Antigen antibody reactions

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 )

17. 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

18. Marrack’s hypothesis

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

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28. 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

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30. 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

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

32. Elek’s gel precipitation Dr Neethu Asokan

33. 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

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35. Immunoelectrophoresis Dr Neethu Asokan

36. 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 Counterimmunoelectrophoresis (One dimensional double electroimmunodiffusion ) Rocket electrophoresis (One dimensional single electroimmunodiffusion ) Dr Neethu Asokan

37. 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

38. 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

39. Rocket electrophoresis Dr Neethu Asokan

40. Laurell’s two dimensional electrophoresis It is a v ariant 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

41. 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

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

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44. ELISA can be maily classified into 3 namely Indirect Sandwich Competitive Dr Neethu Asokan

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48. 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

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

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51. 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

52. 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

53. 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

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55. Major steps in RIA Radio labelling of the Antigen or radio labelled production Preparation & characterisation of the Antigen [Ligand to be analysed ] Preparation of the Specific Antibody Development of Assay System or separation techniques Two types of RIA: Solid phase RIA and Liquid phase RIA Dr Neethu Asokan

56. 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