Antigen-Antibody Interactions - Antigen-antibody interactions depend on four types of noncovalent interactions: hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions.  The affinity constant, which can be determined by Scatchard analysis, provides a quantitative measure of the strength of the interaction between an epitope of the antigen and a single binding site of an antibody. The avidity reflects the overall strength of the interactions between a multivalent antibody molecule and a multivalent antigen molecule at multiple sites.  The interaction of a soluble antigen and precipitating antibody in a liquid or gel medium forms an Ag-Ab precipitate. Electrophoresis can be combined with precipitation in gels in a technique called immunoelectrophoresis.  The interaction between a particulate antigen and agglutinating antibody (agglutinin) produces visible clumping, or agglutination that forms the basis of simple, rapid, and sensitive immunoassays.  Radioimmunoassay (RIA) is a highly sensitive and quantitative procedure that utilizes radioactively labeled antigen or antibody.  The enzyme-linked immunosorbent assay (ELISA) depends on an enzyme-substrate reaction that generates a colored reaction product. ELISA assays that employ chemiluminescence instead of a chromogenic reaction are the most sensitive immunoassays available.  In Western blotting, a protein mixture is separated by electrophoresis; then the protein bands are electrophoretically transferred onto nitrocellulose and identified with labeled antibody or labeled antigen.  Fluorescence microscopy using antibodies labeled with fluorescent molecules can be used to visualize antigen on or within cells.  Flow cytometry provides an unusually powerful technology for the quantitative analysis and sorting of cell populations labeled with one or more fluorescent antibodies.

1. MADE BY -
JAYSHREE CHATTERJEE
MSc 2 nd sem
( BIOTECHNOLOGY )
Place - Jabalpur (MP)

2. CONTENTS
 Antigen And Antibody
 Antigen – Antibody Interaction
 Stages Of Ag – Ab Reactions
 S a l i e n t F e a t u r e s O f An t i g e n – An t i b o d y R e a c t i o n
 Chemistry Of Antigen Antibody Binding
 Types Of Antigen – Antibody Interaction
 Application Of Antigen – Antibody Reaction
 Summary

3. ANTIGEN - Molecules that can be
recognized by the immunoglobulin receptor of B
cells or by the T-cell receptor when complexed
with major histocompatibility complex (MHC)
are called antigens. The word antigen is a
shortened form of the words “antibody
generator.” The antigens that are not
immunogenic but can take part in immune
reactions are termed as haptens.
ANTIBODY - Antibodies also known as
Immunoglobulin are antigen- binding proteins
present on the B-cell membrane and secreted by
plasma cells. Three types of globulins are present
in the blood: alpha, beta, and gamma. The
antibodies are the gamma globulins. Antibodies
are one of the major plasma proteins, and
against infection often referred to as “first line of
defense”. The most important function of
antibodies is to confer protection against
microbial pathogens.
What Is Antigen & Antibody ?

4.  The Antigen – Antibody interaction is a bimolecular association similar to an
enzyme-substrate interaction, with an important distinction: it does not lead to an
irreversible chemical alteration in either the antibody or the antigen. The association
between an antibody and an antigen involves various noncovalent interactions
between the antigenic determinant, or epitope, of the antigen and the variable-region
(VH/VL) domain of the antibody molecule, particularly the hypervariable regions, or
complementarity - determining regions (CDRs).
 They form the basis for humoral/antibody mediated immunity. They are used for
detection of disease causing agents & some non-specific Ag’s like enzymes. When Ag-
Ab reaction occurs in-vitro they are known as ‘serological reactions’. The reactions
b/w Ag & Ab occurs in 3 stages:
1st = formation of Ag-Ab complex.
2nd = leads to visible events like precipitation, agglutination etc.
3rd = destruction of Ag or its neutralization.

5. STAGES OF Ag – Ab REACTIONS
Primary stage:
a) Initial interaction between Ag
& Ab – INVISIBLE
b) Rapid, occurs at LOW
TEMPERATURES.
c) Reaction is REVERSIBLE.
d) Ag & Ab is bound to each
other by WEAK VAN-DER
WAAL’S FORCES, IONIC
BONDS & HYDROGEN
BONDING.

6. Secondary stage
IT IS AN IRREVERSIBLE INTERA-
-CTION WITH VISIBLE EFFECTS.
Demonstrable events –
a) Precipitation
b) Agglutination
c) Lysis of cells
d) Killing of live antigens
e) Neutralization of toxins
f) Complement fixation
g) Immobilization of motile
organisms.
h) Enhancement of phagocytosis.

7.  Specificity of Antigen – Antibody Reaction.
 Immune complex.
 Binding Site of Antigen – Antibody Reaction.
 Binding Force of Antigen – Antibody
Reaction.
SALIENT FEATURES OF
ANTIGEN – ANTIBODY
REACTION

8. Specificity refers to the ability of an individual antibody
combining site to react with only one antigenic determinant or
the ability of a population of antibody molecules to react with
only one antigen.
Each antibody binds to a specific antigen; an interaction
similar to a lock and key.
1). SPECIFICITY

9. An immune complex is formed from the integral binding of
an antibody to a soluble antigen.
The bound antigen acting as a specific epitope, bound to an
antibody is referred to as a singular immune complex.
Ag + Ab  Ag-Ab complex
2). IMMUNE COMPLEX

10. 3). Binding Site of Antigen – Antibody
Reaction In antigen - antibody reaction, the antibody attaches with the
antigen. The part of antigen which combines with antibody is called
Epitope.
 An epitope, also known as antigenic determinant, is the part of an
antigen that is recognized by the immune system, specifically by
antibodies, B cells, or T cells. The part of an antibody that recognizes
the epitope is called a paratope.

11. 4). Binding Force of Antigen – Antibody
Reaction
 The noncovalent interactions that
form the basis of Ag-Ab binding
include hydrogen bonds, ionic bonds,
hydrophobic interactions, and van der
Waals interactions. Because these
interactions are individually weak, a
large number of such interactions are
required to form a strong Ag-Ab
interaction.
 Furthermore, each of these
noncovalent interactions operates
over a very short distance, generally
about 1 x 10¯ mm (1 angstrom, Å);
consequently, a strong Ag- Ab
interaction depends on a very close fit
between the antigen and antibody.
7

12. Chemistry Of Antigen Antibody
Binding :-
The interaction of an antigen antibody is a reversible binding
process that requires several non-covalent interactions like
hydrogen bonds, electrostatic forces and hydrophobic interactions.
Affinity and Avidity between the antigen antibodies also play a
major role in their interaction.
Normally antigen-antibody binding site on antibodies are more or
less flat and hence spacious so that they can attach large complexes
or structures.

13. AFFINITY - Affinity denotes the intensity of attraction between
antigen and antibody. Low-affinity antibodies bind antigen weakly and
tend to dissociate readily, whereas high-affinity antibodies bind antigen
more tightly and remain bound longer.
[Ab-Ag]
Affinity K =
[Ab] × [Ag]
AVIDITY - Avidity is a measure of the overall strength of binding
of an antigen with many antigenic determinants and multivalent
antibodies. The various antibodies produced by a single Ag combine with
the different antigenic determinants of the Ag.
nAb+ mAg ↔ AbnAgm
Where nAb is no. of Ab’s and mAg is no. of Antigenic determinants.

14. Agglutination
reaction
PRECIPITATION
REACTION
Radio-immuno
assay
Immobilization
test
Neutralization
reaction
ImmunofluorescenceOpsonisation
Complement
fixation test
Enzyme
Immunoassay
Types Of Antigen – Antibody
Interaction

15. PRECIPITATION REACTION
 Precipitation refers to an antigen-antibody reaction between a
soluble antigen & its antibody resulting in the formation of
insoluble precipitate. The antibody causing precipitation is called
PRECIPITIN.
 Precipitation occurs in two media:
a) Liquid or solution,
b) Gel - agar, agarose or polyacrylamide.
 Formation of an Ag-Ab lattice depends on the valency of both
the antibody and antigen.
 The antibody must be bivalent; a precipitate will not form with
monovalent Fab fragments. The antigen must be either bivalent
or polyvalent; that is, it must have at least two copies of the
same epitope, or have different epitopes that react with different
antibodies present in polyclonal antisera.

16. Precipitation In Liquid Or Solution
 Soluble antigen + antibody (in proper proportions)  visible precipitate
 Ring test and flocculation test are examples of precipitation in solution.
a) Ring test :- In this test, antigen solution is layered over antiserum in a
test tube or capillary tube. Precipitation between antigen & antibodies in
antiserum solution is marked by the appearance of a ring of precipitation at
the junction of two liquid layers. C-reactive protein (CRP) & streptococcal
grouping by the Lancefield methods are the examples of the ring test.
Test - tube Capillary - tube

17. 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 (PROZONE) -
precipitation is inhibited and antibody
not bound to antigen can be detected in
the supernatant.
2. Zone Of 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 (POSTZONE)
- precipitation is inhibited & Ag. not
bound to Ab. can be detected in the
supernatant.

18. Precipitation in Gels
 The precipitation test in agar gel is termed as immunodiffusion test. In this test,
reactants are added to the gel and antigen – antibody combination occurs by
means of diffusion. The rate of diffusion is affected by the size of the particles,
temperature, gel viscosity, amount of hydration, and interactions between the
matrix and reactants.
 Immunodiffusion reactions have the following advantages:
■ In this test, the line of precipitation is visible as a band, which can also be
stained for preservation.
■ The test can be used to detect identity, cross-reaction, and nonidentity between
different antigens in a reacting mixture.
 Immunodiffusion reactions are classified based on the -
(a) number of reactants diffusing and
(b) direction of diffusion,
as follows:

19.  Single Diffusion In One Dimension
(Oudin Procedure) :-
 Ab is incorporated in agar gel in a test tube
& Ag solution is layered over it.
 Ag diffuses downward through the agar gel
– forming a line of precipitation.
 The number of precipitate bands shows the
number of different antigens present in the
antigen solution.
 Double diffusion in one dimension
(Oakley-Fulthorpe procedure) :-
 Ab is incorporated in agar gel. Above
which is placed a column of plain agar.
 The Ag is layered over it. The Ag & Ab
move towards each other through the
intervening column of plain agar & form
the precipitate.
Ag (High Concentration)
Two different precipitates (two Ag
with different molecular weights)
Agar with Antiserum which
reacts with Two Ag’s
Ag – Ab
Reaction
Visible
Precipitation
Antibody

20.  Single Diffusion In Two Dimension
(Radial Immunodiffusion) :-
 In this method antiserum solution containing
antibody is incorporated in a agar gel on a RID
slide or petri plate.
 Wells are cut and antigen is applied in the gel.
Then Ab present in the gel reacts with the Ag
which diffuses out of the well. Precipitation rings
are formed around the wells.
Double Diffusion In Two Dimensions
( O ucht erl ony Procedure) :-
 Ab is incorporated in agar gel.Above which is
placed a column of plain agar. The Ag is layered
over it. The Ag & Ab move towards each other
through the intervening column of plain agar &
form the precipitate.
 Antiserum – central well. Different Ags in the
surrounding wells.
Reaction of identity
Lack of relatedness
P a r t i a l i d e n t i t y
RID
DD

21. AGGLUTINATION REACTION
 The interaction between
antibody & a particulate
(Insoluble) antigen results in
visible clumping called
agglutination. The antibodies
that cause agglutination are
called Agglutinins & the
particulate antigens
aggregated are called
Agglutinogens.
 Particulate antigen
include:
a) bacteria,
b) white blood cells,
c) red blood cells,
d) latex particles .
Agglutination Test
antige n antibody
positive negative

22. Types of agglutination reactions
Direct Agglutination Passive Agglutination
Tube Agglutination
Antiglobulin
(Coombs’) Test
Slide Agglutination
Heterophile
Agglutination
Direct Coombs Test Indirect Coombs Test
Latex Agglutination Test
Coagglutination Test
Hemagglutination Test

23. APPLICATIONS :-
- Blood typing.
- Bacterial infections.
- Viral Infections.
LIMITATIONS :-
- Time consuming (1 day)
- Cannot distinguish IgG from IgM.

24. COMPLEMENT FIXATION TEST

25. Enzyme – Linked
Immunosorbent Assay
(ELISA)
•In 1971, enzyme labeled Ag’s
and Ab’s were developed as
serological reagents for the
assay of Ab’s and Ag’s.
• These are very simple,
sensitive, economic and less
hazard when compared to RIA.
•The ligand used here is a
molecule which can detect the
Ab and is covalently coupled to
an enzyme such as peroxidase,
betagalactosidase, alkaline
phosphatase etc.
ENZYME IMMUNOASSAY
Types Of Enzyme-linked Immunosorbent
Assay (ELISA)

27. OPSONIZATION

28. Application of Antigen – Antibody Reaction
The chief use of antigen-antibody reactions
are:
a. Determination of blood groups for transfusion.
b. Serological ascertainment of exposure to infectious
agents.
c. Development of immunoassays for the quantification
of various substances.
d. To detect the presence or absence of protein in
serum.
e. Determining the characteristics of certain immuno-
deficiency disease.

29. SUMMARY
 Antigen-antibody interactions depend on four types of noncovalent interactions: hydrogen bonds,
ionic bonds, hydrophobic interactions, and van der Waals interactions.
 The affinity constant, which can be determined by Scatchard analysis, provides a quantitative
measure of the strength of the interaction between an epitope of the antigen & a single binding site
of an antibody. The avidity reflects the overall strength of the interactions between a multivalent
antibody molecule and a multivalent antigen molecule at multiple sites.
 The interaction of a soluble antigen and precipitating antibody in a liquid or gel medium forms an
Ag-Ab precipitate. Antibody and soluble antigen interacting in aqueous solution form a lattice that
eventually develops into a visible precipitate. Antibodies that aggregate soluble antigens are called
precipitins.
 The interaction between a particulate antigen and agglutinating antibody (agglutinin) produces
visible clumping, or agglutination that forms the basis of simple, rapid, and sensitive Immuno-
assays.
 The enzyme-linked immunosorbent assay (ELISA) depends on an enzyme-substrate reaction that
generates a colored reaction product. Other than these many types such as complement fixation
test, opsonization, neutralization etc. are the reactions which takes part in Ag – Ab interaction.

30. References :-
 Textbook of Microbiology and Immunology (2nd Ed) - Subhash
Chandra Parija .
 Kuby Immunology (5th Ed) - Judith A. Owen, Jenni Punt,
Sharon A. Stranford, Patricia P. Jones .
 Immunology & Microbiology – A.Mani, L.M.Narayanan, Dulsy
Fatima, A.M.Selvaraj, N.Arunugam (Saras Publication)
 https://www.google.co.in/search?q=complement+fixation+test+
procedure&source
 www.google.co.in