This topic describes about antigen-antibody reaction in detail including their classification, mechanism of action, various examples of each reaction with labelled diagrams.

1. DR ARUNA RANI BEHERA
MD MICROBIOLOGY

2. INTRODUCTION
 ANTIGEN-ANTIBODY
REACTION-
-USES
-CHARACTERISTICS
-STAGES
-MEASUREMENT
 TYPES
-PRECIPITATION
-AGGLUTINATION
-COMPLEMENT FIXATION TEST
-NEUTRALISATION
-OPSONISATION
-IMMUNOFLUROSCENCE
-RADIOIMMUNOASSAY
-ENZYME IMMUNO ASSAY
-IMMUNOCHROMATOGRAPHY
-CHEMILUMINESCENCE IMMUNO
ASSAY
-IMMUNOBLOTTING

3. GENERAL FEATURES
 Antigens and antibodies combine with each other
specifically and in an observable manner.
 Ag-Ab reaction in –vitro  serological tests.
A.Uses
 In-vivo
-basis of immunity against infectious diseases.
-l/t tissue injury in HSN reacns & AI diseases.
 In-vitro:
-For diagnosis of infection.
-for epidemiological studies.
-For identification of non-infectious agents such as enzyme.
-Detection and quantitation of either antigens or antibodies.

4. Characteristics
1.The reaction is specific.
2. Entire molecule react and not fragments.
3. There is no denaturation of the antigen or antibody during the reaction.
4. The combination occurs at the surface.
5. The combination is firm but reversible. Influenced by Affinity and Avidity of the
reaction
AFFINITY: Refers to the intensity of attraction between the antigen and antibody
AVIDITY: It is the strength of the bond after the formation of antigen and antibody
complex.
6. Both antigen and antibody participates in the formation of the agglutinates and
precipitates.
7. Antigens and antibodies can combine in varying proportions.

5. PRIMARY STAGE
-Initial interaction
without any visible
effects
-Rapid & Reversible
reaction
-weaker intermolecular
forces-
Vander waal’s forces
H2 bonds
Ionic bonds
SECONDARY STAGE:
. leading to visible effects such as
 Precipitation
 Agglutination
 Lysis of cells
 Killing of live antigens
 Neutralization of toxins
 Fixation of complement
 Immobilization of motile
organisms
REACTIONS OCCUR IN 3 STAGES

6. Antibodies are designated by the reactions
Antibody causing agglutination as Agglutinin
(Antigen called as Agglutinogen)
Antibody causing precipitation as Precipitin
(Antigen called as Precipitinogen)
TERTIARY STAGE:
Antigen-antibody reactions lead to neutralization or
destruction of injurious antigens or to tissue
damage.

7. COMPARATIVE EFFICIENCY OF THE
IMMMUNOGLOBULIN CLASSES IN
DIFFERENT SEROLOGICAL REACTIONS:
REACTION IgG IgM IgA
Precipitation Strong Weak Variable
Agglutination Weak Strong Moderate
CFT Strong Weak Negative
Lysis Weak Strong Negative

8. MEASUREMENT OF ANTIGEN AND ANTIBODY
 Measurement as units or titre
 The antibody titre of a serum is the highest dilution of
the serum which shows an observable reaction with the
antigen in the particular test.
SENSITIVITY: Refers to the ability of the test to detect
even very minute quantities of antigen or antiboby.
False –ve results are absent or minimal.
SPECIFICITY: Refers to the ability of the reactions
between homologous antigens and antibodies only
and with no other. False +ve reactions are absent or
minimal.

9. PRECIPITATION REACTION
 Take place in liquid media or
in gels (Agar, Agarose,
Polyacrylamide)
 When a soluble antigen
combines with its antibody in
presence of electrolytes
(NaCl) at a suitable
temperature and pH, the
antigen-antibody complex
forms an insoluble
precipitate.
 If the precipitate remain
suspended as floccules, the
reaction is known as
Flocculation.

10. :
MECHANISM OF PRECIPITATION:
Marrack proposed the lattice hypothesis
PROZONE PHENOMENON-In the zone of antibody excess,
form a large lattice
ZONE OF EQUIVALENCEAg & Ab are in optimum
proportions.
POSTZONE PHENOMENON-In the zone of antigen excess,.

11. GraphicalRepresentation
If amount of precipitate
in the different tubes are
plotted on a graph, the
resulting curve will have
three phases.
Prozone – imp. in clinical
serology.
- Several dilutions are
tested.

12. Antibody excess
Optimal
proportions Antigen excess
Serial Dilution of Serum containingAbs-
decreasing concentration ofAb

13. APPLICATIONS OF PRECIPITATION REACTION:
- qualitative or quantitative test.
- Sensitive for detection of antigens than antibodies.
- Forensic application Identification of blood & seminal stains
- Testing for food adulterants
- Grouping of streptococci
- VDRL test for syphilis
- Standardise toxins & antitoxins
- Test toxigenicity in diptheria bacilli

14. Typesof Precipitation &Flocculation
1. RING TEST
-Simplest
-Layering Ag solution over a column of
antiserum in a narrow tube
- Ppt forms at the junction.
e.g. Ascoli’s thermopreciptin test
Lancefield grouping of streptococci
2. SLIDE TEST – A drop each of Ag &
antiserum are placed on a slide &mixed –
floccules appear
e.g. VDRL test for syphilis

15. 3. TUBE TEST - Tube flocculation
e.g. Kahn test for syphilis
Standardisation of toxins & toxoids
4. IMMUNODIFFUSION (Precipitaiton in gel)
Advantage – Distinct band of ppt form
- Reaction is visible
-Number of different Ags in the reacting
mixture can be identified.
- Indicates identity, cross-reaction and
nonidentity between different antigens

16. 1.SINGLE DIFFUSION IN ONE DIMENSION
(OUDIN PROCEDURE)
Antibody incorporated in agar gel in a test tube
antigen is layered over the solution
Antigen diffuses downward
Form a line of precipitation
Number of bands indicates the number of different antigens
present

17. Modifications ofImmunodiffusion
Single diffusion in one dimension (Oudin
procedure)
Antigen
Precipitation band
Ab in agargel

18. 2. DOUBLE DIFFUSION IN ONE DIMENSION
(OAKLEY-FULTHORPE PROCEDURE)
Antibody incorporated in gel
A column of plain agar placed above it
Antigen layered on top of agar
Antigen and antibody move towards each other
through the intervening column of plain agar
Band of precipitate at optimum proportion

19. Modifications
Double diffusion in one dimension (Oakley-
Fulthorpe procedure)

20. 3.SINGLE DIFFUSION IN TWO DIMENSIONS
(RADIAL IMMUNODIFFUSION)
Antiserum incorporated in agar gel poured on a flat
surface
Antigen is added to the wells
Ag diffuses radially and forms ring shaped bands of
precipitation-Halo
Diameter of the halo concentration of
antigen

21. Modifications
Single diffusion in two dimensions (Radial
immunodiffusion)
Uses : estimation of Ig classes in sera.
screening sera for Abs to Influenza viruses

22. 4. DOUBLE DIFFUSION IN TWO DIMENSIONS
(OUCHTERLONY PROCEDURE)
Agar gel is poured on a slide
Wells are cut
Antiserum placed in the central well
Antigens in the surrounding wells
If two adjacent antigens are
identical Line of precipitate fuse
If unrelated Lines cross each other
Partial identity Spur formation
Example: Elek’s gel test

23. Double diffusion in two
dimensions (Ouchterlony
procedure) e.g. Elek’s test
for C.diphtheriae
- most widelyused.
- helps to compare different
Ags &antisera directly.

24. IMMUNOELECTROPHORESIS:
Electrophoresis followed by immunodiffusion.
Agar or agarose gel on a slide
Ag well and Ab trough cut on it
Test serum Ag well
Electrophoresed
Antibody Trough
Diffusion For 18 – 24 hours
Precipitin lines Photographed, stained and preserved
Uses: Testing for normal and abnormal serum and proteins in urine.

25. IMMUNOELECTROPHORESIS

26. ELECTROIMMUNODIFFUSION:
Development of precipitin lines speeded up by
electrically driving the Ag and Ab.
COUNTERIMMUNOELECTROPHORESIS (CIE):
Simultaneous electrophoresis of the Ag and Ab in
opposite directions
Precipitation
Uses:
- Detection of alphafetoprotein in serum
- Detection of antigens of Cryptococcus and
Meningococus in CSF.

27. COUNTERELECTROPHORESIS (CIE)

28. Electroimmunodiffusion
b. Rocket electrophoresis –
one dimensional single
electroimmunodiffusion
- Ag in increasing
concentration
Uses : quantitative estimation
ofAgs.
Antiserum is incorporated in
agarose
Ag in increasing concentrations
is placed in wells
Ag is electrophoresed into the
Ab containing agarose
Pattern of immunoprecipitaiton
resembles rocket.

29. Electroimmunodiffusion
c. Laurell’s two dimensional electrophoresis – variant
of Rocket electrophoresis.
- can quantitate each of several Ags.

30. AGGLUTINATION REACTION
-When insoluble or particulate antigen is mixed with its antibody
in presence of electrolytes at a suitable temperature & pH ,the
particles are clumped or agglutinated
-More sensitive than precipitation for the detection of antibodies.
Applications of Agglutination Reaction:-
Slide Agglutination:- Drop of antiserum
Particulate Antigen in a drop of saline
Positive: Clumping of the particles &
Cleaning of the drop.
USES:-
- Identification of bacterial isolates.
- Blood grouping & cross – matching.

32. Tube agglutination:-
Fixed volume of a particulate antigen
Serial dilutions of antiserum in test tubes
Agglutination titre of the serum → estimated.
USES:- Diagnosis of Typhoid → widal test
Brucellosis
Typhus fever → weil felix reaction

33. THE ANTIGLOBULIN (COOMBS) TEST:-
- Devised by Coombs, Mourant & Race (1945)
- Detection of anti – Rh antibodies.
.
Direct - Sensitisation occurs in vivo
Coombs test - Haemolytic disease of newborn
Indirect - Sensitisation performed in vitro.
USES:-
-For the detection of anti – Rh antibodies
-For demonstrating any type of incomplete or non
agglutinating antibody.

35. Heterophile agglutination tests
 Weil-felix reaction-based on sharing of common
antigen , serodiagnosis of typhus fever by using
proteus bacilli.
 Streptococcus MG test-for primary atypical
pneumonia
 Paul-Bunnel test-detection of IMN

36. Passive Agglutination Test:-
Soluble Antigen + carrier particles Particulate Ag.
(Precipitation Test) (Agglutination test)
Carrier particles:
Red cells(haemagglutination test),
Latex particles(latex agglutination test) or bentonite
Detection of ASO,CRP, RA factor, HCG.
Ex:- Rose waaler test:- For RA factor

37. Coagglutination(Reverse passive
agglutination)

38. COMPLEMENT FIXATION TEST
.
 Principle: The ability of Antigen – Antibody
complexes to fix complement is made use of in the
complement fixation test.
- Consist of two steps and five reagents –
Ag, Ab, Complement, Sheep
erythrocytes & Amboceptor (Rabbit Ab to sheep
RBC)
- Antigen- soluble / particulate
- Antiserum - heated at 56 degree c
- Source of complement - Guinea pig serum

39.  Standardisation of complement-
 Guinea pig serum should be titrated for activity.
• 1 unit of MHD of complement- highest dilution of the serum
that lyses one unit volume of washed sheep erythrocytes in
presence of excess hemolysin (amboceptor) in a fixed timeat
a fixed temperature
 Titration of amboceptor-
 1 unit of MHD of amboceptor -highest dilution of the
inactivated amboceptor that lyse one unit volume of washed
sheep erythrocytes in presence of excess complement in a
fixed time at a fixed temperature.
 Classical example of CFT: Wassermann reaction

41. Indirect complement fixation test
•used for certain avian and mammalian sera not fixed
by guinea pig complement.
 Negative Test-
1.antigen+ test serum(-
ve for Ab)+ guinea pig
complement
2.standard
antiserum(known to
fix complement) will
react with Ag & fix free
complement
 Indicator system-no
hemolysis
 Positive test-
1.antigen+test serum(+ve
for Ab)+ guinea pig
complement
2. standard antiserum will
not react with antigen as
Ag is used in first step by
Ab.
 Indicator system-
hemolysis occurs as
complement is free to act
on system.

42. Conglutination
 Horse complement is used.
 Indicator system- sensitised sheep erythrocytes with
bovine serum(contain a β globulin component named
conglutinin, act as Ab to complement)
 Conglutinin causes agglutination of eryrhrocytes if
combined with complement(conglutination).
 1.Ag+antiserum(+ve)+horse complementfixed
 2.sheep eryrhrocyte with conglutinin no agglutination
 Result- No agglutination-+ve
Agglutination- -ve

43. NEUTRALISATION TESTS
Ability of the Ab to neutralize various effects
of micro organisms mediated through toxins,
enzymes ormicrobial Ags.
e.g. Nagler’s test for
Clostridium perfingens
(test to detect alpha toxin)

44. OPSONISATION TEST:
Process by which a particulate Ag becomes more susceptible to
phagocytosis
Opsonin combines with antigen Facilitates
phagocytosis
Opsonic Index: Ratio of phagocytic activity of the
patients blood for a given bacterium, to
that of a normal individual
Phagoytic Index: Average number of phagocytosed
bacterium per polymorphonuclear
leukocyte from blood films.

45. IMMUNOFLUORESCENCE
FLUORESCENCE – property of certaincompounds
to absorb light of shorter (UV) wavelength &
emit light of higher (visible)wavelength.
Fluorescent dyes (Fluorochromes) are conjugated
to Abs – LabelledAbs.
Fluoresce when binds to specific Ag in tissues.
Can be direct or indirect.
Detected by FluorescentMicroscope.

46. Fluorochromes
FITC- Fluorescein
Isothiocynate :–Green
Rhodamine- Auramine :-
Red
Acridine orange :-Orange
Calcofluor white :- fungal
elements
Applications
Direct IF – detection of
bacteria, viruses or other
Ags using specific
antiserum labelled with
fluorescent dye e.g.
detection of Rabies virus
Ag in brain smears.
Indirect IF – fluorescent
treponemal Ab test

48. RADIOIMMUNOASSAY
Radio isotopes are conjugated to Abs
or Ags.
A competitive binding assay in which
fixed amounts of Ab &radiolabelled Ag
react in the presence of unlabelled Ag
( test sample)
High levels of unlabelled Ag– less
radioactivity
Applications – quantitation of
hormones, drugs, tumor
markers, IgE &viralAgs.

49. ELISA
Enzyme linked immunosorbentassay.
Can be used for the detection of Ag or Ab.
Corresponding Ag or Ab is conjugated with an enzyme.
The enzyme is then detected by its ability to convert acolorless
substrate to a colored product.
 The color is then measured in a machine called ELISA reader.
 The test can be done in polystyrene tube (Macro – ELISA)
or Polyvinyl Microtiter plates (Micro – ELISA)

51. Indirect ELISA: (Ab detection)
Wells coated with antigen
Sera added
Ab present – binds to coated antigen
To detect, goat antihuman Ig conjugated with an
enzyme added
Substrate added
Colour production

52. SANDWICH ELISA: (Antigen detection)
wells coated with specific antibody.
Specimen added.
Antigen present – binds to coated antibody
To detect this Ag – Ab reaction,
Ab conjugated with an enzyme added.
Conjugated Ab binds to Ag
Substrate added
Positive result:- Colour production
Read by spectrtophotometer or ELISA reader.

54. CASSETTE OR CYLINDER ELISA
Specific Type 1 and 2 Ag immobilized on the
nitrocellulose membrane
Serum added
Positive serum – Ab bind to appropriate Ag
Washing – Remove unbound Ab, conjugate added
Washing - Remove unbound conjugate, substrate
added
Positive result – coloured spot

55. ADVANTAGES:
 Testing one or few samples of sera at a time
 Test is rapid (10 mins)
 For detection of HIV type 1 and 2 Ab
Uses:-
1. Detection of HIV antibodies in serum.
2. Detection of Mycobacterial Ab in TB.
3. Detection of Rotavirus in faeces.
4. Detection of Hepatitis – B markers in serum.

56. Immunochromatographic tests
One step test.
HBsAg detection
Membrane impregnated with anti-HBsAg Ab
colloidal gold dyeconjugate.
Presence of a colored band is +ve.

58. ImmunoelectronMicroscopy
Viral particles + specific antisera
EM
clumping of viruses

59. Immunoelectronmicroscopic Tests:
Immunoferritin Test:- To detect antigen.
Ferritin (electron dense substance) conjugated Ab + Ag
Visualized under electron microscope
Immunoelectronmicroscopy:
Ag mixed with specific Ab
Electron microscope
Clumps seen
USES:-
Diagnosis of Hepatitis – A and viruses causing diarrhea.

60. Immunoblotting:
Western blotting: (To detect proteins)
Proteins electrophoretically separated in a gel.
Transferred to a nitrocellulose paper.
Reacted with test sera (Ab) and enzyme conjugated
anti human Ig
Substrate added
Colour produced
Detection of DNA – Southern Blotting.
Detection of RNA – Northern Blotting

61. Immunoblotting