A clear view on antigen antibody reaction

1. ANTIGEN-ANTIBODY REACTIONS
Dr Neethu Asokan

2. Introduction
• Interaction between antigen and antibodies are
called as antigen-antibody reactions
• When this reaction occur in vitro it is called as
Serological reactions
• An antibody or Immunoglobulin is a large Y-
shaped protein in immune response that identify
and neutralize any foreign body that enters the
body.
• An antigen is biological molecule or a structure or
a foreign particle like pathogens that may bind to
antibody triggering an immune response
Dr Neethu Asokan

3. • Antigen antibody reactions are highly specific
• These reaction in a host that is in in vivo may
cause tissue injury in hypersensitivity
reactions and autoimmune diseases
• The antigen and antibody is reversible and can
be prevented or dissociated by high ionic
strength or extreme pH
Dr Neethu Asokan

4. Properties of Ag-Ab reaction
• There should be a good fit between antigen and antibody
• Intermolecular forces
– The forces that are involved in these reactions include
electrostatic force, hydrogen bonding, van der Waals bonds and
hydrophobic reactions
• Affinity
– Affinity is the intensity of attraction between antigen and
antibody
– Low affinity antibodies bind antigen weakly and dissociate
readily
– High affinity antibodies bind antigen tightly and bound for long
period. This is a result of a very close fit between antigen
binding sites
Dr Neethu Asokan

5. • Avidity
– This is a measure of the overall strength of binding of
an antigen with several antibodies and antigenic
determinants
– It is an indicator of the strength of interactions than
affinity
– It is dependent on valencies of both antigen and
antibody and greater than sum total of affinities
• Specificity
– It is the ability of an individual antibody combining
site to react with only one antigenic determinant
– It can be described as the ability of an antibody
population to react with only antigen
Dr Neethu Asokan

6. • Cross-Reactivity
– Even though Ag-Ab reactions are highly specific
sometimes antibody of one antigen can cross
react with an unrelated antigen
– This occurs when two unrelated antigen share the
identical or similar epitope
Dr Neethu Asokan

7. Stages of Ag-Ab reaction
• The Ag-Ab reaction occur in three stages- First,
Second and Third stage.
• The stages are also known as Primary stage,
Secondary stage and Tertiary stage
– In first stage, the formation of Ag-Ab complex
– In second stage leads to visible events like
precipitation, agglutination etc
– In third stage the antigen is destroyed or
neutralized
Dr Neethu Asokan

8. • Primary stage
– It is initial interaction between antigen and antibody
– The reaction is rapid and reversible
– Weaker intermolecular forces are involved namely ionic bonds,
hydrogen bonds, van der Waals forces, and hydrophobic
interactions
• Secondary stage
– It is an irreversible interaction with visible effects like
agglutination, precipitation, neutralization, complement
fixation, and immobilization of motile organisms
– The binding of Ag-Ab occurs by covalent bond which is a
stronger intermolecular force
• Tertiary stage
– This stage includes neutralization or destruction of injurious
agents
– Usually involves humoral immunity against diseases or allergy
Dr Neethu Asokan

9. Antigen Antibody reaction types
• These involves the different types of serological tests
• These tests are used for the detection of either serum
antibodies or antigens for diagnosis of diseases
• The types of tests involves- (a) precipitation,
(b)agglutination, (c) complement-dependent
serological tests, (d)neutralization test, (e)
opsonization, (f ) immunofluorescence,(g) enzyme
immunoassay, (h) radioimmunoassay, (i) western
blotting, ( j) chemiluminescence assay, and (k)
immunoelectronmicroscopic tests.
Dr Neethu Asokan

10. There are few other commonly used tests
• Flocculation tests which is used for the detection
of reagenic Ab in syphillis by VDRL test
• Radial Immunodiffusion which is used for the
detection of fungal Ag and Ab
• Counter current immunoelectrophoresis which is
used for the detection of both Ag and Ab of
bacteria, virus, fungus and parasites
• Tube agglutination tests which is used for the
detection of Ab in bacterial infections
• Slide agglutination test which is used for the
identification of bacterial isolates
Dr Neethu Asokan

11. • Latex agglutination tests which is used for the
quantification and detection of Ag and Ab
• Hemagglutination test which is used for the
detection of both Ag and Ab in virus and parasite
infection
• Coagglutination test for the detection of
microbial antigens
• Complement fixation test for quantification and
detection of Ab
• Immunofluorescence tests for the detection,
localization of Ag in cell/tissue and of specific Ab
in serum
Dr Neethu Asokan

12. • ELISA test for the detection of Ag and Ab and
their quantification
• Radioimmunoassay for quantification of
hormones, drugs and similar
compounds/molecules
• Western blot for the detection of antigen-
specific Ab
Dr Neethu Asokan

13. Parameters in serological tests
• Sensitivity
– The ability of the test to detect even very minute
quantities of antigen or antibody.
– When the test is highly sensitive, false negative results
may be absent or minimal.
• Specificity
– The ability of the test to detect reactions between
homologous Ags & Abs only, and with no other.
– In highly specific test, false positive reactions are
absent or minimal.
Dr Neethu Asokan

14. MEASUREMENT OF ANTIGEN &
ANTIBODY
• Measurement may be in terms of mass or
more commonly as units or titre.
• The Antibody titre of a serum is the highest
dilution of the serum which shows an
observable reactions with the antigen in a
particular test.
• Higher titer means greater level of antibodies
in serum.
Dr Neethu Asokan

15. PRECIPITATION REACTION
• Antigen usually occur in soluble form
• When a soluble Ag combines with its Ab in the presence of
electrolytes (NaCl) at a suitable temperature & pH, the Ag-
Ab complex forms an insoluble visible precipitate.
• Antibodies that aggregate soluble antigens are called as
precipitins
• If instead of sedimenting the precipitate occur as suspended
floccules it is Flocculation reaction
• The antibody must be a bivalent and the antigen must either
be bivalent or polyvalent.
• Precipitation can take place in liquid media or in gels such as
agar, agarose or polyacrylamide.

16. 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.
• The zone of antibody excess is known as the prozone
phenomenon and the zone of antigen excess is
known as postzone phenomenon.
– Middle tubes – Ag & Ab in equivalent proportions (Zone of
equivalence)

18. Mechanism of precipitation
• The mechanism of precipitation was proposed by
Marrack (1934)
• He proposed the lattice hypothesis to explain the
prozone formation
• The multivalent antigens combine with bivalent
Abs in varying proportions, depending on the Ag
– Ab ratio on the reacting mixture
• Precipitation outcomes when a large lattice is
formed consisting of alternating antigen and
antibody
• He assumed that the number of multivalent sites
of antigen and antibody are equal

19. Marrack’s hypothesis

20. Properties of Precipitation reaction
• It is either quantitative or qualitative test.
• Sensitive for the detection of Ags.
Types of precipitation reactions:
– Ring test
– Slide test
– Tube test
– Immunodiffusion
– Electroimmunodiffusion

21. • Precipitation reactions can also be classified
into
– Precipitation in solution- Ring test , Flocculation
test
– Precipitation in agar- Immunodiffusion
– Precipitation in agar with an electric field-
Immunoelectrophoresis
Dr Neethu Asokan

22. RING TEST:
• Consists of layering of antigen solution over a column
of antisera in a narrow tube/ test tube
Eg: Ascolis thermoprecipitin test, Grouping of
Streptococci by Lancefield technique
Dr Neethu Asokan

23. SLIDE TEST:
• When a drop of Ag & antiserum is placed on a
slide & mixed by shaking, floccules will appear.
Eg: VDRL test & RPR test for syphilis
Dr Neethu Asokan

24. TUBE TEST:
• This is employed for the standardization of
toxins & toxoids.
• Serial dilutions of toxin/toxoid are added to
the tubes containing a fixed quantity of
antitoxin.
• The amount of toxin that flocculates optimally
with one unit of the antitoxin – Lf dose.
Eg: Kahn test for syphilis.
Dr Neethu Asokan

25. IMMUNODIFFUSION (Precipitation in gel)
The technique involves diffusion through a substance / matrix
like agar or agarose used for the detection of antibodies
and antigen.
Different types include:
• Single diffusion in 1D/ Oudin procedure
• Double diffusion in 1D/ Oakley Fulthrope procedure
• Single diffusion in 2D/ Radial immunodiffusion/ Mancini
method
• Double diffusion in 2 D/ Ouchterlony double
immunodiffusion
Advantages of immunodiffusion:
• Reaction is visible as a distinct band of precipitation.
• Stable, can be stained for preservation.
• Indicates identity, cross reactions, non identity between
different Ags.
Dr Neethu Asokan

26. 1. 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.
Dr Neethu Asokan

27. 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.
Dr Neethu Asokan

28. Dr Neethu Asokan

29. 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.
Dr Neethu Asokan

30. Dr Neethu Asokan

31. 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 – central well
• Different Ags in the surrounding wells.
Dr Neethu Asokan

32. Reaction of identity
Partial identity
Lack of relatedness
Dr Neethu Asokan

33. Elek’s gel precipitation
Dr Neethu Asokan

34. 5. Immunoelectrophoresis
• Graber & Williams devised this technique.
• This involves the electrophoretic separation of
composite Ag into its constituent proteins, followed
by immunodiffusion against its antiserum – separate
precipitin lines.
• It is performed on an agarose gel with an Ag well and
Ab through it.
• The test serum is placed in the antigen well and
electrophoresed for about 1 hour. Ab against human
serum is placed in the trough and alowed for
diffusion for 18 – 24 hrs.
Dr Neethu Asokan

35. Dr Neethu Asokan

36. Immunoelectrophoresis
Dr Neethu Asokan

37. ELECTROIMMUNODIFFUSION
• The development of precipitin lines can be
speeded up by electrically driving the Ag &
Ab and this method is specified as
electroimmunodiffusion.
• Two types
1. Counterimmunoelectrophoresis (One
dimensional double electroimmunodiffusion)
2. Rocket electrophoresis (One dimensional single
electroimmunodiffusion)
Dr Neethu Asokan

38. 1. Counterimmunoelectrophoresis (CIE)
• This involves simultaneous electrophoresis of Ag and
Ab in gel in opposite directions resulting in
precipitation at a point between them.
• Produce precipitation lines within 25-30 mins.
• Clinical application: detecting Ags like alphafetoprotein
in serum, Ags of Cryptococcus and Meningococcus in
the CSF.
Dr Neethu Asokan

39. 2. Rocket electrophoresis
• A technique used for quantitative estimation of Ags.
• The antiserum 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 solidified gel.
• The Ag is electrophoresed into the Ab containing
agarose.
• The pattern of immunoprecipitation resembles the
shape of a rocket and hence the name.
Dr Neethu Asokan

40. Rocket electrophoresis
Dr Neethu Asokan

41. Laurell’s two dimensional electrophoresis
• It is a variant of rocket electrophoresis.
• The Ag mixture is electrophoretically
separated in a direction perpendicular to that
of the final rocket stage.
• Used to quantitate each of the several Ags in a
mixture.
Dr Neethu Asokan

42. Measurement of precipitation
– Turbidimetry
– Nephelometry
• Turbidimetry
– precipitation in solution
– measurement of light extraction (precipitate absorption)
– standard curve
• Nephelometry
– precipitation in solution
– measurement of scattered light (proportional to number
of insoluble complexes)
– standard curve
Dr Neethu Asokan

43. PRIMARY ANTIGEN-ANTIBODY
REACTIONS
• Major types of primary Ag-Ab reactions are:
– ELISA
– RIA
– IMMUNOFLUORESCENCE
Dr Neethu Asokan

44. Dr Neethu Asokan

45. • ELISA can be maily classified into 3 namely
– Indirect
– Sandwich
– Competitive
Dr Neethu Asokan

46. Dr Neethu Asokan

47. Dr Neethu Asokan

48. Dr Neethu Asokan

49. IMMUNOFLUORESCENCE
• Fluorescence is the property of absorbing light
of a specific wavelength and emitting rays
with a different wavelength.
• Coons and collegues while researching on
antigen detection found that labelled dyes can
be conjugated with antibodies and be used to
detect antigens.
• Most commonly used dyes include
fluorescein, phycoerythrin
Dr Neethu Asokan

50. • The types of this method are:
– Direct method
– Indirect method with labelled antibody
– Indirect method with labelled protein A
Dr Neethu Asokan

51. Dr Neethu Asokan

52. RADIO IMMUNO ASSAY
• Radioimmunoassay (RIA) is a scientific method used to test antigens
(for example, hormone levels in the blood) without the need to use a
bioassays.
• Radioimmunoassay (RIA) is a Radio-analytical technique with
remarkable sensitivity and a high degree of specificity that is widely used
for the estimation of a variety of molecules present in complex matrices.
• Also known as Radio tracer technique and best example of invitro
diagnosis technique using radio isotopes.
•This technique is used over a wide spectra of substances such as
hormones, steroids, vitamins, drugs, tumor markers and viral antigens.
Dr Neethu Asokan

53. •RIA combines the specificity of an antigen-antibody
reaction with sensitivity of radioactivity
measurements.
•This is a technique used for detection of micro
quantities of protein, viral antigens, antibodies,
structural proteins, vitamins and drug and their
metabolites.
Dr Neethu Asokan

54. • RIA is used in place of bioassay in
various branches of science Biochemistry, Microbiology, and
Hematology and Clinical pharmacology.
• RIA works on basic principle of biochemistry that competitive binding
between antigens for same antibody binding site.
• The competition of an analyte with its radioisotopically labeled
counterpart for a limited amount of antibody, the specific reagent, is the
underlying principle of this technique. Increasing the analyte
concentration inhibits the binding of the labeled analyte to the antibody.
Dr Neethu Asokan

55. Dr Neethu Asokan

56. Major steps in RIA
1. Radio labelling of the Antigen or radio labelledproduction
2. Preparation & characterisation of the Antigen [Ligand to be
analysed]
3. Preparation of the SpecificAntibody
4. Development of Assay System or separation techniques
Two types of RIA:
• Solid phase RIA and Liquid phase RIA
Dr Neethu Asokan

57. Application of antigen-antibody
reactions
• Determination of blood groups
• Serological confirmation of infectious agents
• Development 0f immunoassays
• To detect the presence of antigens or
antibodies
• Determine immunodeficiency diseases and
their characteristics
Dr Neethu Asokan