The document discusses antigen-antibody interactions and various serological techniques used to detect them, including precipitation reactions and agglutination reactions. It describes how antigen and antibody bind through non-covalent interactions, and factors that affect the antigen-antibody reaction like concentration, temperature, and pH. It also summarizes different techniques used to detect antigen-antibody interactions like precipitation, agglutination, Coombs test, and flocculation and the principles behind each method.

1. Ag-Ab INTERACTION Nidhi S.
Dept. of Microbiology
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2. INTRODUCTION
• It is association between an antibody and an antigen by noncovalent
interactions between the antigenic determinant (epitope) of the
antigen and the variable-region (VH/VL) domain of the antibody
molecule (CDRs).
• The non covalent forces that hold the reactants (Ag-Ab) together
are:
1. van der Waals forces
2. Electrostatic forces
3. Hydrophobic forces
4. Hydrogen bonds
5. Ionic bonds
• It can be used to detect the presence of either antibody or antigen.
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4. FACTORS AFFECTS ANTIGEN-
ANTIBODY REACTION
• Concentrations of reactants
• Temperature
• Length of incubation
• pH of test system
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5. PHASES OF ANTIGEN/ANTIBODY
REACTIONS
1. Primary Phenomenon: Sensitization – binding of antibody to antigen
(not visible). These tests are Difficult, Complex, Expensive, Require
special equipment and Time consuming. Primary Phenomenon Based
Techniques include: Immunofluorescence, Radioimmunoassay,
Enzyme immunoassay
2. Secondary Phenomenon: Lattice formation - Sensitization taken a
step further to lattice formation. Fab of Antibody molecule binds to
two separate antigens on adjacent antigens: If antigen on large
structures such as RBCs causes agglutination and if both antibody
and antigen are soluble results in precipitation. These tests are easy
to perform, less expensive, less time consuming and do not require
special equipment. Secondary Phenomenon Based Techniques
include: Precipitation, Agglutination and Complement Fixation
3. Tertiary Phenomenon: Reaction not visible, detected by affect of
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6. SOME TERMINOLOGIES
1. Affinity: Measure of the binding strength between an
antigenic determinant (epitope) and an antibody combining
site. It is the sum of the attractive and repulsive forces
operating between the antigenic determinant and the
combining site .
2. Avidity: The cumulative binding strength of all antibody-
epitope pairs which results from multivalent antigen and
antibody.
3. Cross-Reactivity: It is reaction between an antibody and
an antigen that differs from the immunogen. It is sometimes
also referred to as crossimmunity or cross-protective
immunity.
4. Valence: The relative capability of a antibody to react with a
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7. PROZONE EFFECT OR HOOK
EFFECT
• Hook effect or the prozone effect is an immunologic
phenomenon whereby the effectiveness of antibodies to form
immune complexes is sometimes impaired when
concentrations of an antibody or an antigen are very high.
• The formation of immune complexes stops increasing with
greater concentrations and then decreases with extremely high
concentrations, producing a hook shape on a graph of
measurements.
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8. PRECIPITATION REACTIONS
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 (Equivelance).
Antibodies that aggregate soluble antigens
are called precipitins.
Polyclonal antibodies can form lattices, or
large aggregates and monoclonal antibody
can link only two molecules of antigen and no
precipitate is formed.
( Lattices or large
aggregates )
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9. 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:
1. Zone of antibody excess - precipitation is inhibited and
antibody not bound to antigen can be detected in the
supernatant;
2. Zone equivalence - maximal precipitation in which antibody
and antigen form large insoluble complexes and neither
antibody nor antigen can be detected in the supernatant;
3. Zone of antigen excess - precipitation is inhibited & Ag. not
bound to Ab. can be detected in the supernatant
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10. PRECIPITATION CURVE
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11. TYPES OF PRECIPITATION
REACTION
I- Precipitation in Solution
a) Bottom Precipitate
b) Ring Precipitate (Ring Test)
II- Simple Immunodiffusion
(ID) or precipitation in gel
a) Double ID (Ouchterlony or
ODD)
b) Single Radial ID (RID)
(Mancini)
III- Electro-Immnodiffusion
a) Immunoelectrophoresis (IEP)
b) Immunofixation
c) Rocket Electroimmunodiffusion (EID)
d) Counterimmunoelectrophoresis (CIEP)
IV-Measurement of Precipitation by Light
a) Turbidimetry
b) Nephelometry
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12. ADVANTAGES & DISADVANTAGES OF
PRECIPITATION
Disadvantages
1. Time consuming
2. Some costly instruments
are required
3. High technical skill
required
Advantages:
1. Fairly sensitive
2. High specificity
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13. PRECIPITATION REACTION IN FLUID
Precipitation reactions in fluids follow
the same roles of precipitation curve
zones.
A. Bottom Precipitate: Occurs when
Soluble Ag interact with soluble Ab
and form a visible precipitate that
give bottom ppt after centrifugation.
B. Ring Precipitate (Ring Test) (Tube
Precipitation test): Involve soluble
antigens with antibodies in tubes (test
or Capillary tubes). Layer Ag over Ab
Precipitate occurs at the interface of
the two reagents, forming a ring. It is
Test Tube
reaction
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14. PRECIPITATION REACTION IN GEL
•Agar gel used as medium.
•The precipitate is seen in gels as visible
precipitin lines.
a) Single Radial ID
• The technique is quantitative is based
upon the reaction between an Ag, and a
specific Ab during a diffusion period.
• Ag placed in a well diffuses into an agar
containing the Ab (anti-IgG looking for
serum IgG).
• The Ag-Ab interaction is manifested by a
well-defined ring of precipitation around
the Ag well.
The region of equivalence
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15. PRECIPITATION REACTION IN GEL
b) Double Radial ID (Ouchterlony or ODD)
• Both antigen and antibody can diffuse
independently.
• It is based upon the simultaneous
application of Ag and Ab in separate but
adjacent wells of an agar plate.
• As the materials diffuse toward one
another, precipitate lines form resulting
from the Ag-Ab interactions.
The region of equivalenc
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16. If multiple wells of Ag are positioned around an Ab well on the
same plate, several patterns of reactivity may be observed.
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17. If the Ag A (patient)
is the same as the Ag
A (control), the
reaction with the Ab
will be the same and
the result is a solid,
continuous, smooth
line of identity
between the Ag wells
and the Ab well.
If Ag A (patient) is
different from Ag B
(control), and both
react with the Abs to
A & B, the precipitin
lines cross and a
double spur is
formed; this is a line
of nonidentity.
If Ag A (patient) and
Ag A1 (control) share
a common element
but are not exactly the
same (Abs to A), a
single spur is formed.
This is the line of
partial identity.
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18. MEASUREMENT OF
PRECIPITATION BY LIGHT
Principle : Antigen-antibody complexes, when formed, will precipitate
in a solution resulting in a turbid or cloudy appearance that can be
measured by:
1. Turbidimetry: Passing light through a cloudy solution (Net
decrease in light intensity).
2. Nephelometry: Measuring light scattered at a particular angle after
being passed through a solution i.e. indirect measure. Amount of
light scattered correlates to the concentration of the solution
Evaluation: Ag concentration is proportional to the turbidity of
complexes.
• It is used for the measurement of serum proteins concentration
(immunoglobulins, acute-phase proteins, complement components
C3, C4, transferrin, albumin etc.).
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19. AGGLUTINATION REACTIONS
Interaction between Ab & Particulate Ag results in visible clumping called
Agglutination.
When antibodies are mixed with their corresponding antigens on the
surface of large, easily sedimented particles such as animal cells,
erythrocytes, or bacteria, the antibodies cross-link the particles, forming
visible clumps. This reaction is termed agglutination.
Antibodies produces such reactions are called agglutinins.
It depends on cross linking of polyvalent antigens.
Occur best when antigen and antibody are present in optimal proportions
(Equivelance).
Types of particles that participate in such reactions are erythrocytes,
bacterial cells and inert carriers such as latex particles. Each particle must
have multiple antigenic or determinant sites, which are cross-linked to
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20. TYPES OF AGGLUTINATION
REACTION
I- Direct Agglutination
II- Indirect or Passive
agglutination
III- Coomb’s test
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21. ADVANTAGES AND DISADVANTAGES OF
AGGLUTINATION
Advantages:
•Most widely used
•Very simple
•No instrument required
•Cheap
•Sensitive
Disadvantages:
•No highly specificity
•No highly sensitivity
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22. HEMAGGLUTINATION
• It is a type of direct agglutination reaction.
• When antibodies bind antigens on the
surface of red blood cells (RBCs), the
resultant clumping reaction is referred to
as hemagglutination.
• E.g.: Typing of ABO antigen.
• RBCs are mixed on a slide with antisera to
the A or B blood group antigens. If the
antigen is present on the cells, they
agglutinate, forming a visible clumps on
the slide.
• Determination of which Ag are present on
donor and recipient RBCs is the basis for
matching blood types for transfusion.
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23. BACTERIAL AGGLUTINATION
• It is a type of direct agglutination reaction.
• A bacterial infection give rise to the production of serum antibodies
specific for bacterial surface antigen which can be detected by bacterial
agglutination tests.
• It may be qualitative (Slide test) or quantitative (Tube test or serum
agglutination test).
• In SAT, test serum is diluted in a series of tubes, then constant defined
amount of antigen is added to each tube and incubates them for ~20h
at 37°C.
• Particular antigen clumps at the bottom of the test tube
• Test is read at 50% agglutination
• Example: Brucellosis screening , Widal Testing (test of Salmonella
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25. PASSIVE OR INDIRECT
AGGLUTINATION
• It is easy to read visually and give quick results.
• In this technique, antigen coated particles are prepared for agglutination
reaction.
• Particles, such as erythrocytes, latex, gelatin, and silicates are used.
• For example mixing a soluble antigen with red blood cells that have been
treated with tannic acid or chromium chloride (promote adsorption of
antigen on cell surface).
• The use of synthetic beads or particles provides the advantage of
consistency, uniformity, and stability.
• Problems encountered with the use of erythrocytes as carrier particles are
possibility of cross reactivity with heterophile antibody if the cells used are
nonhuman.
• It is used to detect Antinuclear antibody (of lupus erythematosus),
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27. COOMBS TEST
• Coombs test is also known as antiglobulin test (AGT).
• Coombs test is performed using an antiserum containing antibodies that
bridge antibody- or complement-coated RBCs.
• Coombs test used in the preparation of blood for transfusion in cross-
matching, detection of antibodies in the blood plasma of pregnant women
as part of antenatal care, and detection of antibodies for the diagnosis of
immune-mediated haemolytic anemias.
Mechanism:
• Coombs test is based on anti-human antibodies binding to human
antibodies (IgG or IgM). These anti-human antibodies are produced by
plasma cells of non-human animals after immunizing them with human
plasma. These anti-human antibodies will also bind to human antibodies
that may be fixed onto antigens on the surface of red blood cells (RBCs). In
the appropriate test tube conditions, this can lead to agglutination of RBCs
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28. TYPES OF COOMBS TEST
2. Indirect Coombs Test:
•Also known as indirect antiglobulin
test (IAT), used to detect in-vitro
antibody-antigen reactions.
•It is used in
1. Blood transfusion preparation
2. Antenatal antibody screening
1. Direct Coombs test:
• Also known as direct antiglobulin
test (DAT), used to detect if
antibodies or complement system
factors have bound to RBCs surface
antigens in vivo.
•It is used to detect
1. Hemolytic disease of the newborn
(or erythroblastosis fetalis)
2. Autoimmune
hemolysis/Immunohemolytic
hemolysis
3. Drug-induced immune-mediated
hemolysis. E.g.: Penicillin (high
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30. FLOCCULATION TEST
• Flocculation tests are designed for antibody detection and are based on
the interaction of soluble antigens with antibodies, producing a
precipitate of fine particles that can be seen with the naked eye.
• Example: VDRL slide flocculation test, RPR card test, Kahn’s test for
Syphilis.
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31. REFERENCES
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3rd edition. Garland Science Taylor and Francis Group, Newyork
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Immunity. 9th edition. Elsevier
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the immune system in health and Diseases. 6th edition. Garland Science
Taylor and Francis Group, Newyork
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Garland Science Taylor and Francis Group, Newyork
6. J. M.Cruse, R. E. Lewis. (2009). Illustrated Dictionary of Immunology. 3rd
edition. CRC Press Taylor and Francis Group, New York.
7. Google
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