUCTUR

*Classes of antibodies .
IgM , IgG , IgE , IgA & IgD .

CH1
A = COMPLEMENT BINDING
SITE

Constant A

Constant A
CH2

Heavy chain
B = NEUTROPHILS & MACRO- PHAGE
BINDING SITE Hinge bonds

Constant B
Constant B
VARIBLE = ANTIGEN BINDING
SITE . CH3
The basic structure of immunoglobulins
Rotating antibody
 ANTI BODIES

POLYCLONAL ANTI BODIES MONOCLONAL ANTIBODIES

- INDUCED AGAINST WHOLE ANTIGEN . INDUCED AGAINST ONE EPITOPE .

- LESS SPECIFIC (I.E . SMALL PART OF ANTIGEN )
- PRESENT IN SERUM - MORE SPECIFIC .
- PRODUCCED BY HYBRIDOMA
EPITOPE
EPITOPE TECHNOLOGY .

INFECTION
POLYCLONAL Ab. MONOCLONAL Ab MAb MAb MAb
i-Immunization II_ FUSION

antigen + MICE
+
MYELOMA
CELLS
B-CELLS fusion

TUMOR MICE MICE

Ascetic fluid selection of

Desired
Clone

supernatant Tissue culture

Fluid HYPRID CELLS
HybrIdoma technique
 Immunoglobulins
"Humoral antibodies”

They are formed of two identical units each of them is formed of :-

A) heavy chain B) light chain C) hing region

A) Light chain 2( lambda)

but never 1 and 1K
K ( Kappa )
2
B) Heavy chains :
* M-Wt 53.000 - 75.000 Da
*- heavy chains are hold together with (disulphide bonds) .
*- Fixed region contain 2k or 2  .
*- The variable region contain a mixture of K,.

*- both L& H chains contains the following region :

Light chain contain variable (VL) and hyper variable (VH) regions
Heavy chain contain variable and hyper variable regions.
* Amino terminal
* The amino acids differ
on  to another specificity.
* The VL & VH are adjacent to
each other forming paratope .
* They have sub-regions of the
variable region (hypervariable)
These regions have extreme
variability in their A .As
sequence in different antibodies
and they are responsible for
binding with Ag(s)
* carboxyl terminal
* A. As are similar in different
* it contain the effectors domain which
is responsible for the
initiation of the process
by which the body gets-rid of Ag. .
It is responsible for
Designation of Ab class & its
-
distribution.
-
-.

-
)hyper variable (
CDRs}{
comptementary
detemining
regions
C) Hinge region :-

* CH 1, CH 2 , CH 3 : occupies ¾ that of Heavy chains the other ¼ is

VH .
* The Hinge region lies between CH 1 & CH 2 .
* It is flexible & allows movement between the two antibody binding
sites .
* The hinge region is digested by protease (e.g. pa pain ) which splits it
into :-
( i ) antigen binding fragments (fab) = They are 2 identical fragments containing the
antigen binding site .
)ii ) crystallization fragment (FC ):-It contains the effectors (
 Structures and function
Of
Specific Immunoglabulins

*- Ig(s) are glycoprotein's in the gamma globulin fraction of serum proteins (albumin ,
fibrinogen , globulins ( ,  and  ) .
*- they are produced by B- lymphocytes or plasma cells in response on
immunogen (or Ag ).
General Ig structure :-
*- 4 poly peptide chains.
*- they are linked covalently by disulphide bonds
*- the 4 chains , monomeric Ig structure ,are
composed of 2 identical heavy poly peptide chains (H)

2 identical light poly peptide chains (L)

*- Heavy and light chains :
*- H- chain :
*- Have a M.wt of 50-75 KD (Twice that of L chain )
*- H chains contain  400 A.As (Twice that of L chain )
*-A. As differences in the .COOH terminal portion of the heavy chain (CH) identify 5
distinct H-chains isotypes .
* Each H chain has 4 or 5 domains :

1 domain in the variable and 3 or 4 in the constant

3 IgG (  ), IgA( )&IgD( ) Or 4 Igm ( )&IgE(  )

Total = 1 Variable + 3 constant or = 4constant +1 Variable

Notes (1) -Each L- chain has 2 domains 1 VL
1 CL
(2)- Folding of the polypeptides chains brings the hyper
variable regions of the VH and VL domains into close
proximity .
(3)- this folding creates a 3-dimensional structure that is
complementary to the epitope (last figure )

The Hinge region

*- It is the portion of the H-chain between CH 1& CH 2.
*- there is no homology between it and the other H- chain domains, thus .its
sequence is unique (sole) for each Ig type and subclass
IgM & IgE do not possess a hinge region but have one more CH domain.

These structure explain why both IgM & IgE have 4 domains on the CH chains but
not like the other types (which have only 3 domains on CH)

*-In this region (hinge), inter chain disulphide bonds forms between the arms of the
Fab fragments preventing them from folding and therefore
, rendering this portion of the molecule highly susceptible to fragmentation by
enzymatic attach .

* -The hinge region is highly flexible and allows for movement of the Fab arms in
relation to each other .This motility explain why native antibody
molecule do not activate complement , whereas those in an immune
complex do .This is because , the native Ab is not in the appropriate
configuration t1/2 or half life of( Abs) .

*- These heavy chain isotypes form the basis of the 5 Class of Immunoglabulins
molecule IgG () ,IgA ( ) ,IgM ( ),IgD ( )and IgE ( ).

*- H chains Classes  and  are subdivided into subclasses of molecules

 1 , 2 , 3 , and 4
And   1 and  2
The subdivision is based on the greater similarity of A.As sequence shown by
subclasses of the same class
i.e. 1 , 2 , 3 etc,. Than is shown by different classes
(i.e.  ,  , or )

*- The heavy chain subclasses determine immunoglobulin subclasses

e.g. 1 = IgG1
2 = IgG2 , 3 = IgG3 etc,.
*-L-chains :-
* Are composed of  200 A. As .
* They are of 2 types ( K= Kappa or  = lambda ) .
{ based on their structural (antigenic) differences }
* All Igs classes have 2K or 2 chains but not k or  k .
ex. * The proportion of K/ = 3/2 (human Ig) .
- chain Isotypes :-
*- There is no isotypic variations in K chains
*- There are 4 distinct  chains 4 different isotypes .
*- All the 4 subclasses are present in each of the Ig classes
i.e. in IgM , IgE , IgD etc.
*- Disulphide bonds Hold together the 4 polypeptide chains
in Ig molecules .
-*There are 2 types of disulphide bonds-:
 1- Inter- chain disulphide bonds :
occur between

L – L chains
H – H chains H – L chain

Single L-L bond only in

Hinge But also in Ig A2M (1)
region COOH-terminal such bond can
of the H chain occurs in all Ig(s)except occur under path-
Ig A2M (1) which ogenic conditions.
Lacks an inter chain (e.g. Bence Jones
They can be 1:15 depending disulphide bond protein ) seen in
On the class & subclass types urine of some
patients with
multiple myeloma
INTRA CHAIN DISULPHIDE BONDS :
*- occurs within an individual chain .
*- they are stronger than inter chain bonds .
*- they no. of intra-chain disulphide bonds varies depending only on the number of
domains in the molecular .
Light chain have 2 intra-chain bonds .
*- human IgG, IgA, IgD heavy chains have 4 intra_chain bonds
*- human IgM , IgE heavy chains have 5 intra-chain bonds .
*- Each H& L-Chain has a variable (v) and constant (c) region
*- V region lies in the – NH2 terminal portion of the molecule .
*- The V region has a wide variation in it’s A.A composition .
*- The C region lies in the - COOH terminal end of the molecule .
*- The C region has a much more constant A.A Sequence except for minor inherited
changes
*- The variable regions associate with appropiate constant regions .
so that a variable H – Chain regions (VH) does not occur in an variable L – Chain
(VL) and Vise versa .
*- However , a particular VH chain sequence may occur in more than one
H – Chain class ( i.e IgG, IgM , IgD ,IgA and IgE ) .
*- Thus during class switching in an immune response e.g when B – cells change
their production from IgM to IgG heavy chain
only the constant regions of the H (CH) changes and the antibody specificity
remains the same .
HYPER variable regions
*- they are particular areas within the variable regions
That are highly variable in A. As sequence .
*- THESE hyper variable regions often called complentary determining regions
*- THESE regions occurs at simillar A.A positions in an relatively invariant
molecules .
 CDRs :- they are short polypeptide segments lining near A. As positions 30,50
CDR1 CDR2 CDR3
AND 90 in the variable regions of both L and H chains .
FR1 FR2 FR3 FR4
variability
FRs

CDRS 89-97
variable region 24-34 50-56
NO OF AgS
Note :- the variability range ( index ) used is an arbitrary scales of the no. of
different A.AS found in each position if 100 different Light chain were analyzed .
*- the hyper variable regions are important in the structure of the Ag binding site
( paratope ) .
*- L – chain have 3 hyper variable regions ( the last figure )
*- H – chain have 4 hyper variable regions although,
ONLY 3 OF THE 4 have been associated with epitope recognition
*- each Ig chain consists of a series of globular regions or domains enclosed by
disulphide bonds ( intra or inter ) ?? Chain disulphide bond .
*- The A.AS sequence of the domains show a high degree of homology
( i.e the sequences are very similar ) .
Structure of the variable region framework regions
 Properties of Ig :-

IgG IgA IgM IgD IgE

H – CHAIN     
H – CHAIN SUBCLASS 1, 2 1 2 - - -
M.Wt 150 160-400 900 180 190
Carbohydrate (%) 3 7 12 13 11
Serum conc(mg %) 1200 200 120 3 0.05
Seru t ½ ) days( 21 6 10 3 2
Functions :-
Complement activation ++ - ++++ - -
Opsonization ++++ + - - -
Antiviral activation ++ +++ + - ?
Mast cell sensitization - - - - +
Immunoglobulin are glycoproteins :- (3-13 % of their M.WT )
OLIGOSACCHARIGES + PROTEIN
*- THESE oligosaccharides are present in CH2 or CH3 .
*- N -glycosidic bonds usually link N- acetylglucosamine in the carbohydrate moiety
to asparagine residue in the peptide c-chain of Ab
[ linkage with the enzyme N -acetylglucosamine .
- Asparagine transglycosylase ] transferase
*- t ½ of Abs in the circulation depends on the status of oligosaccharide side chain
*- the oligosaccharide side chain of Ab terminate with galactose to which sialic acid
is bind .
*- when Abs have the sialic acid removed by the enzyme neuraminidase , they
become susceptible to degradation in the liver .
*- in this case the terminal galactase bind to a receptor on hepatocytes and the
entire molecule is , then , interenalized to the cell for degradation via
Proteolytic enzymes in lysosomes of the cells .

Immunoglobulin fragments: Structure/function relationships

Digestion of Abs with Restriction enzymes (Immunoglobulin fragmentation) as
well as Structure/function relationships:
S-S

S-S

F(ab)2 FC
Ab
Restriction enzymes digestion of Abs :
1) Papain : digest above hinge region so it leaves 2 Fab fragments each is monovalent
S-S
And crystalline fragment (FC)

papain
Fab FC
Monovalent

2)Pepsin: digest away most of FC

Fragments below the Interchain disulphide bond
(below the hinge region) it give one large fragmentsF(AB)2 which is consist of two Fab
fragments joined by the disulphide bond
Thus , it is bivalent ,possessing the ability to bind and form agglutination

S-S

F(ab)2 FC
Ab
Figure 4 Immunoglobulin fragments:
Structure/function relationships
 Classes of antibodies
They are 5 isotypes
The class of Ab depends on the A.A: sequence
of the constant regions of the heavy chain . IgM

*- Immunoglobulin M (IgM) :-
* it is a pentamer ( 5 molecules ) .
* they are linked together by disulphide bridges at the COOH terminal end
of the heavy chains as well as an additional poly peptide chain ( joining chain)
* this type of Ab account for 8-10% of the total PLASMA ANTIBODIES .
* it is the most abundant Ab produced by the faetus .
* it binds with viruses and bacteria
*- Immunoglobulin g ( IgG ) :-
* it is a monomer
* it accounts of ~ 75 % of the total antibodies .
* it is important for elicit ting the immune response to Ags
* it is only antibody which pass through the placenta to protect the faetus.
*- immunoglobulin D ( IgD):-
* It is a monomer ACCOUNTING FOR < 1% & TOTAL ANTIBODIS .
* Its function is controversial .
*- immunoglobulin E ( IgE):-
* It is a monomer ( below 0.004 % & the total Abs)
* It is present in spleen , tonsils , mucus membrane of lungs GI
* On binding with ag it releases histamine from mast cells leading to
hypersensitivity .
* It provides immunity to intestinal parasites .
*- immunoglobulin A ( IgA):-
* MONOMER , DIMER or TRIMER( mostly dimer )
* Like IgM the units are linked by disulphide and j chain
* it is found in tears , saliva , intestinal treat secretions
* it binds with Ag preventing them from tissue adherence , colonization ,and
making them more phagocytosed .
 Laboratory Methods
Serology
In vitro Ag & Ab reactions called serology
It provide methods for
i) Identification (Diagnosing) ( ii ) quantization of titre of Ab (and \ or) Ag

Titre : or the level of Ab (s) in the serum can be measured by using known Ag
The titre may have diagnostic
or
prognostic
Ex. A rise in Ab titre between acute &convalescent serum can be used as a
diagnostic tool for a specific disease
The titre is defined as the greatest dilution of serum (which contain the Ab under
consideration ) that reacts which the antigen ( i.e. gives +ve result ) .
- the forces involved in Ag-Ab reactions are greatly affected by various
environmental factors :-

*- The Ag- Ab complex is not bound firmly together .

*_This complex may even dissociate spontaneously .
* physiologic ph & salt concentration promote optimal union of them .
*- the force of attraction tend to be weaker in
a) very acidic .e.g. 0.01M
b) very alkaline medium
i.e pH 4 and alkaline ( i.e. above pH 10 )
- temperature :- it plays an important role :
* the higher the temp ( up to 50 – 55 0 c ) , the more rapid is the rate of reaction
between Ag & Ab .
* the reason is the increase in kinetic motions of the reactants ( Ag & Ab )
various forces act to hold the Ag-Ab complex together :-

* The maximum attractive forces stabilizing Ag-Ab complexes

Are van der weal forces
Ionic bonds

1- van der weal forces :-

* occurs because of spatial fit ( the below fig )
* these forces of attraction hold Ag to Ab only
When the two molecules have complementary shapes (a)

puratope 2
puratope 2 Epitope 2

(a) (i) significant changes

Epitope 2a
In the shape of epitope 2 (b)
Into 2a
 these change precludes its ( 2a ) interactions with the matching binding site of the
original Ab .
* When the molecules have less similar shapes ( b) , these forces are less effective
(b)
2-Ionic bonds :-
* They are patterns of complementary electric charges on the molecule .
* The electrostatic interactions tend to hold the molecules together .
COO
NH3
COO
NH3+

COO +NH3
Affinity :- the strength of attraction between a single epitope and its matching
paratope is the referred to as the affinity of the reaction between the two reactants .
Ag-Ab complex of low affinity dissociate readily
Avidity :-
* It is a related term to affinity
* It refers to the strength of the interactions between multivalent antigens and the
population of Abs that they have included .
*- Avidity is influenced by the affinity of individual Abs for their
(A) epitope
(B) the valency of Ag and
(C) the valency of Ab
tertiary structure of protein :
*- the ability of Ab to bind with Ag can be affected by altering the tertiary
structure of any of them
ex.
insulin which is composed of A&B chains Ab to either one of these chains can
be produced by
(a) splitting the chains
(b) purifying tem
(b) injecting ) .e.g. a pig)
them into foreign host
the pig will produces Ab to the particular chain that was injected
*- if the host (pig) Abs are injected back into the animal species that supplied the
original insulin (man) , the abs will not react with intact insulin molecules .
*- This is because the tertiary structure of native insulin is such that the
on the A & B chains are not accessible .. epitopes
Now , it is generally accepted that in a given poly peptide
the A .As that are spatially accessible because of
Tertiary structure of this protein are only immune
reactive
*- The physical state of the antigen is responsible for the identification of Ag –Ab
reactions and the naming of Abs .
*- The same Ab molecule could , in fact , be described by each of the following
terms :
(1) Agglutinins are Abs that aggregate cellular Ags.
(2) Lysins are abs that cause dissolution of cell membrane .
(3) Precipitins are abs that form precipitate with soluble Ags .
(4) Antitoxins are abs that neutralize toxins .
procedures must be involving direct demonstration and observation of
reaction ..
The relative sensitivities of the tests for Ags and Abs are
Presented in table 8.1 page 156 [ immunology , 3rd edn ].
A- Agglutination Reactions :- a b
Serve to detect and quantities Agglutinins and identify cellular Ags

Bacterial cell white blood cells red blood cells .

**-- when the cells intact with the appropriate Ab , they clump together and eventually

Large enough to be visible

form masses
with naked eye
*- When Ab agglutinates bacteria in the body opsonization occur .
*- Agglutination occurs because Abs and at least bivalent .
*- Two sites on the Ab and multiple sites on the Ag

Ag – Ab lattice formation
that can

build up into increasingly larges coupled

lattice structure

Example widal test :- (diagnostic test of typhoid )

*-Ab of patient serum is measured by adding a constant mount Ag
(e.g. salmonella typhi ) to serially diluted serum .
*- After incubation , the test tubes are examined for visible agglutination .
*- the last tube (i.e the highest dilation of serum ) showing agglutination
is referred as the titre.
B- lyses Reactions :-
In the presence of a complement an Ag – Ab reaction , on a cell
membrane , may result in membrane damage that leads to cell lyses

This phenomenon is important in the host's defense against condition

such as microbial infection or cancer ( graft cell , virus infected cells
, etc…………….(

*i)- Haemolysis :-
In which the Hemoglobin is released from R.B.C, is a requisite
phenomenon for the complement fixation test .

*ii)-- bacteriolysis :-
cells of gram (– ve) bacteria are undergoes immune lyses under certain
condition .
*iii)-- cytolysis :-
involves the destruction of other cells types (e.g. lymphocytes ).

C- precipitation:-
* occurs when the Ag is soluble instead of cellular
*therefore a large number of molecules are required for lattice formation and a
large no .of lattice must be formed for an aggregate to be formed and visibly seen.
*when soluble Ag (s) come intact with specific Ab. They aggregate
(i.e precipitate )
Three conditions are present
A- where the (Ag) is very low with excess Ab (zone of Ab excess ),
Formation of complex occurs
But
Residual Ab remains in the supernatant

B- As more Ag is added , large aggregate is formed

In the (zone of equivalence) ,
maximal Ag-Ab complex are formed and precipitated

C- Instead of reaching a plateau , this curve comes back down to zero

with increasing the mount of Ag (zone of Ag excess )
* this is because the lattice size becomes too small to precipitate .
* In extreme Ag excess . the complex will be trimmer
i.e one Ab +2Ag
Note:- the soluble Immune complex are not processed efficiently by
the reticuloendothelial system ,and ,this cause damage (how??)
 Amount of precipitate
Zone
Of
equivalence

Effects of increasing amounts of Ag on the total immune precipitate

obtained from a mixture of soluble Ag and its homologous Ab
INDIRECT HCG :
Examples 1 :- determination( and\ or) detection of HCG by using indirect methods .
(i) an Ag will be added ( HCG ) .(from the kit)
(ii) Urine will be added ( excess Ag ) from a female may be pregnant .
(iii) Ab to HCG will be added
In case of positive In case of negative pregnancy
A state of Ag excess a state of equivalence will be reached
Therefore, no precipitation therefore, precipitation occur
Direct HCG assay :
* (i) Ab to HCG will be added (from the kit)
* (ii) Ag ( HCG of the test sample will be added) .
If precipitation occur ( positive) if, no precipitation occur ( negative )
Hyaluronic acid (HA) assay using excess
HA binding protein (HABP) :-
* HABP will be added in excess ( known excess ) (ACT AS Ab)
* Sample will be added ( containing HA) (ACT AS Ag)
* [ A state of Ab excess no ppt ]
* An radiolabelled HA will be added ( Ag )
Thus, precipitation occur ( IF +ve sample) and immune complex will be separated
and quantities by radio- immune assay technique ,in case of no precipitation, the
sample is negative
Immune diffusion
* It used for quantization of Ag (s)
* Thus, precipitate will also be demonstrated .
* If an Ag – Ab reaction takes place in semisolid medium (e.g. agar ) , band of
precipitate will be formed .
* The reason of precipitation , is the diffusion of the components (Ag & Ab ) towards
each other .
* A useful example is a double immune diffusion technique :-
Procedure :-
* Ag& Ab preparations are placed in separate wells that are cut into a thin layer of
agar in a Petri dish .
* The reactants diffuse towards each other through the agar until they meet an
optimal proportions [ zone of equivalence ] and forms
( ppt ) bands
Solid
Chevron Fig (a)
Ag
PPT Ag

Zone of Ab

equivalence

The advantages of the procedure is that antigenic relationship

can be detected by the precipitation pattern (s)
3 basic patterns are given :
(a)- in reaction of identity , the 2 Ags are similar , they will diffuse at the same rate
and the two precipitations bonds merges into a solid chevron ( fig b)

Aga Aga Fig b

Ab

2- in reactions of non-identity , the two Ags are completely different and the lines
of the precipitate cross (fig c)
Ayab Agac
Aga Agb
Fig c
Aba Abb
Aba Abb

3- reaction of partial identity :-

* It is indicated by spur formation indicating that one of the
Ag(s) is cross-reactive ( but not identical ) to the other one .
* The spur occurs because one the Abs (b) does not react with the cross-reacting
Ag (Ag ac) but migrate past that Ag (Ag ac ) until it reaches an Ag (Ag ab ) that
Has an epitope for which it has specificity .
B- quantitative radial immune diffusion
* It is used routinely to quantities Ab in serum .
* For this purpose , an agar coated slide is used .
* The agar being impregnated by anti sera ( antibody to human IgG )
* SERUM samples are placed in wells in the sugar .
* As it diffuse through the agar and encounters the Ab, the IgG in the sample form a
concentric ring or halo precipitate .
* The diameter of the halo of precipitate directly correlate with the [ IgG] in the
sample .
Thus , the levels of IgG in the sample can be determined by referring a standard
curve based on halo diameter (s) of known concentration (s) of IgG
C-immune electrophoresis :-
* It was developed because the double immune diffusion technique.
(i) Could not resolve high complex mixtures of Ags .
(ii) and, a more sophisticated technique was needed .
 In this procedure :
(a) Ag is placed in wells in agar on a glass slide and then , subjected to
electrophoresis through application of an electric current .
(b) Under these conditions , the individual Ags or antigenic components ( in
the same sample ) migrate through the agar at variable rates .
(c) If Ab is placed in a well that runs the length of the slide parallel to the path
of migration , the reactants will diffuse towards one another and form separate arcs
of precipitate for each antigenic determinant
D- counter-immune ectrophoresis (CIE)
This technique Is
the double diffusion method + an electric current
Which plays as the migratory force which:
(i) – amplify the speed of reaction ( 24 hrs to 30 min )
(ii) Intensifies the precipitation bonds .
(iii) Increasing the sensitivity of the assay about 10 fold .
Procedure :-
(i) Ag & Ab are placed in wells and the current is applied .
(ii) in suitable buffer ( eg ph 8.6 ) the negativity charged Ags migrate
towards the anode , whereas the Ab [ which has no sufficient net charge ] migrate in
the opposite or counter-direction , as a result of endosmosis .
Precipitation occurs where the reactants melt .•

D- Antitoxin :-
* If a serum contain an antitoxin ( i.e. antibody to a toxin ) , the Ab . Will neutralize
the toxin examples :-
- Suppose serum containing antitoxin is mixed with toxin ( in vitro ).
- Then , after a few minutes , a small amount of the mixture is injected into an
experimental animal ( in vivo ) .
- The animal will be protected against the introduced toxin , and thus , its deleterious
affects disappear because of antitoxin is present .
Clinical example :- ( the virus haemagglutinate R.BCs)
* To examine the serum of a patient suspected of having influenza ,
*1) The patient serum is mixed with known influenza
2)add red blood cells
i- if Ab is present haemagglutination will be prevented
.i.e the sample is positive
this is due to the ability of Ab to bind with the virus and block its ability to
haemagglutinate the R.B.Cs
ii- if no Ab is present , haemagglutinate will occur .
virus + R.B.Cs haemagglutination occur
.i.e the sample is negative
E-Flacculation :-
it is another form of Ag –Ab reaction that
occurs if the Ag is neither cellular
nor soluble
but it is an insoluble particulate
VDRL TEST FOR Syphilis
The venereal disease research laboratory (VDRL) test is a slide flocculation test
used for the diagnosis of syphilis .
The VDRL make use of heterogenetic ( heterophillic ) antigen shared between the •
Spirochete of syphilis & normal beef heart .
* The Ag used is a water insoluble cardiolipin that had coated the surface of
cholesterol particles that were added to the system .
* These form visible aggregate indicate to the presence of Ab ( reagin ) in the serum
of patient for syphilis
[ reagin is Ab type which flocculate (or ppt) an Ag that is neither cellular nor soluble
but it is insoluble ]
* The test can be performed on a glass slide .
Technique :-
cholesterol particles + normal beef heart extract
( inert support ) ( antigen like substance )

Insoluble antigen
serum
( A.Bsource )
visible aggregate

Which can either seen by

The naked eye Using a microscope

and green filter
COMPLEX SEROLOGICAL PROCEDURES
Ag-Ab reactions in which the visible manifestation requires Participation of:
a) Accessory factors
b) Indicators system
c) Specialized equipment
A- fluorescent dyes :-
e.g fluorescein isothiocyanate ( FITC)
* FITC can be conjugated to Ab. Molecules to visualize of the molecule under (uv) or
(b)
a fluorescence microscope .
* such labeled Ab. May then be used to identify Ag(s)
(i) Direct immunofluorescence assay :-
* The method uses Ab. That is specific for a particular Ag
or
parasite
* This Ab is labeled with a fluorescent dye (FITC)
* This conjugate is allowed to react with unknown tissue or organism .
* IF the Ab reacts ( i.e +VE the result ) , it will visualized as green stain on the
specimen when it examined under the fluorescence microscope
by
using uv light
Examples :-
Identification of Trepenoma palladium ( syphilis ) in an extracted from a patient
suspected of having syphilis .
- Procedure :-
*1) The slide is coated with the Ag .
*2) Ab tagged with FITC is added .
* 3)Excess Ab is then washed .
* 4)Then , the slide is examined with uv fluorescent microscope .
* Trepenoma palladium is fluoresce against the black back ground
this methods can be extended for other pathogens .
(ii)-immune peroxides technique :
If viral antigen in tissues will be detected, horse radish peroxides
is conjugated with the Ab .

*-(1) After the enzyme – Ab complex has reacted with the tissue (Ag) .
*- (2) Excess Ab is washed .
*-(3) And , an appropriate enzyme substrate is added to the tissue
section .
*- the bound Ab. Is detected by the presence of a dark precipitate

*- Advantage of immune peroxides technique over the immune fluorescent

technique:-
*- The specimen can be stained with conventional Histochemical dyes
So structural details can be seen ( noted )
*- the tissue con be examined by standard light microscope .

(iii)- Indirect immune fluoresce technique:-

*- The procedure use Ab ( secondary ) ( against ) another Ab
( primary ) of patient .
*- the primary Ab is the patient' serum detection of Abs.
*- the secondary Ab is covalently conjugated whit fluorescent
compound (FITC)

*-*- ex. Of secondary Ab is rabbit antihuman( INJECTED Ab in host is FROM

HUMAN ) gamma globulin anti sera .
.i.e Produced against Ab used to immunize rabbit and that Ab
which will be examined latter on (unknown conc.)

Technique :- (this procedure allows for detection of Abs,)

Example serodiagnosis of syphilis by the fluorescent trepenomal antibody
absorption (FTA-Abs) test
*-(i) -T. pallidum is fixed to a slide
*-(ii)- the slide is flooded with the patient serum (staining Ab )
*-(iii)-If Ab to spirochete are present, the Ab will reacts (bind) with the
organism on the slide
* (iv)-Excess Ab (serum) must be removed with washing , to detect the bound Ab
only .
*-(v) the Ag-Ab complex formed is them treated with the fluorescein – tagged Ab to
human gamma globulin, the excess Ab is washed carefully.
*-If the patient's serum contains Ab (+ v e) against the T. pallidum, fluorescein
organism will be seen when the slide is examined with fluorescence microscope
 FITC

**-- Ab fluorescence
conjugate is
Patient’s binded .
Slide coated
with ag in Serum is Microscope (uv ) is used
(excess)
added
- Indirect Immune fluorescence assay is also used for
Detection of Antinuclear Antibodies (Ana) :-
( e.g. DNA , RNA & His tone)
ANA are present in systemic lupus erythroMatosis ( SLE ) , some
Times in rheumatoid arthritis and other autoimmune collagen –
Vascular diseases .
Example (SLE ) :-
* The procedure is similes to that of T . palladium .
* The Ag is ( DNA ) histone in form such as

Animal Buffy coat calls Rat kidney section

Human Buffy coat calls beef thymus
Lymphoid Thymus

Lymphoid organs
 Haem agglutination Inhibition teat :-
It involves The agglutination of R.B.cs by

Or
(haemagglutinin(s)) Certain virus other
Ab(s) particles (influenza) substances

It demonstrates the presence of serum Ab to haemagglutinating viral substance .

Technique :-
R.B.C s

serum sample Ab from the

Which contain Ab kit that make
Prevent haemagglutination haemagglutination

Agglutination occur no agglutination

The sample is negative the sample is positive
Similar test can be used to detect soluble Ag(s) which able to
react with and neutralize a haemagglutinating Ab .

R.B.Cs from kit

serum Ag which
Abs from kit which
Prevent haemagglutination
Capable of haemagglutination

haemagglutination haemagglutination
takesplace
Sample is negative
Inhibition
Sample is positive
- passive agglutination:-
in the conversion of a reaction system from one that
precipitate one that agglutinate
Thus yields a more sensitive indication of
the presence of antibodies .
Example :RHEUMATOID ARTHRITIS-
The use of latex particles in the diagnosis of rheumatoid arthritis (soluble Antibodies
) is an example of passive agglutination .
Principle :-
In this disease ,the patient produces an Ab (Mainly IgM) to his own IgG
Technique :-
*-(i) latex particles were coated with IgG.
*-(ii) patient 's serum is added (which contains antibodies IgM)
*-(iii) Agglutination indicates the presence of Antibodies (Ig?)
(i.e The test is positive )
The detectable antibody is called rheumatoid factor
 Bis-diazotized diphenyl :-
it is a coupling reagent that can be used to
proteins
conjugate : or to R.B.Cs
Haptens

and thus
Passive haemagglutination
Occur.

Thus :
*- Addition of serum containing Antibodies to these substances (proteins or
haptens ) allow the detection of these specific antibodies to these substance by a
technique called Passive Haemagglutination

rose –waaler Test :

which detect rheumatoid factor in serum of the patients suspected to
have an anti-IgG auto-antibody .i.e. .(IgM to an accumulated IgG
Tannic acid –treated a sheep R.B.Cs (S R.B.Cs) are coated with rabbit IgG -
Antibodies specific for these S. R.B.Cs
 from
Tannic acid the kit

coated by IgG

serum from a patient suspected

R.B.Cs to have autoantibodies

haemagglutination

the sample is positive IgM

Coomb's (antiglobulin) Test :-

* In certain people .Abs directed against antigenic determinants (e.g. R.B.Cs

antigens )are able to form visible aggregates when subjected to :

1- precipitation 2- Agglutination
*-To demonstrate the presence of Abs in such cases the coomb's (antiglobulin)test
may be used .

*- The test involves the addition of Ab direction against gamma globulin : which
provides a bridge between two antibodies coated call or particle
Thus ,The major use of the coomb's test is to detect the Non agglutinating
(haemagglutinating )anti-red blood Cs Abs.

(1) – Direct coomb's test

* It is used to detect call bound antibodies
Technique :-
You must use EDTA BLOOD then centrifugation
*-(1) The red blood calls (bound antibodies )are washed free from serum and the
unbound antibodies (to be leaving the bound ones ).
(2) Antiglobulin serum is added directly to this call suspension -*
*- The direct coomb's test is of value in the detection of antibodies to
R.B.Cs associated with hemolytic disease of new born (e.g.
erythroblastosis fetalis ) and auto immune anemia or disease .

*- The Abs associated with these diseases have the ability to attach to but
not agglutinate the target R.B.Cs .
*- These absorbed Abs can be detected by the use of Ab (i.e. coomb's Kit
serum ) to This human gamma globulin .
(2) indirect coomb's test :-
*- It is used to detect the presence of Circulating Antibodies.
*- It is of value in detecting IgG - associated antibodies in the serum of
woman who is though to be (a) sensitized to Rh antigen And
(b) at risk for carrying an erythoblastbotic febus .

Technique :-
*(1)- Serum sample (containing Ab ) is incubated with donor R.B.Cs( contain
Rh antigen).
Faetus like blood

*- (2) Then ,the cells R.B.Cs are washed off ( to remove excess Ab ) .
*- (3) The anti globulin (coomb reagent) reagent is added ( kit(
 Serum Ab is absorbed haemagglutination ( + ve ).

serum No Ab .(No absorption ) haemagglutination ( - ve ).

Anti globulin commb reagent

Viral Neutralization :-
It is very similar to haemagglutination Inhibition on ( i.e. it is a
neutralization event ) .
*- Principle :-
The assays is based on the ability of specific Abs to interfere with
Some biological function of the virus under consideration ( usually
The infective property is blacked ) .
( 1 )- Cyotpathic effect ( CPE ) :--
certain virus + cells ( in tissue culture ) cell destruction .
2 )- the CPE is useful in the search for virus neutralizing Abs in serum sample.
*-Technique:-
**_ serum suspected of containing Ab is added to a virus suspension .
**_ Susceptible cell culture is inoculated with the mixture .

CPE developed (killing )

If the culture fail to develop
no Neutralizations
CPE (no killing ) ( + ve )
( culture Cell death ) ( - ve )
Abs are interfere with the
Ability of the virus to kill
Tissue culture cells
– Radio immune assay (RIA ):-
* - It is and extremely sensitive method for quantization
of any substance that is Immunogenic or heptenic and can be
labeled with radioactive isotope e .g. I (25I) .
Liquid phase RIA :-
It depends on the competition between labeled (Known ) and
Unlabeled ( unknown )antigen for the same antibody .
 Labeled Ag (known amount) Unlabled Ag[unknown amount]

Ab specific known amount

Immune reaction product

(Immune complex)

( i ) Separation of this complex by immunological method by

secondary Ab or by Precipitating agent . [ (NH4)(SO4)]
ii) Separation of this complex by physiological methods
(a) centrifugation
(b) Decantation .

PPT (commonly used)

The radioactivity of
either . The supernatant
Is then determined

CPM
A Calibration curve based on using serial dilution of known
unlabeled standard ( instead of the serum ) is used for
calculating of un known samples conc.
*- Solid phase RIA :-
Liquid phase is modified by :-
( i ) adsorption or covalently linkage of Ab to solid matrix ( solid phase
RIA) .
( ii ) The unlabeled Ag (sample ) is added followed by the labeled one
( Antigen or Antibody ) .
Then
The bound versus free Ag can be determined by
Using reference calibration curve ( as before)
[ washing steps]
Enzyme linked Immune Sorbet Assays { ELISA } :-
*- ELISA is both highly sensitive ( > 99% ) and specific ( > 99% in
high – risk populations ) .
*- It can be for the assay of either Ag(s) or Ab(s) .
*- Ag or Ab can be attached to solid phase support ( plastic
surfaces , paper disks ) and still retains its immunologic activity.
*- Either Ag or Ab can linked with an enzyme e.g. ( horse
reddish peroxidease alkaline phosphates ) .
*- substrate is added and the color absorbed by the enzymatic
procluct is then quantization and compared with a calibration
curve .

Example :- detection of Ab(s) to the human HIV :-

*- The virus is grown in vitro in a human T-cell culture .
*- purified whole virus is disrupted 8 viral proteins are immobilized
onto plastic beads or multi well trays .
*- Abs to any of these antigens will bind with them & immobilized .
*- Excess proteins are removed by washing the beads ( or wells ) and
an enzyme linked anti human gamma globulin antibody is added .
*- The presence of this second Ab can be detected calorimetrically
by adding a substrate for the enzyme that will yield a colored end
product .
*- The rate of substrate degradation is determined by the amount of
enzyme – labeled Ab that is bound which is proportional with the
amount of Ag in the solution being tested.
*- the color change can be measured quantitatively in a
spectrophotometer .
* Double Antibody sandwich ELISA :-
It is used for the assay of Ag ( e.g. HBSAg ) uses tow Abs as
below :-
( i ) first Ab ( specific e.g. HBs Ag ) is coated on a plastic surface
( poly styrene), the solution being tested for HBs Ag is then
applied to the surface .
( ii ) Washing of any un reacted material .
( iii ) The second Ab ( ie enzyme linked anti HBs Ag specific Ab is
then applied .
( iv ) Any excess conjugate is rewove by washing .
(v) finally substrate is added to the detest the present of En2

ABO group & Transfusion Reactions

*- ALL human erythrocytes contain all antigens ( i.e. Antigens that vary
among individual members of a species ) of the ABO group .
*- This is important system , which . is the basis for blood typing &
transfusions .
*- The A & B antigens are carbohydrates that differ by a single sugar .
*- Despite this small difference , A & B antigens do not cross – react
*- R.B.Cs have 3 terminal sugars ( in common ) on there are surface..

N – acetyl glucose amine

Galactose Fucose H antigen
**-- Type A cells have an additional N –acetylgalatose .
**-- Type B cells have an additional galactose.
N.B . Type A & B genes code for transferaes that add
the respective Sugar .
*-* Type O have only the H antigen :
To avoid Ag – Ab reactions that would result in transfusion, all blood
for transfusion must be carefully cross matched .
*-* So , Ag the corresponding Ab do not coexist in the some person's
blood .
*-* Transfusion reactions result when incompatible donor's R.B.Cs
are transfused e . g . group A In to group B .
 Ag – Ab – Reaction Involving R . B . Cs antigens :-

ABO blood group . Structure of the terminal sugars that

Determine ABO blood groups .
Group Antigen on R.B.Cs Antibody in plasma

A A Anti B

B B Anti A

AB A&B No Anti A NOR Anti B

O No A nor B Anti A & Anti B

4
POSSIBILITES
OF
CONBINATION