Antigen-antibody 9
reactions
9.1 Antigen-Antibody Reactions
9.2 Types of Antigen-Antibody Reactions
Introduction:
Serology as a science began in 1901. Austrian American immunologist Karl Landsteiner
(1868-1943) identified groups of red blood cells as A, B, and O. From that discovery
came the recognition that cells of all types, including blood cells, cells of the body and
microorganisms carry proteins and other molecules on their surface that are recognized
by cells of the immune system.
Antigens are those substance that stimulates the production of antibodies which, when
enter into the bodyreacts specifically in a manner that are clearly visible.
Some antigens may not induce antibody production, but instead creates immunological
tolerance.
An antigen introduced into the body produces only specific antibodies and will react with
only those specific antigens.
These antibodies appear in the serum and tissue fluids. All antibodies are considered
as immunoglobulin. They are mainly of five classes; IgG, IgA, IgM, IgD and IgE.
Antigen-antibody reactions are known as serological reactions and are used as
serological diagnostic tests for the identification of infectious diseases.
9.1 Antigen antibody reactions (Serological reactions):
A) Key features:
1) It explains how antibodies are used to diagnose the disease.
2) Tests based on the interactions of antibodies and antigens have been developed to
determine the presence of antibodies or antigens in a patient.
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S. Y. Pharm D Pharmaceutical Microbiology
3) These tests require both specificity and sensitivity of the antibodies. Sensitivity is the
ability to recognize and bind to the antigen, specificity is the characteristic of binding
only to one antigen and no others.
4) All tests based on Ag/Ab reactions will have to depend on lattice formation or they
will have to utilize ways to detect small immune complexes
5) All tests based on Ag/Ab reactions can be used to detect either Ag or Ab.
B) Role of Ag-Ab Reaction:
In Vivo:
1) Basis of Ab mediated immunity in infections
2) Tissue injury- hypersensitivity / autoimmune diseases
In Vitro:
3) Diagnosis of infections
4) Epidemiological surveys
C) Stages of Ag-Ab reactions:
The reaction occurs mainly in three stages:
1) Primary stage:
The initial interaction between the antigen and antibody, which produces no visible
effects. It is a reversible and rapid reaction.
2) Secondary stage:
Usually, but not always following primary.
Visible effects: precipitation, agglutination, lysis, killing, immobilization killing,
neutralization, or immobilization.
3) Tertiary stage:
(in only in vivo )
Destruction of injurious Ags (humoral immunity)
Tissue damage (allergy/ immune diseases)
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Antigen–Antibody Reactions
Fig.9.1: Antigen and antibody reaction
D) Characteristics of Ag-Ab Reactions:
There are certain characteristics for antigen-antibody reactions;
1) Reaction is specific but ‘Cross reactions’ may occur due to antigenic similarity
2) Entire molecules react
3) No denaturation of Ag or Ab occurs during the reaction
4) Combination occurs on the surface.
5) Combination is firm but reversible, Firmness influenced by:
Affinity & Avidity of the reaction
6) Antigens in Antigens and antibodies can combine in varying proportions
7) Antigens multivalent
8) Antibodies usually bivalent (IgG – 5 or 10)
9.2 Types of Ag-Abs Reactions
1) Precipitation Reaction.
2) Agglutination reaction.
3) Neutralization reaction.
4) Complement fixation.
5) Immunofluorescence.
6) ELISA and RIA.
1) Precipitation reaction:
When sufficient antigen and antibody molecules interact, they precipitate out of
solution
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S. Y. Pharm D Pharmaceutical Microbiology
– Too few antigen molecules, little ppt.
– Too many, Ag-Abs cross links not made
One of the easiest of serological tests
• Soluble Ag & Ab interact and form a lattice that develops into a visible precipitate.
• Occur best when antigen and antibody are present in optimal proportions
(Equivalence).
• Antibodies that aggregate soluble antigens are called precipitins.
• Polyclonal antibodies can form lattices, or large aggregates.
• However, monoclonal antibody can link only two molecules of antigen and no
precipitate is formed.
Fig.9.2: A) Polyclonal Antiserum B) Monoclonal Antibody
A) Precipitation Curve:
Plots of the amount Ag/Ab complexes precipitated when increasing Ag
concentrations are added to constant concentration of Ab. It reveals 3 zones:
i) Zone of antibody excess:
Precipitation is inhibited and antibody not bound to antigen can be detected in
the supernatant.
ii) Zone equivalence:
Maximal precipitation in which antibody and antigen form large insoluble
complexes and neither antibody nor antigen can be detected in the
supernatant.
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Antigen–Antibody Reactions
iii) Zone of antigen excess:
Precipitation is inhibited & Ag not bound to Ab can be detected in the
supernatant.
Fig.9.3: A) Test Tube Reaction B) Precipitation
2) Agglutination Reaction:
The interaction between antibody and a particulate antigen results in visible
clumping called agglutination.
Particulate antigen include: bacteria, white blood cells, red blood cells, latex
particles
Antibodies that produce such reactions are called agglutinins
The interaction of particulate antigens (cells that carry antigens) with antibodies
leads to agglutination reactions.
i) Agglutination is the basis for multiple serological reactions including :
• Blood grouping.
• Diagnosis of infectious diseases.
• And noninfectious diseases.
Diseases may be diagnosed by combining the patient’s serum with a known
antigen.
Diseases can be diagnosed by a rising titer (antibody concentration in serum) or
seroconversion (from no antibodies to the presence of antibodies).
A) Types of Agglutination:
i) Direct agglutination:
Dr. Vividha Dhapte
S. Y. Pharm D Pharmaceutical Microbiology
• In this reaction the antigen is an intrinsic component of the particle.
• The agglutination test is used to determine whether antibody, specific for the
antigen is present in the biological fluids
• serum, urine or CSF.
• Direct agglutination tests are used primarily for diagnosis of infectious diseases.
Direct agglutination reactions test patient serum against large, cellular antigens to
screen for the presence of antibodies.
Direct agglutination reactions can be used to determine antibody titer.
Fig.9.4: Direct agglutination reactions
ii) Indirect agglutination:
• Antigen has been affixed or adsorbed to the particle surface.
• Red blood cells were used as particles for indirect agglutination, however, their
use for indirect agglutination has decreased.
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Antigen–Antibody Reactions
• The commercial availability of latex or other inert particles to which antigen is
affixed has replaced RBCs.
• Indirect agglutination allows testing of organisms that are very pathogenic
without the need to maintain cultures because only the immunologically
dominant molecules are affixed to the particles, not the intact organism.
To test patient serum for the presence of antibodies against soluble antigens
serum is mixed with latex spheres with the soluble antigens attached.
Antibodies will then cause visible agglutination of the latex spheres with the
soluble antigens attached.
Alternatively, antibodies may be attached to the latex spheres to test for the
presence of soluble antigens in patient serum.
Fig. 9.5: Indirect agglutination
Hemagglutination reactions involve agglutination reactions using red blood cells. \
Hemagglutination reactions are used in blood typing, the diagnosis of certain
diseases, and the identification of viruses.
Viral hemagglutination occurs when spikes on the virus cause agglutination of red
blood cells - there is no antigen-antibody interaction.
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S. Y. Pharm D Pharmaceutical Microbiology
• Qualitative
agglutination test
– Ag or Ab
Y
Y + ↔Y
Fig. 9.6: Hemagglutination reactions
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Antigen–Antibody Reactions
B) Advantages:
1) The agglutination reaction has wide spread use in the clinical laboratory. The reason
for the popularity of agglutination tests is that:
i) They are simple,
ii) Inexpensive, and
iii) Reliable.
2) The visible manifestation of the agglutination reaction eliminates the need for
complex procedures and expensive instrumentation.
3) Numerous techniques have been described for agglutination tests. These
techniques may be performed using:
i) slides,
ii) test tubes,
iii) or micotiter plates, depending on the purpose of the test.
4) However the principle of the agglutination remains the same.
3) Neutralization Reactions:
In neutralization reactions, the harmful effects of a bacterial exotoxin or virus are
eliminated by a specific antibody.
An antitoxin is an antibody produced in response to a bacterial exotoxin or a toxoid that
neutralizes the exotoxin.
In a virus neutralization test, the presence of antibodies against a virus can be detected
by the antibodies’ ability to prevent cytopathic effects of viruses in cell cultures.
Antibodies against certain viruses can be detected by their ability to interfere with viral
hemagglutination in viral hemagglutination inhibition tests.
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S. Y. Pharm D Pharmaceutical Microbiology
Fig.9.7: Neutralization Reactions
4) Complement-Fixation Reactions:
The complement fixation test is one of the major traditional tests for the
demonstration of presence of specific antigen or antibodies. It requires a veritable zoo
of reagents and numerous preparatory steps. There are almost as many versions as
there have been users; the microtitre version developed at the Centers for Disease
Control and Prevention (LBCF Test) includes rigorous controls and is commonly used.
Complement-fixation reactions are serological tests based on the depletion of a fixed
amount of complement in the presence of an antigen- antibody reaction.
Good for detecting very small amounts of antibody, when the amount of antibody is too
low to cause a precipitation or agglutination reaction.
Complement fixation was once the basis of the Wasserman test, a test to diagnose
syphilis. It is still used to diagnose some viral, fungal, and rickettsial diseases.
There are two steps, the complement fixation step and the indicator step.
A) Complement Fixation Step:
Add antigen and complement to serum. If the serum contains antibodies against the
antigen they will bind to the antigen and fix the complement.
This ties up all the free complement so it can't participate in the next step, the
indicator step.
B) Indicator Step:
Add sheep red blood cells and anti-sheep red blood cell antibodies to the serum.
Antibodies to the sheep red blood cells bind and can fix complement, if any is available.
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Antigen–Antibody Reactions
If complement is available it will be fixed by the sheep red blood cell antigen-
antibody complex and the sheep red blood cells will be lysed. This indicates that the
serum did not contain antibodies against the antigen added in the complement
fixation step and complement remained free.
If no complement is available the sheep red blood cells will not be lysed. This
indicates there were antibodies against the antigen added in the complement
fixation step and all the complement was tied up when it was fixed by the original
antigen-antibody complex.
Fig.9.8: Complement Fixation reaction
Dr. Vividha Dhapte
S. Y. Pharm D Pharmaceutical Microbiology
C) Advantages of Complement Fixation Test:
i) Ability to screen against a large number of viral and bacterial infections at the same
time.
ii) Economical
D) isadvantages of Complement Fixation Test:
i) Not sensitive – cannot be used for immunity screening
ii) Time consuming and labor intensive
iii) Often non-specific e.g. cross-reactivity between HSV and VZV
5) Fluorescent-Antibody Techniques:
Fluorescent-antibody techniques use antibodies labeled with fluorescent dyes.
Direct fluorescent-antibody tests are used to identify specific microorganisms.
Indirect fluorescent-antibody tests are used to demonstrate the presence of antibody in
serum.
Fig.9.9: Reaction in Fluorescent-Antibody Test
A fluorescence-activated cell sorter can be used to detect and count cells labeled with
fluorescent antibodies.
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Antigen–Antibody Reactions
Fig.9.10: Fluorescent-Antibody Technique
6) Enzyme-Linked Immunosorbent Assay (ELISA):
ELISA techniques use antibodies linked to an enzyme, such as horseradish peroxidase
or alkaline phosphatase.
Antigen–antibody reactions are detected by enzyme activity. An antibody linked to the
indicator enzyme is added to the test well and is bound in the well if the antigen it is
specific for is present. To determine whether or not the enzyme-linked antibody is
bound in the well substrate for the enzyme is added. If the enzyme linked antibody is
present the substrate is converted to a product that causes a color change.
A) Direct:
The direct ELISA is used to detect specific antigens bound in a test well.If a patient's
serum contains a specific antigen.First you coat the bottom of the test well with an
antibody against that antigen. Then you add patient serum. If the antigen you're
looking for is present in the patient's serum it will stick to the antibody that you
coated the bottom of the well with.
So let the serum sit in the well for a few minutes to give the antigen a chance to
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S. Y. Pharm D Pharmaceutical Microbiology
stick and then wash the excess serum away.
Next you add your enzyme-linked antibody, which is also specific for the antigen - its
the same antibody that you coated the bottom of the well with except you linked and
enzyme to it. If the antigen is present you'll end up with a sort of antibody-antigen-
enzyme-linked antibody sandwich.
You have to wash again to get rid of any enzyme-linked antibody that didn't bind to
antigen.
Now add substrate. If there is any enzyme-linked antibody present (and it should
only be there if it is bound to antigen) a product will be formed that causes the color
change.
B) Indirect:
The indirect ELISA is used to detect antibodies against an antigen bound in a test
well.
In this case you want to know if a patient's serum contains an antibody against a
specific antigen (the opposite situation from the one in the direct ELISA test).
First coat the bottom of the well with the antigen that the antibody would be specific
for.
Next add patient serum and allow it to sit in the well for a few minutes to allow any
antibody a chance to bind to the antigen.
Now you have to wash away the serum and any unbound antibodies.
Add your enzyme-linked antibody. This time the enzyme-linked antibody is specific
for human immunoglobulins (its an anti-human antibody antibody). Let it sit for a few
minutes and wash the excess away. If the patient's serum had antibodies against
the antigen you coated the well with you'll end up with an antigen-antibody-enzyme-
linked anti-antibody antibody sandwich.
Now add substrate and look for the color change just like in the direct ELISA.
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Antigen–Antibody Reactions
Fig.9.11: ELISA techniques
ELISA kits are available for both clinical diagnostics and home use. These tests are
used for everything from screening blood for anti-HIV antibodies to home pregnancy
tests.
You can use fluorescent dyes instead of enzymes on your test antibodies but you'll
need a fluorescence microscope or scanner to read the results.
Fluorescence is much easier and more sensitive for lab work but not practical for use
outside of a well-equipped lab.
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