This document provides an overview of antigen-antibody reactions, including definitions, general features, measurement techniques, types of reactions such as precipitation, agglutination, neutralization, immunofluorescence, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA). It describes techniques like precipitation reactions in liquids and gels, single and double diffusion, electrophoresis, latex agglutination, complement fixation, and microtitration agglutination tests. The document outlines the applications and uses of these various antigen-antibody reaction techniques.

1. Dr. Pulipati Sowjanya
Professor & Head
Dept. of Pharm. Biotechnology
Vignan Pharmacy College
Vadlamudi, Guntur (Dt)

2. INTRODUCTION
DEFINITION : Antigen-antibody reaction, is a specific chemical
interaction between antibodies produced by B cells of the WBC and
antigens during immune reaction.
This reaction occurs in three stages.
1.The primary stage - rapid , reversible.
2. The second stage - precipitation , agglutination, lysis of cells.
3.The tertiary stage – destruction of antigens.

3. GENERAL FEATURES
✓The reaction is specific.
✓There is no denaturation of the antigen – antibody
during reaction.
✓The combination is firm but reversible.
✓ Both antigen & antibodies are multivalent.
✓Both antigen & antibodies participate in the formation
of agglutinates or precipitates.

4. MEASUREMENT
➢Antigen- antibody participating in the reaction may be measured
in terms of mass or units or titer.
➢The antibody titer is the highest dilution of serum that gives an
observable reaction with the antigen in the particular test. It
influenced by the nature and quantity of the antigen.
➢Antigens may also be titrated against sera.

5. The two important parameters of serological test are:
SENSITIVITY: It is the ability to detect even very minute
quantities of antigen or antibodies. When a test is highly sensitive,
false negative results will be absent.
SPECIFICITY: It is the ability to detect reaction between
homologous antigen & antibodies only. In a highly specific test, false
positive results will be absent.

6. TYPES OF ANTIGEN-ANTIBODY REACTIONS
PRECIPITATION REACTIONS:
DEFINITION:- When a soluble antigen combines with its
antibody in the presence of an electrolyte at a suitable
temperature(37oC) and PH (7.4) it forms an antigen – antibody
complex in the form of an insoluble precipitate.
▪The antigen -“precipitinogen” and antibody - “precipitin”.
▪If the precipitate remains suspended as floccules, the reaction is
known as “flocculation”.

7. MECHANISM
❖Marrack(1934) proposed the lattice hypothesis to explain
the mechanism of antigen – antibody reaction.
❖According to this hypothesis multivalent antigen combine
with bivalent antibodies in varying proportions, depend on
the antigen- antibody ratio in reaction mixture.

9. APPLICATIONS
a. These are highly sensitive for the detection of antigen.
b. Diagnosis of infectious disease.
c. Forensic medicine in the identification of blood &
semen stains on weapons & clothing or in other criminal
investigations.
d. Detection of food adulterants.
e. Detection of unknown antibody.

10. TYPES OF PRECIPITATION REACTIONS
Single / double diffusion in
one dimension.
PRECIPITATION
TESTS
IN LIQUIDS IN GELS
Single / double diffusion in
two dimensions.
Counter- current & rocket
electrophoresis.
Ring test
Slide test
Tube test

11. PRECIPITATION IN LIQUIDS
RING TEST:
▪ In a positive case a ring of white
precipitate is formed at the junction
of two liquids.
Ex :Ascolis thermo precipitin test

12. SLIDE TEST:-
A drop each of antigen and antibody are placed on
a slide and mixed by rotation , floccules appear.
Ex: test for syphilis
TUBE TEST-:
The antigen and antibody are taken in a test tube
mixed, incubated and then observed for precipitation
Ex: Kahn test is a tube flocculation test used for the
diagnosis of syphilis.

13. PRECIPITATION IN GELS
Gel precipitation tests are more advantageous than the
liquid precipitation test.
The bands are stable, can be stained & preserved.
The gels has got minute pores through which the antigens
and antibodies migrate & form bands where they meet in
optimum proportion.
Since each antigen – antibody reaction gives rise to band of
precipitation, the number of antigens in the reaction mixture
can be detected.

14. TYPES
1.SINGLE DIFFUSION IN ONE DIMENSION:
It is also called as Oudin method.
The antibody is incorporated in agar gel in
a test tube and the antigen solution is layered
over it.
USES:
The number of bands indicates the number of
different antigens present.

15. DOUBLE DIFFUSION IN ONE DIMENSION:
It is also called as Oakly – Fulthorpe
procedure.
This method separates antigen and antibody
solution with a layer of clear agar .
USES :
This test is used to determine the number of
antigens in a mixture.

16. 3. SINGLE DIFFUSION IN TWO DIMENSIONS:
It is also called as radial immuno diffusion.
Antiserum is incorporated in agar gel at 560C and poured on a glass
slide or into petridishes.
The gel is allowed to set. Wells are cut and Antigen is added.
Diameter of the ring gives an estimate of the concentration of
the antigen.

17. 4.Double diffusion in two dimensions:
• This method is also known as Ouchterlony method.
• Antiserum is placed in the central wall and antigen in the
surrounding wells.
USES:
•Used to detect & compare difference between antigen & antibody.
•In the diagnosis of bacterial, viral, fungal and parasitic infection.

19. ELECTRO IMMUNODIFFUSION
•It was discovered by Graber and Williams, 1953.
1. Counter – current immunoelectrophoresis: This involves
simultaneous electrophoresis of Ag & Ab in gel in opposite direction &
form a precipitation line within few minutes is called “cross- over”.
Antibodies tend to migrate towards cathode while most antigenic
protiens move towards anode.
USES:
❖ Detection of antigen in various body fluids.
❖ Diagnosis of bacterial, viral, fungal infections
Ag Ab
- +

20. 2. Rocket electrophoresis
• It is a simple technique described by Laurell
in 1965.
• Antibody is mixed with agarose gel and
poured on a slide.
• Increasing concentration of antigen is added
to wells.
• Antigen moves toward the anode.
• Based on the height of the rocket ,
concentration of antigen is estimated.
USES:
• Estimation of proteins and other antigens in
various body fluids.
Direction
Of current
Increasing concentrations of
antigen in wells
cathode
anode

22. Immunoelectrophoresis
• Method
– Ags are separated by electrophoresis
• Interpretation
– Precipitin arc represent individual antigens
Ag
-+
Ag
Ab
Ag
Ab
– Ab is placed in trough cut in the agar

23. AGGLUTINATION REACTION
• When a particulate antigen is mixed with its antibody in the
presence of an electrolyte at a suitable temperature and PH.
• Antigen - agglutinogen and antibody - agglutinin.
TYPES:- 1. Slide Test:
A drop of antiserum is added to the suspension & mixed.
The clumping together of the particles & clearing of the drop
within few seconds indicate positive results.
USES:
Blood grouping and cross matching can be detected.

24. 2.TUBE TEST
Serum is diluted in saline serially by double dilution in a
series of test tubes.
An equal volume of standard antigen suspension is added
to all the tubes.
Highest dilution of serum at which agglutination occurs is
recorded as antibody titre.
USES:
Widal test for diagnosis of enteric fever.
Tube agglutination for brucellosis.
Weil- Felix test for Typhus fever.

27. Microtitration Agglutination Tests
These tests are done in microtitre plates
1. Indirect passive haemagglutination test:
Carrier particle: Ag is coated on red cells. Turkey red cells are
often selected as red carrier red cells. The formalin fixed red
cells are treated with tannic acid to make the antigen adhere.
These are called sensitized cells.
Procedure: Serially diluted patient serum is mixed with sensitized
cells.
Result: Positive result shows agglutination of red cells

28. Microtitration Agglutination Tests
2. Haemagglutination inhibition antibody (HAI) test:
detects antibodies against viruses (arbo, influenza, measles, rubella,
orthomyxo & paramyxo)
Procedure:
❑ Patient serum is treated with kaolin or heparin-magnesium chloride (to
remove nonspecific inhibitors)
❑ The serum is allowed to react with a suspension of known viral
antigens
Result: If antibodies to virus are present, it will coat the haemagglutinins
on the viral particles and will form complexes which will block the
binding sites on the viral surface preventing haemagglutination.

30. Microtitration Agglutination Tests
3. Reverse passive haemagglutination (RPHA) test:
❑ This technique utilizes stabilized red blood cells coated with
specific antibody.
❑ If the corresponding antigen is present, the red cells will
agglutinate.
❑ It is useful for non-agglutinating viruses.

31. LATEX AGGLUTINATION TEST:
➢ Latex is an inert particle of mineral
origin with a uniform diameter of 0.08 – 1
micron.
➢ Antigen molecules are adsorbed on to
the surface of particles like bentonite clay
or latex.
USES:
• C-Reactive Protein test.
• Antistreptolysin O test.

32. Complement fixation test
• It tests for the presence of either specific antibody or specific
antigen in a patient's serum.
• It uses sheep red blood cells (sRBC), anti-sRBC antibody and
complement, plus specific antigen or specific antibody.
• If either the antibody or antigen is present in the patient's serum,
then the complement is completely utilized, so the sRBCs are not
lysed. But if the antibody (or antigen) is not present, then the
complement is not used up, so it binds anti-sRBC antibody, and
the sRBCs are lysed.
• The is one form of complement fixation test

34. The neutralization reaction are the reactions of the antigen –
antibody that involves the elimination of harmful effects of bacterial
exotoxins or a virus by specific antibodies. The neutralizing substance
i.e., antibodies are known as antitoxins.
USES:
1.Diagnosis of viral infection:
2.Shick test
NEUTRALISATION TEST

35. NEUTRALISATION TEST

36. IMMUNOFLUORESENCE
Fluorescence is a property exhibited by certain substances which
absorb light rays of one particular wavelength and then release the
absorbed energy by emitting rays of different wavelength.
These are of 2 types:
1.DIRECT: Ab to tissue Ag is labeled with fluorochrome
USES:
To identify microorganisms present
in clinical specimens
Ag
Fluorochrome
Labeled Ab
Tissue Section

37. 2.INDIRECT:
To Known tissue Ag attached on slide unknown serum is added
Fluorochrome-labeled anti-Ig is used to detect binding of the first
Ab.
USES:
Diagnosis of Syphilis.
Ag
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled
Ab

38. .
.
.
DIRECT TEST IMMUNOFLUORESENCE
antigen
fluorescent conjugated -
antibody
ANTIGEN – ANTIBODY COMPLEX
INDIRECT TEST -
IMMUNOFLUORESCENCE

39. RADIOIMMUNO ASSAY:
The most sensitive technique for detecting antigen or antibody.
First developed by two endocrinologist, S.A Berson and Rosalyn
Yalowin in 1960 to determine levels of insulin – anti insulin complexes
in diabetics.
1.Liquid Phase RIA:
❑Increase amount of antigen (unlabeled) of unknown concentration is
added .
❑The amount of labeled antigen in solution is measured & the
construction of unlabeled antigen can be determined.
2.Solid Phase RIA:
It is simple easy in handling as compared to liquid Phase RIA.

41. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
• Uses an enzyme system to show the specific combination of
antigen antibody
• An enzyme labeled or linked to a specific antigen
• A substrate
• A color reader
• Double antibody technique to detect and assay antigen
• Indirect technique to Assay and antibody
TYPES
a. Indirect ELISA
b. Sandwich ELISA
c. Competitive ELISA

42. • Done on Polyvinyl microtitre plate
Enzyme substrate color
• Horse radish o-phenyl-diamine red/
peroxidase dihydrochloride orange
• Alkaline p-nitro phenyl yellow
phosphatase phosphatase
USES:
a. Detection of viral antigens like Rota virus, Hepatitis.
b. Detection of hormones & toxins.
c. Pregnancy test.
Detection of antibodies in various bacterial & viral infection.
Bacterial – Salmonella, Vibrio cholera, Haemophilus.
Viral - Rubella , HIV, Herpes simplex, Hepatitis B.
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)