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Serological Reactions.pptx
- 1. Serological Reactions- Precipitation Dr.Arun Kumar Parthasarathy Ph.d Dept. of Microbiology D.Y Patil Medical College, Kolhapur
- 2. • Definition • Purpose •General Features of antigen-antibody reactions • Types 1.Precipitation 2.Agglutination 3.Complement Fixation Tests(CFT) 4.Neutralisation 5.Opsonisation Serological Reactions
- 3. Definition • Antigen-antibody reactionsin vitro in a specific & observable manner
- 4. Purpose In-vivo :Basis of • Antibody mediated immunity • Tissue injury in hypersensitivity • Autoimmune diseases In-vitro : For • Diagnosis of infections • Epidemiological surveys • Identification of infectious agents & non-infectious agents such as enzymes • Detection /quantitation of antigens/antibodies
- 5. General Features • Reactionis specific • Entire molecules react ,not fragments • No denaturation • Combination occurs at surface • Combination is firm but reversible • Both antigens and antibodies participate • Combination is in varying proportions
- 6. Precipitation Reaction Whena soluble antigen combines with its antibody in the presence of electrolytes (NaCl) at a suitable temperature & pH, the antigen – antibody complex forms an insoluble precipitate • Flocculation – when precipitate remains suspended as floccules.
- 7. Mechanism of precipitation •1954 : Marrack proposed lattice hypothesis • Multivalent antigens combine with bivalent antibodies in varying proportions ,depending on the antigen –antibody ratio in the reacting mixture • Precipitation results when a large lattice is formed consisting of alternating antigen and antibody molecules
- 8.
- 9. Zone phenomenon – Prozone– Zone of antibody excess – Zone of equivalence – Post zone – Zone of antigen excess. Prozone Sera rich in antibody may sometimes give a false negative precipitation or agglutination result, unless several dilutions are used.
- 10.
- 11. Applications of precipitation •Ring test Ascoli’s thermoprecipitation test Grouping of Streptococci by Lancefield technique. • Slide test – VDRL test for syphilis • Tube test – Kahn test for syphilis
- 12. Immunodiffusion- Precipitation in gel •1%soft Agarose gel used in Immunodiffusion •Results in formation of visible bands instead of floccules and can be preserved for longer time •It can differentiate individual antigens from a mixture , each antigen forms a separate bands after reacting with specific antibody
- 13. Principle Based on 2principles 1. Whether only antigen diffuses (single diffusion) or both Ag and Ab diffuse (Double diffusion) 2. When Ag or Ab diffuses in one dimension (vertical diffusion when test is done on a tube layered with gel) or 2 dimension (diffusion in both X and Y axis which occurs when test is done on a slide or petri dish layered with gel)
- 14. Applications of precipitation(cont) – Immunodiffusion 1.Single diffusion in one dimension 2.Double diffusion in one dimension 3.Single diffusion in two dimensions 4.Double diffusion in two dimensions 5.Immunoelectrophoresis
- 15. 1. Single diffusionin one dimension when Ag solution is poured over a layer of gel containing Ab, only Ag diffuses in one direction towards Ab to form a band 2. Double Diffusion in one Dimension (Oakley-Fulthorpe Procedure), Column of Plain agar is placed between the antigen layer and layer of gel incorported with Ab, then Ag-Ab move towards each pother in opposite directions and precipitate band is formed at the line they meet in the plain agar
- 17. 3. Single diffusionin two dimension (Radial Immunodiffusion):- Gel is incorporated with Ab is placed on a slide and several wells are cut when drop of Ag is placed in the wells they diffuse radially in all directions to meet with Ab and ring shaped bands are formed around the well The diameter of the ring is directly proportional to the concentration of the antigen in the well 4. Double dimension in two dimension (Ouchterlony procedure):- on a slide poured with agar gel, Ab is placed in the central well and surrounding wells are filled with Ags both Ag and AB moves towards each other in all the directions and forms a band
- 18. The adjacent precipitatebands interact in 3 ways 1. They may fuse with each other (indicates both antigens are identical) 2. They may cross one another (indicates antigens are unrelated) 3. They may cause spur formation (indicates cross reaction or Partial Identity)
- 19. Precipitation reaction inGel in presence of Electric Field 1. Electro-immunodiffusion – The development of precipitin lines can be speeded up electrically driving the antigen & antibody. 2. Counter-immunoelectrophoresis ( one dimensional double electroimmunodiffusion) 3. Rocket electrophoresis (one dimensional single electroimmunodiffusion)
- 20. Electroimmunodiffusion When electric currentis applied to a slide layered with gel the serum proteins placed in a well are separated into individual antigens antiserum is present in trough moves towards the antigen components resulting in formation of separate precipitin lines in 18-24 hours
- 21. Counter current Immunoelectrophoresis Takes30 minutes, Faster More sensitive because it involves simultaneous electrophoresis of the Ag and Ab in gel in opposite direction Uses:- alpha feto protein in serum and Capsular Ags of Cryptococcus neoformans and Meningococci in CSF.
- 22. Rocket Immunoelectrophoresis One dimensionsingle Immunodiffusion test, mainly done in the past for quantitative estimation of antigens