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Antigen - Antibody Reactions

Antigen - Antibody Reactions. Antigen – Antibody Reactions. Antigen combines with its specific Antibody in observable manner . Reaction between Ag & Ab specific. Ag – Ab reactions in – vitro Serological tests. In the Body or in Vivo :

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Antigen - Antibody Reactions

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  1. Antigen - Antibody Reactions

  2. Antigen – Antibody Reactions • Antigen combines with its specific Antibody in observable manner. • Reaction between Ag & Ab specific. • Ag – Ab reactions in – vitro Serological tests.

  3. In the Body or in Vivo: Forms the basis of antibody mediated (humoral) immunity against infectious diseases. May lead to tissue injury in some hypersensitivity reactions & autoimmune diseases. In the Lab.or in Vitro: For diagnosis of infection. Helpful in epidemiological studies. For identification of non infectious agents such as enzymes. Detection & quantitation of either Ag or Ab. Uses of Antigen – Antibody Reactions

  4. Lock & Key concept. Non covalent bonds. - Hydrogen bonds - Electrostatic bonds - Van der Waal forces - Hydrophobic bonds Nature of Ag/Ab Reactions

  5. Stages of Antigen – Antibody Reactions • Ag – Ab Reaction occurs in two stages 1. PRIMARY 2. SECONDARY • Primary Stage: • Initial, rapid, reversible, reaction between Ag & Ab without any visible effect, occurring at low temp. • Binding between Ag & Ab by weaker intermolecular forces. • No covalent bonding

  6. Secondary Stage • In most but not all primary stage followed by secondary one. • Leads to demonstrable (visible) effects ------- * Precipitation. * Agglutination. * Lysis of cell. * Killing of live antigens. * Neutralisation of toxins. * Complement fixation. * Immobilisation of motile organisms. * Phagocytosis.

  7. Role of Immunoglobulin Classes in different Serological Reactions.

  8. Characteristics of Ag – Ab Reaction • Specific: Ag combines only with its homologous Ab & vice versa. • Specificity not absolute. Cross reactions occur due to antigenic similarity or relatedness. • Entire molecules of Ag & Ab & not fragments react. • No denaturation of Ag or Ab during reaction. • Ag – Ab combination firm but reversible. • Firmness of combination depends on - Affinity & Avidity

  9. Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site.

  10. Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs.

  11. Precipitation Tests

  12. Precipitation Reaction • When a soluble antigen combines with its antibody in presence of electrolytes ( NaCl) at suitable temperature & pH, the Ag – Ab complex forms an insoluble precipitate. • The Ag – Ab complex forms an insoluble precipitate which remains suspended instead of settling down ‘ Flocculation’.

  13. Precipitation reactions: • 1. Zone of antibody excess - precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant; • 2. Zone equivalence - maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant; and • 3. Zone of antigen excess - precipitation is inhibited & Ag. not bound to Ab. can be detected in the supernatant.

  14. Applications • Qualitative & Quantitative test. • Very sensitive in detecting antigens & as little as 1g of protein. Less sensitive to detect antibody. • Ring test - Ascoli’sthermoprecipitin test , Grouping of streptococci by Lancefield technique. • Slide test – VDRL test for syphilis = eg of flocculation test. • Tube test – Kahn test eg of tube flocculation test. • Quantitative tube flocculation test – used for standardisation of toxins & toxoids.

  15. Immunodiffusion ( Precipitation in Gel) • Several advantages in allowing precipitation to occur in gel than in liquid. • Reaction visible as distinct & stable band of precipitation . • Can be stained for preservation.

  16. Immunodiffusion ‘Oudin procedure’ ‘Oakley – Fulthorpe procedure

  17. Method Ab in gel Ag in a well Interpretation Diameter of ring is proportional to the concentration Quantitative Ig levels Screening sera for antibodies to influenza virus Radial Immunodiffusion

  18. Oucheterlony procedure e.g. : Elek’s test for toxigenicity in C. diphtheriae

  19. Immunoelectrophoresis

  20. - + Ab Ag Countercurrent electrophoresis • Method • Ag and Ab migrate toward each other by electrophoresis • Used only when Ag and Ab have opposite charges • Qualitative • For Hepatitis B Ag & Ab , Antigens of Cryptococcus in C.S.F.

  21. Rocket Electrophoresis • One dimensional single immunodiffusion : Mainly applied for quantitation of antigens.

  22. Agglutination Reactions

  23. Agglutination Reactions • Particulate antigen combining with its antibody in presence of electrolyte at optimal temp. & pH, resulting in visible clumping of particles. • Incomplete or monovalent Ab do not cause agglutination. • More sensitive than precipitation for detection of Ab. Better with IgM than IgG.

  24. Agglutination Tests • Agglutination occurs due to the cross-linking of particulate antigens by antibody molecules. • Agglutination is the visible clumping of insoluble particles, whereas precipitation involves the aggregation of soluble molecules

  25. TYPES • Slide agglutination • Tube agglutination • The antiglobulin (Coomb’s) test • Passive agglutination test • Heterophile agglutination test • Haemagglutination test

  26. Y + ↔ Y Y Applications Slide Agglutination : - • Routine procedure to identify bacterial strains from clinical specimens e.g. Salmonella sp. • Blood grouping & cross matching.

  27. Applications Tube Agglutination test : - standard quantitative method for measurement of antibodies. Fixed volume of particulate Ag+ Equal volume of serial dilutions of antiserum= Agglutination 1. Enteric fever ( Widal test). 2. Typhus fever (Weil Felix test). 3. Infectious mononucleosis ( Paul Bunnel test). 4. Brucellosis. 5. Primary atypical pneumonia .

  28. 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

  29. 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 Detection of Anti Rh Ab & Autoimmune hemolytic anemia.

  30. Passive Agglutination Test • A precipitation reaction can be converted to agglutination test by attaching soluble antigens to the surface of carrier particles, bentonite, latex particles, red blood cells. Latex Agglutination test : • For detection of Hepatitis B Ag. • ASO, • RA factor ( Rose Waller test)

  31. Reverse passive Agglutination test • Antibodies are bound to the surface of carrier particles, instead of antigen. • Human chorionic Gonadotropin (HCG) • C Reactive Protein (CRP)

  32. ImmunoFluorescence • Fluorescence is the property of absorbing lighter of one particular wavelength (UV) and emitting rays of different wavelength (Visible). • Uses fluorescent dyes as labels • Dyes: Fluorescein, isothiocyanate, lissamine, rhodamine. chemically linked to an antibody • Glows bright green when exposed to fluorescent light • Types: • Direct fluorescent antibody test • Indirect fluorescent antibody tests

  33. Direct Immunofluorescence tests • Microbe (Unknown antigen) fixed to a slide • Known Antibody to tissue Ag is labeled with fluorochrome • Incubated and rinsed (remove free Ab) • Labeled Antibodies that attach can be viewed using a fluorescence microscope (yellow green fluorescence). • Used to detect specific antigen under a fluorescent microscope. Antigen Slide Fluorescent dye Antibody Y

  34. IndirectImmunofluorescence test Used to demonstrate the presence of antibody in serum. Ab to tissue Ag is unlabeled Fluorochrome-labeled anti-Ig is used to detect binding of the first (serum) Antibody Fluorochrome Labeled Anti-Ig Unlabeled Ab Y Y Ag Tissue Section

  35. Complement mediated serological reactions • Comp. fixation test • Immune adherence • Immobilization test • Cytolytic or cytocidal reaction

  36. Complement fixation test • Highly sensitive and diagnostically important test for serum antibody. • Capable of detecting as little as 0.04 microgram of Ab and 0.1 microgram of Antigen. • Consists of 2 steps and 5 reagents: Ag, Ab, Complement, Sheep erythrocytes, Amboceptor (Rabbit Ab to sheep RBC)

  37. Principle • (Ag+Ab) Complex Complement is activated and there by consumed or utilized. • Indicator system (sheep red cells + Anti-sheep red cell Ab) is then added to detect the presence of remaining complement. • If is the comp. has been utilized by Ag+Ab reaction No haemolysis (positive) • If comp. has not been consumed (sheep RBC+Ab to sheep RBC) +comp. Haemolysis (Negative)

  38. Examples: Wasserman reaction Diagnosis of syphilis. Reagents: i) Ag (cardiolipin from Bovine heart) ii) Serum to be tested for Ab. Decomplemented at 56o C for 30 mins. iii) Pooled guinea pig serum (source for complement) iv) Haemolytic system (amboceptor)

  39. Complement Fixation Ag mixed with test serum to be assayed for Ab No Ag Ag Patient’s serum Y Y Y Y Y Y Y Y Y Y • Methodology • Standard amount of complement is added • Erythrocytes coated with Abs is added • Amount of erythrocyte lysis is determined Ag Ag

  40. Indirect Complement fixation test • Certain avian (duck, turkey) and mammalian (cat, horse) sera don't fix guinea pig complement. • Test set up in duplicate, and after first step std antiserum known to fix complement is added to one set. • If test serum contains Ab the Ag has been used up in the first step and therefore Std Antiserum added later will not be able to fix the complement. • Hemolysis indicates positive test.

  41. Other comp. mediated serological reactions • T P I (Treponema pllidum Immobilisation test TP (Nichols strain) + serum (Ab) + complement Immobilization of living treponemes seen by dark field microscope. • Immune adherence Bacteria (V. cholera + Tr. pallidum) + Sp Ab + Comp. + particulate materials (RBC, platelets etc.) Bact. are aggregated and adhere to cells Phagocytosis. • Cytolytic cytocidal Test: (V. Cholera + Sp Ab + Comp.) Bacterium is killed.

  42. OPSONIZATION • Opsonins are the substances (Complement and IgM) which coats on the target cells and make them palatable for phagocytosis. The process initiated by opsonin is called Opsonization

  43. NEUTRALIZATION TEST • Virus neutralization: - Neutralization of bacteriophage can be demonstrated in plaque inhibition. - Ab prevents virus adsorption to the receptor. - Viral Heamagglutination inhibition (HI) test HI is used in the diagnosis of influenza, measles, mumps etc. • Toxin Neutralization: ATS, ADS in vivo, Schick test, (Invivo), ASO: Invitro

  44. NEUTRALIZATION

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