Antigen-antibody reactions can be observed in vitro as serological reactions which are used to identify and quantify antigens or antibodies. There are three stages of antigen-antibody reactions - primary, secondary, and tertiary. The sensitivity and specificity of serological tests are inversely proportional, with sensitivity referring to detecting small quantities and specificity only detecting complementary antigens and antibodies. Common serological reactions include precipitation, agglutination, complement fixation, neutralization, and immunoassays. Measurement of antigens and antibodies involves titration of serial dilutions.

1. Antigen Antibody Reactions
Dr. Himanshu Khatri
Email: himanshubkhatri@yahoo.co.in

2. • Antigen and antibody combine with each
other specifically and in an observable manner
• Antigen-antibody reactions which can be
observed in vitro are called serological
reactions

3. Uses of serological reactions
• Identification of antigens or antibodies
• Quantification of antigens or antibodies

4. Stages of antigen-antibody reactions
• Primary stage: weak binding, no demonstrable
events
• Secondary stage: firm binding, demonstrable
events like precipitation, agglutination,
complement fixation, neutralization,
enhancement of phagocytosis, etc.,
• Tertiary stage: occurs in vivo and includes
hypersensitivity and immunity

5. Measurement of antigen and antibody
• They are measure in units or titer
• The titer of an antibody is reciprocal of the
highest dilution of the serum that shows an
observable reaction with the antigen in that
test
• For example, if dilutions of 1/20,1/40 and
1/80 of serum give a positive Widal test, then
the titer is 80
• Antigens may also be titrated in a similar way

6. Sensitivity and specificity
• Sensitivity of a test is its ability to detect even
minute quantities of antigen and antibody. In
such a case, false positive results may be possible
• Specificity of a test is its ability to detect reactions
only between absolute complementary antigens
and antibodies. In such a case, some reactions
may not be detected, and false negative results
are possible
• In general, sensitivity and specificity of a test are
in inverse proportion

7. Serological reactions
• Precipitation reaction
• Agglutination reaction
• Complement fixation test (CFT)
• Neutralization
• Opsonization
• Immunofluorescence
• Radioimmunoassay (RIA)
• Enzymeimmunoassay (EIA)
• Chemiluminescence assay (CLIA)
• Immunoelectronblot techniques
• Immunochromatographic tests
• Immunoelectron microscopic tests

8. Precipitation and Agglutination
reactions
• They are similar reactions, the only difference
being, that the antigen is soluble is
precipitation, and insoluble in agglutination
• Precipitation can take place in liquid media or
in gels
• When the precipitate, instead of sedimenting,
remains suspended as floccules, the reaction
is known as flocculation

10. Marrack’s lattice hyphthesis for
precipitation and agglutination
• Antigens having multiple valencies combine with
antibodies having two valencies to give a lattice
• The lattice becomes large enough to give a visible
precipitation or agglutination, only when the
antigens and antibodies are present in optimal
proportions (zone of equivalence)
• If the antibodies are in excess (prozone), or the
antigens are in excess (post zone), enlargement
of the lattice does not take place, and there is no
visible precipitation or agglutination

11. Importance of zone phenomenon
• If the levels of antibody are too high in the
serum, it may result in prozone phenomenon,
and the result may be falsely negative
• This can be overcome by testing serial
dilutions of serum to dilute the antibody
concentration used for testing

12. Slide flocculation
• When a drop of antigen
and antiserum are placed
on a slide, and mixed by
shaking, floccules appear
• For example: Venereal
Disease Research
Laboratory (VDRL) test for
syphilis
• Rapid Reagin Reaction
(RPR) is a modification of
VDRL where charcoal is
added to enhance the
visualization of floccules

13. Slide agglutination
• When a drop of
homogenous suspension of
particulate antigen in saline
and antiserum are placed
on a slide, and mixed by
shaking, agglutinates
appear and there is clearing
of the fluid
• It is used for identification
of bacterial isolates grown
on solid media
• It is also used for blood
grouping and cross-
matching

14. Tube agglutination
• It is used for
quantification of
antibodies
• When a fixed volume of
particulate antigen is
mixed with equal volumes
of serial dilutions, the
agglutination titer of the
serum can be estimated
• For example: Widal test,
Weil-Felix test etc.,

16. Coomb’s test
• Initially used to detect anti-Rh antibodies, it is
now used to detect incomplete antibodies in a
variety of conditions
• Incomplete antibodies are antibodies, which can
bind to antigens , but cannot cause agglutination
of erythrocytes
• When such RBCs are treated with complete
antibodies such as those against human
gammaglobulin (anti globulin or Coomb’s serum),
agglutination occurs

17. Direct Coomb’s test
• The RBCs are sensitized with antibodies in vivo
in conditions like hemolytic disease of the
newborn, auto-immune hemolytic anemia
etc.,
• When these RBCs are treated with Coomb’s
serum, agglutination occurs

18. Indirect Coomb’s test
• It is used to detect incomplete antibodies
• The incomplete antibodies which are formed against
RBCs inside the patients body are present in the serum
• The serum of the patient is extracted, and then treated
with RBCs in vitro
• This mixture is then treated with Coomb’s serum
• If antibodies are present inside the serum,
agglutination occurs
• Indirect Coomb’s test is used for cross-matching and to
screen women for antibodies which cause hemolytic
disease of the newborn

19. Passive agglutination
• The precipitation reaction can be converted to
agglutination reaction by attaching soluble antigens on
carrier molecules.
• This is the principle of passive agglutination
• Initially, RBCs were used as carrier molecules
• Nowadays, latex particles are used as carrier molecules
• Examples of latex agglutination include tests for ASO,
CRP, RA factor, etc.,
• When antibodies are adsorbed on the surface of carrier
molecules for estimation of antigens, the technique is
known as reverse passive agglutination

21. Immunofluorescence-principle

22. Immunofluorescence
• It is the property of absorbing light rays of one
particular wavelength, and emitting them with
a different length
• Special dyes like fluorescein isothiocynate and
lissamine rhodamine have this property
• They show up brightly as blue-green and
orange-red fluorescence, respectively
• These dyes can be conjugated to antibodies to
increase the specificity of the test

24. Direct and Indirect
immunofluorescence
• Fluroescent dye is conjugated with primary
antibody in direct immunofluorescence
• It is conjugated with secondary antibody in
indirect immunofluorescence

25. Immunofluorescence under
microscope

26. Enzyme linked immunosorbent assay
(ELISA)
• The antigen or antibody
(against the antibody or
antigen, which is to be
detected) is usually
absorbed on a solid phase
material like polystyrene,
polypropylene or
polycarbonate
• ELISA microtiter plates are
made out of these materials
• These plates contain 96-
wells, each of which can
hold several milliliters of
liquid

27. • The binding of the antigen or antibody ( to the
antibody or antigen fixed on the solid phase) can be
detected by conjugating them with an enzyme, and the
subsequent addition of a substrate.
• If the binding of the conjugating antibody takes place
with the fixed antigen (or vice versa), the conjugated
enzyme acts on the substrate, and changes the color.
• If the binding does not take place, the conjugated
antigen or antibody is washed off in the subsequent
step, and it cannot thus act on the substrate to give a
change in color

29. • ELISA process can be
partially automated by
using ELISA washers
• The color change is
estimated quantitively
by ELISA readers

30. Cassette or cylinder ELISA
• It is a modification of ELISA
• It is used for testing one or
few samples at a time
• The test takes around 5
minutes, as against
conventional ELISA, which
takes 2-3 hours
• There is no need for ELISA
washers or readers, and the
result is read visually
• Example: HIV tri-dot, HCV
tri-dot

31. Chemiluminescence Immunoassay
(CLIA)
• The label used here emits
light
• Luminol or acridinium
esters are used as
chemiluminescent
compounds in CLIA
• The light can be amplified
and measured, and thus
the concentration of the
analyte can be calculated
• The process can be
automated

32. Immunoelectroblot techniques
• It consists of three steps;
1. The antigen components are separated by
polyacrylamide gel electrophoresis (PAGE)
2. The antigen fractions are blotted on
nitrocellulose membrane strips
3. Antibodies in sera are detected for these antigen
fractions using ELISA
• Example: Western blot for detection of
antibodies against various antigenic components
of HIV

34. Immunochromatography-principle

35. Immunochromatography cassette

36. Immunochromatography-results

37. Immunochromatographic test (ICT)
• An example of ICT is HBsAg detection using a single use cassette
• The serum sample is dropped into the window
• The serum travels along the membrane due to capillary action
• It comes across the conjugate pad. In the conjugate pad, are present anti-
HBsAg antibodies, which are conjugated with a label, usually protein A
• If the sample contains HBsAg, it combines with the conjugated antibody
• At the test (T) line, anti-HBsAg antibodies are immobilized. This antigen-
antibody-conjugate is captured, and the line becomes red
• At the control (C) line, recombinant HBsAg is immobilized. If the sample does
not contain antigen, the antibody-conjugate is captured here, and the line
becomes red. It becomes red even in a postive test due to the binding of
conjugated antibody
• The C line serves as a procedural control, and is indicative of a properly
performed test. The T line becomes red only if the sample contains HBsAg. If
both the T and C lines do not become red, the procedure is considered invalid

38. Immunoelectronmicroscopy
• When antisera is added to
the sample, the viral
particles get attached to
the antibodies, and they
appear to be clumped
• This increases their
visualization in the
sample
• Example: it is used in
detection of viruses
causing diarrhoea in stool
samples

39. Thank you