Humoral Immunity

Antibody Mediated

1
Humoral Immunity
Results in
production of
proteins called
immunoglobulin's
or antibodies.
Body exposed to
foreign material
termed antigen
which may be
harmful to body:
virus, bacteria, etc.
Antigen has bypassed
other protective
mechanisms, ie, first
WHAT ARE ANTIBODIES?
Antigen specific proteins produced
by plasma cells
Belong to immunoglobulin
superfamily
Located in blood and extravascular
tissues, secretions and excretions
Bind pathogenic microorganism
and their toxins in extracellular
compartments
Secreted form of immunoglobulin's
CLASSES (ISOTYPES) OF
IMMUNOGLOBULINS
Classes based on constant region of
heavy chains
Immunoglobulin A (IgA)
Immunoglobulin D (IgD)
Immunoglobulin E (IgE)
Immunoglobulin G (IgG)
Immunoglobulin M (IgM)
Differentiation of heavy chains
Length of C region, location of disulfide
bonds, hinge region, distribution of
carbohydrate
Classes have different effector
Different classes of Antibodies
Structural configuration of
Antibody

Dr.T.V.Rao MD 6
 THREE DIMENSIONAL
STRUCTURE OF ANTIBODIES
Antibodies function in setting of infectious
process
Proteolytic enzymes, salt and pH differences
Antibodies remain stable based on
Sequence of domains
Single domain consists of
100 110 amino acids folded into compact and
stable conformation
Domains
Variable (V)
Single V domain in H and L chains
Constant (C)
Single C domain in L chains
Three to four (C) domains in H chains
Antibodies are Produced by B
Lymphocytes

Dr.T.V.Rao MD
 Properties of stem cell
1. They are capable
of dividing and
renewing
themselves for
long period.
2. They are
unspecialized.
3. They can give rise
to specialized cell
types.
 Specialization of Stem Cell:
Differentiation
Differentiation: unspecialized stem cell give
rise to specialized (differentiated) cell in
response to external and internal chemical
signals.
Internal signals: specific genes causing
differential gene expression.
External signals:
Chemicals secreted by other cells such as
Growth factors, cytokines, ect.
Physical contact with neighboring cells.
 Stem Cell types
MULTIPOTENT
Can differentiate into multiple specialized cells of a
closely related family of cells (ex. Hematopoietic stem
cell)
OLIGOPOTENT
the ability to differentiated into a few cells (ex.
Lymphoid)
UNIPOTENT
these cells only produce one cell type, but have
property of self renewal which distinguishes them
from the non stem cells (ex. Muscle stem cells,
cardiac stem cells).
 Cord blood
Umbilical cord blood is also know as
placental blood.
It is the blood that flows in the
circulation of the developing fetus in
the womb.
After the baby`s birth the left over
blood in the umbilical cord and
placenta is called cord blood.
This blood is a rich source of stem
cells.
Embryonic vs adult stem
cells
ESC
Totipotent
Differentiation into ANY cell
type
Large number can be
harvested from embryos.
May cause immune
rejection.
Potential for undesired
tumor formation (teratoma)
High ethical controversy &
uncertain legal status.
ASC
Multi or pluripotent
Differentiation into SAME cell
types, limited outcomes.
Limited numbers, more
difficult to isolated.
Less likely to cause immune
rejection, since the
patients own cell can be
used.
Less likely to form tumors.
Less moral & legal
controversy
 Immuno-markers for
Hematopoietic Stem Cells (HSC)
CD 34
CD133
C-kit receptor CD 117
Thy-1 CD 90
CD 59
CD 110
WHAT DISEASES CAN BE CURED
BY STEM CELL THERAPIES ?
1. Any disease in which there is tissue
degeneration can be a potential
candidate for stem cell therapies:
Alzheimers disease
Parkinsons disease
Spinal cord injury
Heart disease
Severe burns
Diabetes
WHAT DISEASES CAN BE CURED
BY STEM CELL THERAPIES ?
2. Cord blood stem cells have been
used to treat more than 45
malignant and genetic disease.
Leukemia
SCID
 Immunoglobulin Classes
I. IgG
Structure: Monomer
Percentage serum antibodies: 80%
Location: Blood, lymph, intestine
Half-life in serum: 23 days
Complement Fixation: Yes
Placental Transfer: Yes
Known Functions: Enhances phagocytosis,
neutralizes toxins and viruses, protects fetus and
newborn.
 Immunoglobulin Classes
II. IgM
Structure: Pentamer
Percentage serum antibodies: 5-10%
Location: Blood, lymph, B cell surface (monomer)
Half-life in serum: 5 days
Complement Fixation: Yes
Placental Transfer: No
Known Functions: First antibodies produced
during an infection. Effective against microbes
and agglutinating antigens.
 Immunoglobulin Classes
III. IgA
Structure: Dimer
Percentage serum antibodies: 10-15%
Location: Secretions (tears, saliva, intestine,
milk), blood and lymph.
Half-life in serum: 6 days
Complement Fixation: No
Placental Transfer: No
Known Functions: Localized protection of
mucosal surfaces. Provides immunity to infant
digestive tract.
 Immunoglobulin Classes
IV. IgD
Structure: Monomer
Percentage serum antibodies: 0.2%
Location: B-cell surface, blood, and lymph
Half-life in serum: 3 days
Complement Fixation: No
Placental Transfer: No
Known Functions: In serum function is unknown.
On B cell surface, initiate immune response.
 Immunoglobulin Classes
V. IgE
Structure: Monomer
Percentage serum antibodies: 0.002%
Location: Bound to mast cells and basophils
throughout body. Blood.
Half-life in serum: 2 days
Complement Fixation: No
Placental Transfer: No
Known Functions: Allergic reactions. Possibly
lysis of worms.
CLASSES (ISOTYPES) OF
IMMUNOGLOBULINS
Additional
classification based on
light chains
Kappa
Lambda
Each IG has either
kappa or lambda, not
both
IgG kappa
IgG lambda
No functional
differences between
light chains
 B Cell Receptors for
Antigens
B cell receptors
Bind to specific, intact antigens
Are often called membrane antibodies or
membrane immunoglobulin's
Antigen- Antigen-
binding binding site
site Disulfide
bridge
Light Variable
chain regions

Constant
C C regions
Transmembrane
region

Plasma
membrane
Heavy chains

B cell Cytoplasm of B cell

(a) A B cell receptor consists of two identical heavy

Dr.T.V.Rao
chains MD light chains linked by
and two identical
several disulfide bridges.
Antibodies bond to antigenic determinants
Antigenic determinants are portions of the antigen
Antibodies are Proteins that
Recognize Specific Antigens
 Epitopes: Antigen Regions
that Interact with Antibodies
Dynamics of Antibody
Production
Primary
immune
response
Latent period
Gradual rise in
antibody
production
taking days to
weeks
Plateau reached
Antibody level
Secondary Response
Second exposure to
SAME antigen.
Memory cells are a
beautiful thing.
Recognition of
antigen is immediate.
Results in immediate
production of
protective antibody,
mainly IgG but may
see some IgM
Dynamics of Antibody
Production
Antibody production
Initial antibody produced in IgM
Lasts 10-12 days
Followed by production of IgG
Lasts 4-5 days
Without continued antigenic
challenge antibody levels drop off,
although IgG may continue to be
produced.
 In the secondary immune
response
Memory cells facilitate a faster, more
efficient response

1 Day 1: First 3 Day 28: 4 Secondary response to anti-

2 Primary
exposure to Second exposure gen A produces antibodies
response to
antigen A to antigen A; first to A; primary response to anti-
antigen A
produces anti- exposure to gen B produces antibodies to B
bodies to A antigen B

104
Antibody concentration

103
(arbitrary units)

102 Antibodies Antibodies

to A to B
101

100
0 7 14 21 28 35 42 49 56
Time (days)
THE PRIMARY HUMORAL
IMMUNE RESPONSE
Immune response initially produces IgM
antibodies then switches to IgG
antibodies
Question
Why switch from IgM to IgG?
Answer
Limited effector mechanisms for IgM
Range of effector mechanisms for IgG
Mechanism
Isotope or class switching
Humoral (antibody-mediated) Immunity

IL 1

Autocrine IL 2
stimulation
ISOTYPE OR CLASS SWITCHING

Process by which B cell changes class of

IG produced while preserving antigenic
specificity
Involves somatic recombination which
attaches different heavy chain constant
region to variable region
Occurs only during active immune
response
Mechanisms involves recombination
between
Switch sequences (regions)
 Clonal selection of B cells
Generates a clone of short-lived activated effector
cells and a clone of long-lived memory cells
Antigen molecules
Antigen molecules bind to the antigen
B cells that receptors of only one
differ in of the three B cells
antigen shown.
specificity
Antigen
receptor

The selected B cell

proliferates, forming
a clone of identical
cells bearing
receptors for the
selecting antigen.

Some proliferating cells
develop into long-lived
memory cells that can
respond rapidly upon
subsequent exposure
to the same antigen.
Some proliferating
cells develop into
Antibody short-lived plasma
molecules cells that secrete
antibodies specific
for the antigen.
Clone of memory cells
Clone of plasma cells
Humoral (antibody-mediated) Immunity

Memory
Cells
Benefits of Immunological Memory
Clonal Selection
Only one type of
antibodyand one
type of B cell
responds to the
antigenic determinant

That cell type

then produces a
large number of
clones
FUNCTIONS AND PROPERTIES OF
ANTIBODY
Neutralization
Direct inactivation of pathogen or toxin
thereby preventing its interaction with
human cells
Opsonization
Coating of pathogens for more efficient
phagocytosis
Activation of complement
More efficient phagocytosis
Direct killing
DIVERSIFICATION OF ANTIBODIES
AFTER B-CELLS ENCOUNTER ANTIGEN

Mature, nave B cell has membrane bound IgM

and IgD antigen receptors
Binding of antigen initiates proliferation and
differentiation of B-cells into plasma cells
During differentiation, B cells switch from
making immunoglobulin to antibody M and D
isotypes
IgM
Produced in large amounts
Provides protective immunity
IgD
Produced in small amounts
No known function
 DIVERSIFICATION OF ANTIBODIES
AFTER B-CELLS ENCOUNTER ANTIGEN
Following antigen activation of B-cells,
additional diversification occurs in V
domain by
Somatic hyper mutation
Somatic hyper mutation
Introduction of random single nucleotide
substitutions (point mutations) throughout V
regions of H and L chains
Mechanism poorly understood
More common in hyper variable regions (CDRs)
 OUTCOME OF SOMATIC
HYPERMUTATION
Gives rise to some antibodies
with higher
Affinity for antigen
Affinity
Strength of binding of one molecule
to another by a single binding site
Higher affinity antibodies are
produced as immune response
proceeds
 IgM ANTIBODY OF THE
IMMUNE RESPONSE
First isotype produced in primary response
May or may not be produced in secondary response
Produced before B cells undergo somatic
hypermutation
Occurs as pentamer with J chain
Found primarily in blood and lymph
Multiple binding sites confers high avidity and
compensates for low affinity of monomers
Highly effective in complement activation
Functions as rheumatoid factor
 IgG ANTIBODY OF THE
IMMUNE RESPONSE
Second isotype produced in primary response
Primary isotype of
Secondary immune response
Memory immune response
Represents approximately 75% of total serum
IG
Four subclasses (1-4)
Different effector functions
Transported across placenta
Functions as rheumatoid factor
 IgA ANTIBODY OF THE
IMMUNE RESPONSE
Two subclasses (IgA1 and IgA2) and
two forms (monomeric and dimeric)
Monomeric
Located in blood and extracellular spaces
Predominately IgA1
Ratio of IgA1 to IgA2 is 10:1
Functions as rheumatoid factor

Dimeric
Located in mucous membranes and secretions
Predominately IgA2
Ratio of IgA2 to IgA1 is 3:2
J chain like IgM
 IgE AND IgD ANTIBODIES
OF THE IMMUNE
RESPONSE
IgE
Binds with high affinity to receptors on mast
cells, basophils and activated Eosinophils
Longer half-life when cell bound
Initiates a strong inflammatory reaction to
parasites
Involved in allergic reactions
IgD
Antigen receptor on mature B-cells
No other known function
 Immunological
Memory
Antibody Titer: The amount of antibody
in the serum.
Pattern of Antibody Levels During Infection
Primary Response:
After initial exposure to antigen, no antibodies
are found in serum for several days.
A gradual increase in titer, first of IgM and then
of IgG is observed.
Most B cells become plasma cells, but some B
cells become long living memory cells.
Immunological Memory
(Continued)

Secondary
Response:
Subsequent exposure to
the same antigen displays
a faster and more intense
antibody response.
Increased antibody
response is due to the
existence of memory
cells, which rapidly
produce plasma cells
upon antigen stimulation.
ANTIBODIES AS DIAGNOSTIC
AND THERAPEUTICS AGENTS
Based on specificity and affinity of
antibodies
Both applications require large quantities
of identical antibodies
Monoclonal antibodies
Monoclonal antibodies are produced using
hybridoma cell line
Hybridoma cell line
Derived from single antibody producing cell
fused with myeloma cell (neoplastic plasma
cell)
Methods for analysis of
hummoral immunity
CURVE OF HEIDELBERG
Imunoprecipitation reaction

1. Used for qualitative and

quantitative detection
of antigens and
antibodies:
phase one formation of
primary complexes with
low MW
Phase two
interconnection of Ag and
Ab to the three
dimensional network
(formation of insoluble
aggregates )
Precipitation and immunodiffusion
in gels
2. Double diffusion is utilized as a
rough estimation of antigen or
antibody purity.
3. Double diffusion in agar can be used
for semiquantitative analysis in
human serological system.
 Precipitation and immunodiffusion
in gels

Double diffusion in two dimension

Similar precipitin Precipitin lines Precipitin lines

lines do not form a completely cross
complete cross

Ouhterlony
 Radial immunodifusion-Mancini
(immunoglobulins, complement, haptoglobins etc)

In radial immunodiffusion antibody is incorporated into the agar gel as

it is poured and different dilutions of the antigen are placed in holes
punched into the agar. As the antigen diffuses into the gel, it reacts
with the antibody and when the equivalence point is reached a ring of
precipitation is formed.
Radial immunodifusion-
Mancini
 Immunoelectrophoresis

Immunoelectrophoresis
Plasma
combines electrophoresis
(mixture of
antigens) separation, diffusion and
precipitation of proteins.
Electrophoresis
In immunoelectrophoresis, a
complex mixture of antigens is
Antiserum
(mixture of placed in a well punched out of an
antibodies) agarose gel and the antigens are
electrophoresed so that the antigen
Imunodiffusion 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 agarose,
precipitin lines are produced in the
Immunoelectrophoresis
 Western blot

A technique in which proteins are

separated by gel electrophoresis
and transferred to a membrane
sheet. A specific protein is then
identified through its reaction
with a labeled antibody.
Where is this technique
used?
The HIV Test known as "Western Blot" uses
the same technique, where the goal is to
detect the presence of antibody in a
sample. Known HIV infected cells are
opened and their proteins separated and
blotted on a membrane. Then the serum
to be tested is applied. Free antibody is
washed away, and a secondary antibody is
added that binds to human antibody and is
linked to an enzyme. The stained bands
then indicates the proteins to which the
patient's serum contains antibody.
Western Blot Banding

Image reproduced from Commercial Methods in Clinical Microbiology, 2000. ASM Press.
Interpretation of Results
(General Consensus)

Negative: No bands present

Positive: 2 ENV band present

(WHO
Guidelines)

Indeterminate: Any bands present but

do not meet criteria for
positive
Measurement of Precipitation by Light Scattering-
Nephlometry & turbidimetry

Antigen-antibody complexes, when formed

at a high rate, will precipitate out of a
solution resulting in a turbid or cloudy
appearance.
Turbidimetry measures the turbidity or
cloudiness of a solution by measuring
amount of light directly passing through a
solution.
Nephelometry indirect measurement,
measures amount of light scattered by the
antigen-antibody complexes.
Turbidimetry versus
Nephelometry
Turbidimetry measures light which
PASSES through.
Nephelometry measures light which
is SCATTERED.
Fluorescent tests

Use fluorescent dyes as labels

Fluorescein is the most important dye
used in these test
Fluorescein-labeled antibodies used in
two types of tests
 There are two ways of doing IF staining
Direct immunofluorescence
Indirect immunofluorescence

1. Direct immunofluorescence
Ag is fixed on the slide
Fluorescein labeled Abs are layered over it
Slide is washed to remove unattached Abs
Examined under UV light in an fluorescent
microscope
The site where the Ab attaches to its specific Ag
will show apple green fluorescence
Use: Direct detection of Pathogens or their Ags in
tissues or in pathological samples
Direct immunofluorescence
indirect

[INSERT FIGURE 17.14]

Indirect immunofluorescence
The different patterns of fluorescence on HEp-2 cells include (A)
peripheral, (B) homogeneous, (C) nucleolar, (D) centromere,
and (E) speckled.
A B C

D E
Anti ds DNA antibodies
 ANCA

p-ANCA

c-ANCA
 Clinical relevance of humoral immunity
investigation

Immunoglobulines

1. Low igG
. Primary and secondary ID
- SCID
- -linked agammaglobulinemia (Bruton)
- selective IgA deficiency, igA myeloma
. Lymphomas
2. High IgG - infections, autoimmunity, liver diseases
IgG 1 is the most abundant of the four subclasses and reaches
'adult' levels in early childhood.
IgG1 provides the largest immune response and the dominant
response to protein/polypeptide antigens.

IgG2 is the most common IgG subclass deficiency and may also
be associated with IgA deficiency. Recurrent respiratory
infections in children (Haemophilus influenzae type b,
Streptococcus pneumoniae) are particularly associated with IgG2
deficiency. 'Adult' levels of IgG2 are not usually reached until 6-7
years of age.

IgG3 deficiency can manifest itself in recurrent respiratory

infections with obstructive lung disease. It may be seen in
association with IgG1 deficiency. Usually patients suffering
from Chronic Fatigue Syndrome and Fibromyalgia will have
low IgG3 deficiency.

IgG4 deficiency may be associated with IgG2 and IgA deficiency

in ataxia telangiectasia, frequently masked by normal levels of
total IgG. Subclass deficiencies can also be associated with T-cell
dysfunction. Elevated levels of IgG4 are seen in atopic
dermatitis, asthma and some parasitic diseases.
In a recent study of 56 adults with IgA deficiency the authors
Ig
high:
Ethanol abuse, primary biliary cirrhosis, damage of
mucosal surfaces, acute and sub- acute infections
low:
Chronic lung diseases, PID, third pregnancy trimester,
immunosuppression

Ig
high:
macroglobulinemia waldenstrom, parasitic infections
(malaria), actinomycosis, infectious mononucleosis, SLE,
RA, hyper ig syndrome
low:
agammaglobulinemia, lymphoproliferative disease, igG
IgA myeloma
igE

high:
dermatitis atopica(>100 IU/ml), ig myeloma, Wiskott-
Aldrich, hodgkin's lymphoma, aspergilosis broncopulmona,
parasitic diseases, AIDS, Buckley syndrom
low:
-linked agammaglobulinemia, deficiency, ataxia-
teleangiectasia
 Cryoglobulinemia
is a medical condition in which the blood contains large amounts of
cryoglobulins - proteins that become insoluble at reduced
temperatures. Cryoglobulins typically precipitate at temperatures
below normal body temperature (37 degrees Celsius) and will
dissolve again if the blood is heated. Cryoglobulinemia can be
associated with various diseases such as multiple myeloma and
hepatitis C infection.

Type I isolated monoclonal immunoglobulins/10-15% of the total cases

These are composed of a single monoclonal immunoglobulin paraprotein
(usually IgM). Sometimes, these are represented by light chains only and
can be extracted from the urine, or they will accumulate in blood serum in
the event of renal failure.
Type II immunocomplexes formed by monoclonal IgM /50-60% of reported
cases/They usually have a polyclonal component, usually IgG, and a
monoclonal component, usually IgM or IgA, which has an RF function. The
IgM can recognize intact IgG or either the Fab region or Fc region of IgG
fragments. This is why most type II cryoglobulins are IgM-IgG complexes.
Type III immunocomplexes formed by polyclonal IgM /25-30% /of the
reported cases. These have very similar function to the type II
cryoglobulins, however they are composed of polyclonal IgM and IgG
 Complement
Fraction mean level disease

1- inhib. 20 ./ HAE
SLE
chronic vacuities

2 25 ./ dermatomyositis
SLE
chronic vacuities

3 1300 ./ inflamation
gram (-) bacteria
SLE nephritis

4 600 ./ HAE
SLE
chronic vacuities
 Frequency of
Conditions Associated With Positive IF- Positive ANA
ANA Result, %
Test Results
Diseases for which an ANA test is very useful for diagnosis
SLE 95100
Systemic sclerosis (scleroderma) 6080
Diseases for which an ANA test is somewhat useful for diagnosis
Sjogren syndrome 4070
Idiopathic inflammatory myositis (dermatomyositis
or polymyositis) 3080

Diseases for which an ANA test is useful for monitoring or

prognosis
Juvenile chronic oligoarticular arthritis with
Uveitis 2050
Raynaud phenomenon 2060
Conditions in which a positive ANA test result is an intrinsic
part of the diagnostic criteria
Drug-induced SLE 100
Autoimmune hepatic disease 100
MCTD 100
Basic method for assessment of
immune complexes in vasculitis is :

. Immunoturbidimetry.
b. Flow cytometry.
c. Radial immunodiffusion.
d. Immunohistochemical methods
The Heidelberg curve
represents:

a. The amount of antibodies.

b. The quality of antigens.

c. Basic patterns of binding
of antigens and antibodies.