Antigen antibody reactions and methodology with their principles, methods and uses for all students and other professionals.

1. Antigen Antibody
Reactions and clinical utility

2. Objectives
• To know the principles of serological reactions / tests
• To know the technical aspects of serological reactions
• To know the application or clinical use of serological
reactions / tests
• To know various serological tests for clinical diagnosis

3. Classification of antigen-antibody interactions
1. Primary serological tests: (Marker techniques)
e.g. – Enzyme linked immuonosorbent assay (ELISA)
– Immunoflorescent antibody technique (IFAT)
– Radio immunoassay (RIA)
2. Secondary serological tests:
e.g. – Agglutination tests
– Complement fixation tests (CFT)
– Precipitation tests
– Serum neutralization tests (SNT)
–Toxin-antitoxin test
3.Tertiary serological test:
e.g. – Determination of the protective value of an anti serum in
an animal.

4. • Definition: Observable combination of antigen and antibody
• Importance:
A) In vivo – form basis of antibody mediated immunity in infectious
disease, or of tissue injury in hypersensitivity and autoimmune
diseases
B) In vitro – i) Help in diagnosis of infections
ii) Help in epidemiological surveys
iii) Identification of infectious agents
iv) Identification of non infectious agents like
enzymes
Antigen antibody reactions in vitro are called serological reactions

5. General features of Antigen – Antibody Reactions
1. Reactions are specific
2. Entire molecules react and not fragment
3. No denaturation of antigen of Ag/Ab during reaction
4. Combination occurs at the surface
5. Combination is firm but reversible
6. Both the Ag and Ab combine in varying proportion

6. Affinity –
Intensity of attraction between Ag and Ab
molecules
Avidity –
Strength of bond after the Ag – Ab complexes
are formed.
Sensitivity:
ability of a test to detect very small amounts of
a substance
Specificity:
ability of test to give positive result if patient
has the disease (no false negative results)

7. Antigens x Antibody
Bacteria Virus
Ag
Ag
Antigen
binding site
Antigen
binding site
Variable
constant
Light chain
Heavy chain

8. Antibodies (depicted as Y-shaped
structures) form a heterogeneous
population of molecules with different
specificities. A cross-reaction of an
antibody population (an anti-serum)
with a foreign antigen (in the middle)
occurs only, if the homologous and the
foreign antigen are at least partially
equipped with the same determinants.
Every antibody has two identical binding
sites for antigen determinants.

9. Primary stage
• Initial interaction between Ag and Ab
• No visible effect
• Rapid
• Occurs even at low temperature
• Obeys general laws of physical chemistry &
thermodynamics
• Reversible reaction

10. Secondary Stage
Demonstrable effect produces e.g. precipitation,
aggluitnation, lysis of cells, killing of live antigens,
neutralization of toxins, complement fixation,
immobilization of motile organisms, enhancement of
phagocytosis

11. Tertiary stage
Some Ag – Ab reactions in vivo lead to
• Neutralization
• Destruction of injurious agent
• Tissue damage
• Includes humoral immunity, clinical allergy, and other immunological
diseases

12. Measurement of Ag – Ab reaction
Measured in terms of Mass (mg of nitrogen) Units or titer
Titer:
Highest dilution of serum (Ab) which gives an observable reaction with the
Ag in a particular test.
Antigen can also be titrated against sera (Ab).
Sensitivity –
Ability of a test to identify correctly all those who have the disease i.e. true
positive
Specificity –
Ability of a test to identify correctly all those who do not have the disease
i.e. true negative
In general, sensitivity and specificity of a test are in reverse proportion.

13. Serological tests
• Precipitation reaction
• Agglutination reaction
• Complement fixation test
• Radioimmunoassay
• ELISA
• Immunofluorescence
• Western Blot

14. Precipitation Reaction
When a soluble antigen combines with its antibody in
the presence of electrolytes at a suitable temp and pH
the Ag - Ab complex forms an insoluble precipitate. If
instead of sedimenting, the precipitate remains
suspended, the reaction is called as flocculation.
Medium used :
Liquid or gels like agar, agarose and polyacrylamide.

15. Antibody excess Zone of equivalence
Antigen excess
Antigen
Antibody
Zone Phenomenon

16. Mechanism of precipitation
• Marrack (1934) – Lattice hypothesis
Application of precipitation:
- Can be used as qualitative or quantitative test
- Very sensitive test for antigen detection, can detect as little as 1μg
of protein
- Important for forensic application like identification of blood, seminal
stains.

17. APPLICATION OF PRECIPITATION REACTIONS
• Carried out as qualitative and quantitative for detection
of antigen and antibody
• Very sensitive test for detecting antigen
(Relatively less sensitive for detection of antibody)
• Capable of detecting little quantity of antigen proteins as
1ug
• Used for identification of blood and seminal stains and
food adulterants

18. Application in diagnostic bacteriology
• Ring test
– Typing of streptococci and pneumococci
– C- reactive protein
– Ascoli’s thermoprecipitin test for diagnosis of antrax
– Used in detection of adulteration of food stuffs
• Slide test
– VDRL for diagnosis of syphilis
• Tube test
– Kahn test for syphilis and standardization of toxins and
toxoids

19. IMMUNODIFFUSION (precipitation in gel)
When an Ab and its Ag are placed in an
agar gel they diffuse towards each other
and form an opaque band of precipitation
at the junction of their diffusion front.

20. TYPES OF IMMUNODIFFUSION
TESTS

21. Antigen
Precipitin
band
Antibody in
agar gel
Plain agar
Single diffusion (Oudin procedure)
Double diffusion (Okley - Fulthrope procedure)
Single and double diffusion in one dimension

22. Antigen in well
Ring of
precipitation
Antibody in agar
gel
A single diffusion in two dimensions

23. Antiserum in well
Line of precipitate
Antigen 2
Agar gel on a slide
Antigen 1
Double diffusion in two dimensions
(Lines of nonidentity)

24. Antiserum in well
Line of precipitate
Antigen 2
Agar gel on a slide
Antigen 1
Double diffusion in two dimensions
(Partial identity)

25. Antiserum in well
Antigen 2
Agar gel on a slide
Antigen 1
Double diffusion in two dimensions
(Lines of complete identity)

26. Trough cut in agar
Agar covered microscopic
slide
Well cut in agar
Antigen placed in well
Antigen components separated by
electrophoresis
Immunoelectrophoresis

27. Trough filled with antibody
Antibody diffuses towards
separated antigen
components
Immunoelectrophoresis
Precipitin band form where
antibody and antigen meet
at optimal proportions

28. Rocket electrophoresis (One dimensional single electroimmuno diffusion)
Antiserum (Ab)
in agarose gel
Precipitin arcs
(rockets)
Antigen wells
Increasing concentration of antigen
+
_

29. Ag Ab
Agarose
Wells containing antigen and antibody
Precipitin
line
Slide
Electric current
Counterimmunoelectrophoresis
(One dimensional double electroimmunodiffusion)

30. AGGLUTINATION REACTION
When a particulate antigen or an antigen
present on the surface of cell (red cell or
bacterium) or an inorganic particle ( e.g.
polystyrene latex coated with antigen) is
mixed with its antibody in the presence of
electrolytes at suitable temperature and
pH, the particles are clumped or
agglutinated

31. Application of agglutination reaction
• Slide agglutination
– Identification of cultures e.g. Salmonella, Bordetella pertussis
– Typing of pneumococci and streptococci
– Blood grouping
• Tube agglutination
– Serological diagnosis of
• enteric fever (Widal)
• brucellosis
• typhus fever (Weil – Felix)
– Blood grouping
– Coomb’s test

32. Latex particles
Antibody
Antigen
Agglutination
Antibody coated latex particles
Latex agglutination test
ASO, CRP, RA
Passive agglutination test
e.g. Rose Waaler test in Rheumatoid
arthritis

33. Passive agglutination
Precipitation reaction can be converted to agglutination
reaction by coating soluble antigen on the surface of
carrier particles such as RBCs, latex, bentonite, gelatin
More sensitive for detection antibodies
Examples
1. RA factor
2. Latex tests
3. TPHA

34. Negative CFT
Complement Sheep erythrocytes coated with amboceptor
(Indicator system)
Lysis
Positive CFT
Ag Ab Complement
Complement is fixed in
Ag – Ab reaction
Indicator system
No lysis
As complement is not free to act on indicator system
Complement fixation test

35. Cowan strain of S. aureus
Immunoglobulin
Cowan strain coated with immunoglobulin
Antigen
Agglutination
Coagglutination

36. Sandwich ELISA Indirect ELISA Competitive EILSA
Color product
Antigen
Antibody
Conjugate (Ab to Ab)
Conjugate (Ab to Ag)
Conjugate is washed
out as Ag is not free to
bind the conjugate
Substrate
Enzyme (tagged to
conjugate is not there to
act on substrate
ELISA

37. Unknown antigen
Slide
Fluorescein labeled specific
Ab
Fluorescence under UV light
(positive test)
Direct immunofluorescence test

38. Known Ag
Slide
Patient
serum
containing
Ab
Indirect immunofluorescence test
Ag + Ab
Fluorescein
labeled
Antiglobulin
Fluorescence under UV light
(Positive test)

39. Radioimmunoassay
Unlabeled Ag
(Test sample)
Known radio
labeled Ag
Specific Ab
against Ag
Mix
Incubate
Free
fraction
Bound
fraction

40. MCQs
Q1. A large lattice is formed when
a. Antigen is in excess
b. Antibody is in excess
c. Antigens and antibodies are in optimal proportion
d. Non of above
Q.2. Precipitation reaction is relatively less sensitive for detection of
a. Antibodies
b. Antigens
c. Antigen antibody complexes
d. Complement
Q3. Ring test is used for
a. Typing of streptococci and pneumococci
b. C-reactive protein test
c. Ascoli’s thermoprecipitation test
d. All of the above
Q4. Widal test is used to diagnose
a. Syphilis
b. Typhoid
c. AIDS
d. Typhus fever

41. Q.5. VDRL test is an example of
a. ring test
b. Slide test
c. tube test
d. None of above
Q6. Agglutination reaction is more sensitive than precipitation for detection of
a. Antigen
b. Antibody
c. Antigen antibody complexes
d. Complement
Q7. Amount of various immunoglobulin classes can be measured by
a. Single diffusion in one dimension
b. Double diffusion in one dimension
c. Single diffusion in one dimension
d. Double diffusion in two dimension
Q8.Anti- Rh antibodies are
a. IgG type
b. IgD type
c. IgA type
d. IgE type

42. LAQ
Q.1. Define agglutination reaction and discuss the principle, application
of agglutination reactions giving suitable examples
Q.2 Define precipitation reaction and discuss the principle, application
precipitation reactions giving suitable examples
Q.3 Discuss the principle, various types and clinical applications of
ELISA technique
Q.4 Short notes
1. Counterimmunoelctrophoresis
2. ELISA
3. Complement fixation test