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Serological Reactions

Serological Reactions. II BPT Dr Ekta Chourasia Department of Microbiology. Introduction. Ag – Ab reactions in vitro are known as Serological reactions. Help in - the diagnosis of infections - epidemiological surveys - identification of infectious agents

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Serological Reactions

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  1. Serological Reactions II BPT Dr Ekta Chourasia Department of Microbiology

  2. Introduction • Ag – Ab reactions in vitro are known as Serological reactions. • Help in - the diagnosis of infections - epidemiological surveys - identification of infectious agents and of non infectious antigens like enzymes. Dr Ekta, Microbiology, GMCA

  3. Measurement of Ag & Ab • Titre or units - Ab titre of a serum is the highest dilution of serum which shows an observable reaction with the Ag in the particular test. • Sensitivity – accuracy with which a test can detect the presence of infection. • Specificity – accuracy with which a test can detect the absence of infection. Dr Ekta, Microbiology, GMCA

  4. Serological reactions • Precipitation • Agglutination • Complement fixation test (CFT) • Neutralisation • Immunofluorescence (IF) • Radio immunoassay (RIA) • Enzyme immunoassay(EIA) / Enzyme linked immunosorbent assay (ELISA) Dr Ekta, Microbiology, GMCA

  5. PRECIPITATION • 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 precipitate. • When instead of sedimenting, the precipitate remains suspended as floccules, the reaction is called Flocculation. • It can take place in liquid media or in gels such as agar, agarose or polyacrylamide. Dr Ekta, Microbiology, GMCA

  6. Mechanism of precipitation • Lattice formation - lattice hypothesis by Marrack Dr Ekta, Microbiology, GMCA

  7. Zone phenomenon • Precipitation and some other serological reactions are best seen when the antigen and antibody are mixed in optimal (equivalent) proportion. • Since in these test antigen used is in fixed quantity, the only variable is the antibody. Serial dilutions of antibody when treated with antigen show that there is no precipitationwhere the antibody is in excess. This is called as PROZONE PHENOMENON and is caused due to failure to form lattice. Dr Ekta, Microbiology, GMCA

  8. Optimal proportions Antibody excess Antigen excess Serial Dilution of Serum containing Abs- decreasing concentration of Ab Dr Ekta, Microbiology, GMCA

  9. Graphical Representation • If amount of precipitate in the different tubes are plotted on a graph, the resulting curve will have three phases. • Prozone or Zone of Ab excess. • Zone of equivalence (peak). • Post zone or Zone of Ag excess. Prozone – imp. in clinical serology. - Several dilutions are tested. Dr Ekta, Microbiology, GMCA

  10. Applications • Very sensitive in Ag detection (1µg of protein) • Forensic – identification of blood & seminal stains • Food adulteration Dr Ekta, Microbiology, GMCA

  11. Types of Precipitation Reaction • RING TEST - Simplest - Layering Ag solution over a column of antiserum in a narrow tube - Ppt forms at the junction. e.g. Lancefield grouping of Streptococci Dr Ekta, Microbiology, GMCA

  12. Types of Precipitation Reaction • SLIDE TEST – A drop each of Ag & antiserum are placed on a slide & mixed - floccules appear e.g. VDRL test for syphilis Dr Ekta, Microbiology, GMCA

  13. Types of Precipitation Reaction • TUBE TEST - Tube flocculation e.g. Kahn test for syphilis • IMMUNODIFFUSION (Precipitaiton in gel) Advantage – Distinct band of ppt. formed - Number of different Ags in the reacting mixture can be identified. Dr Ekta, Microbiology, GMCA

  14. Modifications of Immunodiffusion • Single diffusion in one dimension (Oudin procedure) Antigen Precipitation band Ab in agar gel Dr Ekta, Microbiology, GMCA

  15. Modifications • Double diffusion in one dimension (Oakley-Fulthorpe procedure) Dr Ekta, Microbiology, GMCA

  16. Modifications • Single diffusion in two dimensions (Radial immunodiffusion) Dr Ekta, Microbiology, GMCA

  17. Modifications • Immunoelectrophoresis– useful for testing normal & abnormal proteins in serum & urine. - identification & quantitation of various proteins in the serum. Dr Ekta, Microbiology, GMCA

  18. ELECTROIMMUNODIFFUSION • Development of precipitin lines can be speeded up by electric current. • 2 methods : Rocket electrophoresis & Counterimmunoelectrophoresis (CIEP) Dr Ekta, Microbiology, GMCA

  19. AGGLUTINATION • When a particulate Ag is mixed with its Ab in the presence of electrolytes at a suitable temperature & pH, the particles are clumped or agglutinated. • More sensitive than precipitation for the detection of Abs. • “Blocking Abs”– incomplete or monovalent Abs, do not cause agglutination. Dr Ekta, Microbiology, GMCA

  20. Types of Agglutination • SLIDE AGGLUTINATION • drop of antiserum + a drop of uniform suspension of Ag clump formation • Visible to the naked eye. • uses : blood grouping identification of bacterial isolates Dr Ekta, Microbiology, GMCA

  21. TUBE AGGLUTINATION • Standard quantitative method for the measurement of Abs. • Uses : WIDAL test for Typhoid fever. • Haemagglutination – RBCs are used as antigens e.g Paul Bunnell test, Cold agglutination test for Mycoplasma pneumoniae Dr Ekta, Microbiology, GMCA

  22. PASSIVE AGGLUTINATION • Soluble Ags are attached to the surface of carrier particles to convert precipitation tests into agglutination tests. • More convenient & sensitive. • Carrier particles : RBC’s, Latex, Bentonite Dr Ekta, Microbiology, GMCA

  23. ANTIGLOBULIN (Coomb’s) TEST • Detection of anti- Rh Abs (incomplete Abs) • Can be direct or indirect. • Direct – in vivo test • Indirect – in vitro test. Dr Ekta, Microbiology, GMCA

  24. COMPLEMENT FIXATION TEST • Ability of Ag- Ab complexes to fix complement. • Very sensitive test but time consuming. • Not done now a days. e.g. Wasserman reaction for Syphilis Dr Ekta, Microbiology, GMCA

  25. NEUTRALISATION TESTS • Virus neutralisation – by their Abs • Demonstrated in animals, eggs or tissue culture. • Toxin neutralisation tests – neutralisation of bacterial exotoxins by corresponding antitoxin e.g. Elek’s test for C.diphtheriae Dr Ekta, Microbiology, GMCA

  26. IMMUNOFLUORESCENCE • FLUORESCENCE – property of certain compounds to absorb light of shorter (UV) wavelength & emit light of higher (visible) wavelength. • Fluorescent dyes (Fluorochromes) are conjugated to Abs –Labelled Abs. • Fluoresce when binds to specific Ag in tissues. • Can be direct or indirect. • Detected byFluorescent Microscope. Dr Ekta, Microbiology, GMCA

  27. Fluorochromes • FITC- Fluorescein Isothiocynate • Rhodamine- Auramine • Acridine orange • Calcofluor white :- fungal elements Applications • Direct IF– detection of Rabies virus Ag in brain smears. • Indirect IF– fluorescent treponemal Ab test Dr Ekta, Microbiology, GMCA

  28. RADIO IMMUNOASSAY • Radio isotopes are conjugated to Abs or Ags. • Applications – quantitation of hormones, drugs, tumor markers, IgE & viral Ags. Dr Ekta, Microbiology, GMCA

  29. ELISA • Enzyme linked immunosorbent assay. • Can be used for the detection of Ag or Ab. • Corresponding Ag or Ab is conjugated with an enzyme. • The enzyme is then detected by its ability to convert a colorless substrate to a colored product. • The color is then measured in a machine called ELISA reader. Dr Ekta, Microbiology, GMCA

  30. Immunoelectron Microscopy • Viral particles + specific antisera EM clumping of viruses Dr Ekta, Microbiology, GMCA

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