Antigen Antibody Reactions

1. Antigen Antibody Reactions Dr Sathya Anandam

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. IMMUNO DIAGNOSTIC PROCEDURES • Based on Ag Ab reactions. What purpose Ag & Ab reaction serves? • In Body: • Basis of humoral immunity • Tissue injury in some hypersensitivity • In Laboratory: • Identification of infectious agents and their antibodies. • Identification of non-infectious antigens. • Epidemiological survey Serological reaction: Ag & Ab reaction invitro

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

5. Ag and Ab interaction occurs in 3 stages: • Primary Stage: - Reaction occurs in low temp. - No visible effect - Reversible being effected by weaker intermolecular forces (vanderwaals, ionic bonding and hydrogen bonding). • Secondary Stage: - Demonstrable effect (precipitation, agglutination….) - Irreversible • Tertiary Stage: - Ag - Ab reaction occurring invivo leading to neutralization, destruction of injurious antigens

6. Role of Immunoglobulin Classes in different Serological Reactions.

7. 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

8. Ag - Ab Reactions • Precipitation • Agglutination • Comp dependant serological reaction. • Opsonization • Neutralization • Immune assay • Immunofluorescent • ELISA • RIA • Immuno Blotting • Immuno – Electron Microscopic tests.

9. 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’.

10. Precipitation Reaction • Zone phenomenon: • Failure of lattice formation: • Immune complex disease.

11. 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.

15. Application of precipitation reaction • Carried out qualitatively quantitatively • Mostly used to detect Ag • Less sensitive to detect Ab • Forensic application Indent seminal stain Food adulteration Types: • Liquid precipitation • Agar gel precipitation (Immuno diffusion)

16. Precipitation in Liquid • Ring Test: • ‘C’ Reactive protein • Lance field grouping of Streptococci • Ascoli thermo precipitin test • Slide test: • VDRL slide flocculation test • Tube test: • Kahn tube Test • Floccules (+ve), uniform opalescence (-ve)

17. Precipitation in gel • Single diffusion in one dimension • Double diffusion in one dimension • Single diffusion in two dimension Quantitative method used for • Estimation of Ig • Hbs Ag • Alpha foeto protein etc • Double diffusion in two dimension (Ouchterlony technique) • Immuno electrophoresis • Immunodiffusion • CIEP • Rocket electrophoresis • Laurell modification

18. 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.

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

20. 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

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

22. Immunoelectrophoresis

23. - + 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.

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

25. 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.

26. Agglutination • Mechanism: lattice hypothesis • Zone phenomenon

27. 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 Types of Agglutination Reactions • Direct agglutination reactions • Indirect or passive agglutination tests • Hemagglutination reactions

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

29. 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

30. 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.

32. 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, CRP, • RA factor ( Rose Waller test) • HCG • N. meningitidis typing

33. Applications 1) Slide agglutination • Bacterial Agglutination • Blood grouping and Rh typing. • Coomb’s Test (Direct and Indirect) • Artificial particle agglutination. • RBC: TPHA, THA • Cold agglutinins at 400 C • Mycoplasmosis, malaria, syphilis, Trypanosomiasis. • Cowan type I (SA) coagglutination • Latex Particle: CRP, ASO, RA etc.(Sol Ags and Ab can be absorbed)

38. Tube Agglutination Test • May be performed in a simple round bottom test tubes. • Doubling dilution of patient’s serum • Constant amount of antigens are added . Incubated 370C • Button formation: Negative • Mat formation: Positive • Titre: Highest dilution which gives observable reaction. • Should be interpreted cautiously • Rise in titre is diagnostic

39. Applications Tube Agglutination test : - 1. Enteric fever ( Widal test). 2. Typhus fever (Weil Felix test). 3. Infectious mononucleosis ( Paul Bunnel test). 4. Brucellosis. 5. Primary atypical pneumonia .

40. 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

42. NEUTRALIZATION TEST • Virus neutralization: - Neutralization of bacteriophage can be demonstrated in plaque inhibition. - Ab prevents virus adsorption to the receptor. - Viral Heamagghutination 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

45. Complement Mediated Serological Reactions • Compliment takes part in many immunological reactions • It is absorbed when there in AG+AB complex • Complement - Lyses erythrocytes - Kills and lysis bacteria - Immobilize motile organisms - Promotes phagocytosis and Imm. Adherence - Contributes to the tissue damage by certain types of hypersensitivity.

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

47. 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.

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

49. 1. Antigen + Test serum (contains antibody) Complement fixed + Complement+ Haemolytic system Result: No hemolysis Positive CF test. 2. Antigen + test serum (contain no Antibody) Complement not fixed + Complement + Haemolytic system Result: Hemolysis Negative CF test.

50. Complement Fixation Test