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ANTIGEN-ANTIBODY REACTIONS

ANTIGEN-ANTIBODY REACTIONS. Lock and Key Concept. Low Affinity. High Affinity. Ab. Ab. Ag. Ag. Affinity. Strength of the reaction between a single antigenic determinant and a single Ab combining site. Affinity =  a ttractive and repulsive forces. Y. Y. Y. Y. Y. Y. Y. 10 4.

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ANTIGEN-ANTIBODY REACTIONS

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  1. ANTIGEN-ANTIBODY REACTIONS

  2. Lock and Key Concept

  3. Low Affinity High Affinity Ab Ab Ag Ag Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site Affinity =  attractive and repulsive forces

  4. Y Y Y Y Y Y Y 104 106 1010 Keq = Avidity Affinity Avidity Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs

  5. Specificity • The ability of an individual antibody combining site to react with only one antigenic determinant. • The ability of a population of antibody molecules to react with only one antigen.

  6. Cross reactions Anti-A Ab Anti-A Ab Anti-A Ab Ag B Ag C Shared epitope Similar epitope Ag A Cross Reactivity • The ability of an individual Ab combining site to react with more than one antigenic determinant. • The ability of a population of Ab molecules to react with more than one Ag

  7. Ab excess Ag excess Equivalence – Lattice formation Factors Affecting Measurement of Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag

  8. Antigen antibody tests • Used in both directions • Qualitative • Quantitative

  9. Qualitative/quantitative • Qualitative • determines antigen or antibody is present or absent • Quantitative • determines the quantity of the antibody • Titer • The highest dilution of the specimen usually serum which gives a positive reaction in the test

  10. Antigen and antibody reactions in the lab • Precipitation tests • Agglutination • ELISA • Radioimmunoassay • Immunofluorscence • Complement Fixation

  11. Y +  Y Y • AGGLUTINATION TESTS • Particulate antigen • Slide agglutination • Tube agglutination • Passive agglutination

  12. Slide agglutination test:

  13. Y Y +  Y Passive Agglutination/Hemagglutination • Agglutination test done with a soluble antigen coated onto a particle • Applications • Measurement of Abs to soluble antigens

  14. Y Y + Y Y Y Y  Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin) Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test • Detects antibodies on erythrocytes

  15. Step 1 Y +  Y Y Y Y Target RBCs Patient’s Serum Y Y Y Step 2 Y Y Y Y Y Y Y Y Y Y Y Y +  Y Y Y Y Coombs Reagent (Antiglobulin) Coombs (Antiglobulin)Tests • Indirect Coombs Test • Detects anti-erythrocyte antibodies in serum

  16. Precipitation Tests(Soluble antigen)

  17. Precipitation tests • Soluble antigen reacts with specific antibody in presence of electrolytes at an optimal temp and pH to form an insoluble precipitate • Flocculation – precipitate remains suspended as floccules

  18. Prozone phenomenon • Precipitation takes place when there is optimal proportions of Antigen and antibodies • Absence of precipitation in presence of excess of antibodies • Such serum should be diluted before doing precipitation tests

  19. ZONE of Equivalence No Soluble Ag or Ab ANTIGEN EXCESS ANTIBODY EXCESS Amount of Precipitate Ab CONC

  20. Equivalence – Lattice formation Mechanism of precipitation • Marrack’s lattice hypothesis • Precipitation occurs whenever multivalent antigens combine with bivalent antibodies to from a large lattice • Same hypothesis applies for agglutination also

  21. Precipitation tests • Precipitation techniques • Tube precipitation test -Ring test – Ascoli’s thermo precipitation test • Flocculation • Slide – VDRL for syphilis • Tube – Khan test for syphilis

  22. Ring test

  23. VDRL

  24. Ab in gel Ag Ag Ag Ag Diameter2 Ag Concentration Radial Immunodiffusion • Interpretation • Diameter of ring is proportional to the concentration • Quantitative • Ig levels • Method • Ab in gel • Ag in a well

  25. - + Ag Ag Ab Ag Ab Immunoelectrophoresis • Method • Ags are separated by electrophoresis • Ab is placed in trough cut in the agar • Interpretation • Precipitin arc represent individual antigens

  26. - + Ab Ag Countercurrent electrophoresis • Method • Ag and Ab migrate toward each other by electrophoresis • Used only when Ag and Ab have opposite charges • Qualitative • Rapid

  27. Complement fixation test (CFT)

  28. Complement plays an imp role in Ag-Ab reaction. • In the presence of appropriate Abs: • Complement lyses erythrocytes • Kills bacteria, • Immobilizes motile organisms • Promotes phagocytosis • Contributes to tissue damage in hypersensitivity reactions

  29. Principle of CFT: Ability of Ag-Ab complexes to fix complement • Very sensitive test, can detect minute quantities of Ag & Ab • All reagents have to be precisely standardised Eg: Wasserman reaction for sero-diagnosis of syphilis

  30. Complement Fixation assay(positive test)

  31. Complement Fixation Assay(negative test)

  32. Interpretation of result: • Lysis of erythrocytes: Negative CFT • Absence of Erythrocyte lysis: Positive CFT Indirect CFT: For testing sera that do not fix guinea pig complement: Hemolysis indicates a positive result

  33. CONGLUTINATING COMPLEMENT ABSORPTION TEST: • Uses horse complement which is non-hemolytic • Indicator system is sheep RBC mixed with bovine serum which contains a globulin called Conglutinin, which acts as Ab to the complement Other complement dependent serological test: • Immune adherence test • Immobilisation test: T.pallidum • Bacterial cytolytic test

  34. NEUTRALIZATION TEST • Virus neutralization tests • Bacteriophage neutralization test • Toxin neutralization test (exotoxin) • Schick test: tests the ability of circulating antitoxin to neutralize diphtheria toxin injected ID • ASO test: • Nagler’s reaction: alpha toxin of Cl.perfringens

  35. IMMUNOASSAYS • RIA • ELISA • IMMUNOCHROMATOGRAPHY • CHEMILUMINSCENCE ASSAY • IMMUNOBLOTTING

  36. Radioimmuoassay (RIA)

  37. Radioimmunoassay • Radioisotopes and enzymes are conjugated to Ag /Ab • BINDER LIGAND ASSAY • Ag whose concentration is to be determined is called Ligand (analyte) • RIA is a competitive binding assay in which fixed amounts of Ab and radiolabelled Ag react in the presence of unlabelled Ag

  38. Labelled and unlabelled Ags compete for the limited binding sites on the Ab Applications of RIA: • Quantitation of hormones. Drugs, tumor markers, IgE, and viral antigens

  39. Immunoflourescence • Immunofluorescence is an antigen-antibody reaction where the Abs are tagged (labelled) with a fluorescent dye and the antigen-antibody complex is

  40. Fluorochrome Labeled Ab Y Ag Tissue Section Immunofluorescence • Direct • Ab to tissue Ag is labeled with fluorochrome

  41. ELISA (types): • Direct indirect • Sandwich • Competetive • Casette

  42. 0

  43. BASIC FORMAT Solid phase = 96 / 384-well microplate

  44. 1. Coat solid phase with antigen when analysing antibody antibody when analysing antigen Analyte = antibody Analyte = antigen Incubate, wash

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