antigen antibody reactions, precipitation reactions

1. Dr DEEPAK CHAUDHARY
SENIOR DEMONSTRATOR
GMC AMBIKAPUR

2.  1. Introduction.
 2. Salient Features of Antigen – Antibody
Reaction.
 3. Strength of Antigen – Antibody Reaction.
 4. Properties of Antigen – Antibody Reaction.
 5. Types of Antigen – Antibody Reaction.
 6. Application of Antigen – Antibody Reaction.
 7. Conclusion.

3.  The antigens and the antibodies combine
specifically with each other in an observable
manner. This interaction between them is called
Antigen-Antibody reaction.
 It may be abbreviated as Ag – Ab reaction.
 In vivo- soluble antibodies present in plasma and
body secretions form the basis for humoral
immunity or antibody mediated immunity.
 In vitro- These reactions form the basis for
detection of infectious disease, epidemiological
surveys, identification of infectious agents and
also some non infectious agents (eg. Enzymes)

4.  When Ag – Ab reactions occur invitro, they are known as
serological reactions
 IMMUNOASSAYS- procedures that measure antigens or
antibodies to determine whether the individual is infected or
responding to immunization.
 2 types- liquid phase immunoassays (LPIA) & solid phase
immunoassays (SPIA)
 LPIA- CHEMISTRY
 SPIA- MICROBIOLOGY
 Important tests for antigen antibody reactions include –
precipiation, agglutination, complement fixation, neutralisation
and labelled assays (immunofluorescence, enzyme

5. General features of Antigen and
antibody reactions.
 Specific reaction – antigen combines with homologus
antibody and vice versa.
 Entire molecule reacts not fragments
 No denaturation of antigen or antibody
 Combination occurs as surface antigens to surface of
antibodies. Antigenic determinant (epitope) makes contact
with hypervariable region of antibody (paratope).
 Molecules are held in lock and key arrangements.
 Combination is firm but reversible depends on affinity and
avidity
 Affinity- intensity of attraction, Avidity- binding strength of
individual antibody with specific antigenic determinant.

6.  Both antigens and antibodies participate.
 Combine in varying proportions. Antibodies-usually
Bivalent and antigen usually multivalent.
 Antibody affinity- the strength of the binding (sum
total of non covalent interactions) between a single
antigen binding site on an antibody and an epitope
is called affinity of the antibody.
 Antibody avidity- strength of multiple interactions
between multivalent antibody and antigen.

7. Antigen binding site of antibody
 Epitope and paratope
 Hypervariable regions
 Also called complementarity determining
regions (CDR’s) as the antigen binding site is
complementary to structure of epitope.

8. ANTIGEN ANTIBODY INTERACTIONS
 The reactions between Ag and Ab occur in
three stages.
 In first stage the reaction involves formation of
Ag-Ab complex.
 The second stage leads to visible events like
precipitation, agglutination etc.
 The third stage includes destruction of Ag or its
neutralization.

9. Primary stage:
 Initial interaction between Ag & Ab –
invisible
 Rapid, occurs at low temperatures & obeys
the general laws of physical chemistry &
thermodynamics.
 Reaction is reversible.
 Ag & Ab is bound to each other by weak
Van der Waal’s forces, Ionic bonds &
Hydrogen bonding.
Stages of Ag – Ab reactions

10. Secondary stage:
 Demonstrable events – Precipitation,
agglutination, lysis of cells, killing of live antigens,
neutralization of toxins, complement fixation,
immobilization of motile organisms &
enhancement of phagocytosis.
 Precipitin – Ab participate in precipitation
 Agglutinin - Ab participate in agglutination
 Precipitinogen – Ag participate in precipitation
 Agglutinogen - Ag participate in agglutination

11. Tertiary stage:
 Includes neutralization or destruction of
injurious agents or tissue damage.
 Also includes humoral immunity against
infectious diseases as well as clinical allergy &
other immunological diseases.

12. Measurement of Antigen and Antibody
reactions Measurement may be in terms of mass or more commonly
as units or titre.
 ANTIBODY TITRE
 The Antibody titre of a serum is the highest dilution of the
serum which shows an observable reaction with the antigen
in a particular test.
 It is usually expressed as the reciprocals of the dilution of
the serum
 To calculate antibody titer, a blood serum sample
containing antibody is diluted in serial ratios (1:2, 1:4, 1:8,
1:16... and so on).
 Using an appropriate detection method (e.g., colorimetric,
chromatographic, etc.), each dilution is tested for the
presence of detectable levels of antibody. The assigned
titer value is indicative of the last dilution in which the
antibody was detected.

13. Procedure
•First tube = (1 mL serum + 1 mL diluent) = 1 : 2
•Second Tube = 1 mL from from ist tube + 1 mL diluent = 1 : 4
•Third tube = 1 mL from tube 2 + 1 mL diluent = 1 : 8
•Fourth tube = 1 mL from tube 3 + 1 mL diluent = 1 : 16

14. the titer is the degree to which the antibody serum solution can
be diluted and still contain detectable amounts of antibody.

15. Terms used in evaluating test
Methodology
Sensitivity
 Analytical Sensitivity – ability of a test to
detect very small minute quantities of antigen or
antibody.
 Clinical Sensitivity – ability of test to give
positive result if patient has the disease.
 When the test is highly sensitive, false
negative results may be absent or minimal.

16. Specificity
 Specificity refers to the ability of the test to
identify reactions between homologus
antigens and antibodies only and no other
substance.
 Analytical Specificity – ability of test to detect
substance without interference from cross-
reacting substances
 Clinical Specificity – ability of test to give
negative result if patient does not have
disease.
 In highly specific test, false positive
reactions are absent or minimal.

17. Types of Antigen – Antibody Reaction:
1. Precipitation reaction
2. Agglutination reaction
3. Neutralization reaction
4. Complement fixation test
5. Immobilization test
6. Opsonisation
7. Immunofluorescence
8. Radioimmuno assay
9. Enzyme immunoassay

18. Precipitation Reaction:
 When a soluble Ag combines with its specific
Ab in the presence of an electrolyte (NaCl) at a
particular temperature and pH, it forms an
insoluble precipitate of Ag-Ab complex.
 The Ab causing precipitation is called Precipitin
and the reaction is called as precipitation
reaction.
 When the precipitate remains suspended it is
called flocculation

19. Mechanism of precipitation
 Marrack (1934) proposed the lattice hypothesis –
mechanism of precipitation
 The multivalent antigens combine with bivalent
Abs in varying proportions, depending on the Ag
– Ab ratio on the reacting mixture.
 Precipitation results when a large lattice is
formed consisting of alternating Ag & Ab.

20. ZONE PHENOMENON
 The amount of precipitate formed is greatly influenced by
the relative proportions of Ags & Abs.
 If increasing quantities of Ags are added to the same
amount of antiserum in different tubes, precipitation is
found to occur most rapidly & abundantly in the middle
tubes.
 Plotting the amount of precipitate against increasing
antigen conc. Yields a precipitation curve having the
following zones
Preceding tubes – Ab excess, uncombined antibody is
present (Prozone)
Middle tubes – Ag & Ab in equivalent proportions (Zone
of equivalence)
Later tubes – Ag excess, uncombined antigen is present
(Post zone)

24. Applications
 Identification of bacteria- eg. Detection of
group specific polysaccaride of streptococcus
(lancefield’s grouping).
 Identification of bacterial component- eg
B.antracis (ascoli’s thermoprecipitin test)
 Detection of unknown antibody- eg VDRL
and KT in syphilis.
 Medicolegal identification of human blood or
seminal fluid.
 Standardisation of toxins and antitoxins.
 Testing for food adulteration.

25. Types of precipitation reaction
 Simple precipitation test- slide and tube test
 Gel diffusion test
 Immunoelecterophoresis
 Electroimmunodiffusion

26. A. Simple precipitation tests
 Ring test
 The reaction is demonstrated by layering
antigen solution over the column of antiserum
in a narrow tube. The precipitate forms at the
junction of two liquids.
 Eg Ascolis thermoprecipitin test, Grouping
of Streptococci by Lancefield technique

28. Flocculation test
 Slide test- VDRL Test for syphilis
 Drop of VDRL antigen solution is added to drop of
decomplemented patient serum on a slide and
mixed and shaken.
 Visible clumps appear in positive cases.

30. Tube test
 This is employed for the standardization of
toxins & toxoids.
 Serial dilutions of toxin are mixed with tubes
containing fixed quantity of antitoxin.
 The amount of toxin that in the shortest time
produces precipitation when mixed with one
standard unit of antitoxin is called LF dose.

31. B.Gel diffusion test
 Precipitation test done in 1% agar or agar gel is
called gel diffusion or immunodiffusion.
 Reaction is visible in the form of distinct bands
of precipitation
 Different antigens in the reacting mixture can be
studied as ag-ab reaction gives rise to single
line of precipitation.
 When only antigen or antibody diffuses it is
referred to single diffusion.
 When both antigen and antibody diffuse toward
each other through the agar gel it is called
double diffusion.

32. 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.

33. 2. 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.

35. 3. Single diffusion in two dimensions (Radial
immunodiffusion)
 Here the antisera is incorporated in a gel &
poured on a flat surface.
 Wells are cut on the surface to which Ag is
added.
 It diffuses radially from the well & forms ring
shaped bands of precipitation concentrically
around the well.
 Higher the conc. Of antigen larger the ring of
precipitation
 Used to detect Immunoglobulins in serum and
for detection of viral antibodies

37. 4. Double diffusion in two dimensions
(Ouchterlony procedure)
 Helps to compare different antisera & antigens
directly.
 Agar gel is poured on a slide & wells are cut .
 Antiserum is placed in the central well.
 Different Antigens in the surrounding wells.
 When two adjacent antigens are identical, lines of
precipitate fuse.
 In the case of unrelated antigens lines of
precipitate cross each other
 Cross reaction or spur formation is observed
when there partial identity of antigens.

39. Elek’s gel precipitation test

41. 5. Immunoelectrophoresis
 Graber & Williams devised this technique.
 This involves the electrophoretic separation of
composite Ag (eg serum) into its constituent
proteins, followed by immunodiffusion against
its antiserum – forming separate precipitin lines.
 Identification and approximate quantitation of
various proteins in the serum can be done.
 It is performed on an agarose gel with an Ag
well & Ab trough cut on it.
 The antigen is placed in the antigen well &
electrophoresed for about 1 hour.

42.  Antigen components migrate in the electric
field according to their charge and size
and get sperated.
 Ab against human serum is placed in the
trough & diffusion is allowed for 18 – 24
hrs.
 Antigen and antibody diffuse towards each
other forming series of precipitate arcs.
 Useful for determining the presence or
absence of serum proteins and detection of
unsual proteins such as human myeloma
protein.

44. ELECTROIMMUNODIFFUSION
 The development of precipitin lines can be
speeded up by electrically driving the Ag &
Ab.
 Two types
1. Counterimmunoelectrophoresis (One dimensional
double electroimmunodiffusion)
2. Rocket electrophoresis (One dimensional single
electroimmunodiffusion)

45. 1. Counterimmunoelectrophoresis (CIE)
 This involves simultaneous electrophoresis of Ag
& Ab in gel in opposite directions resulting in
precipitation at a point between them.
 Produce precipitation lines within 30 mins.
 Clinical application: detecting Ags like
alphafetoprotein in serum, Ags of Cryptococcus &
Meningococcus in the CSF.

48. 2. Rocket electrophoresis
 Used for quantitative estimation of Ags.
 The antibody to the Ag to be quantitated is
incorporated in agarose gel on a slide.
 Ag in increasing concentrations, is placed in wells
punched in the set gel.
 The Ag is electrophoresed into the Ab containing
agarose.
 The pattern of immunoprecipitation resembles
cone like structures i.e appearance of a ROCKET,
the length of which corresponds to the
concentration of antigen.

50. Rocket electrophoresis

51. Laurell’s two dimensional electrophoresis
 Variant of rocket electrophoresis.
 The Ag mixture is electrophoretically
separated in a direction perpendicular to that
of the final rocket stage.
 Several Antigens in a mixture can be
quantitated.

53. THANK YOU