Antigen-antibody reactions occur through specific binding between antigens and antibodies. The primary reaction is reversible and detectable through techniques like radioisotopes, while secondary reactions lead to visible effects like precipitation or agglutination. The strength of antigen-antibody binding depends on factors like affinity, avidity, and ratio. Various serological tests exploit antigen-antibody reactions to detect pathogens or antibodies through precipitation, agglutination, or other reactions.

1. Antigen - Antibody Reactions DWD

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. Uses of Antigen – Antibody Reactions 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.

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

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 Detected by estimating free & bound Ag or Ab separately by radioisotopes, fluorescent dyes or ferritin.

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. * Enhancement of phagocytosis.

7. Secondary Stage contd . --------- Previous belief : different antibodies responsible for each type of reaction. Antibodies named according to type of reaction they produce Antibody causing Agglutination Agglutinin & its corresponding Antigen Agglutinogen . Antibody causing precipitation Precipitin & its corresponding antigen Precipitinogen.

8. Secondary Stage contd . --------- The previous belief replaced by Zinsser’s unitaranian hypothesis (1920). According to this hypothesis Ag give rise to only one class of Ab, capable of producing all different reactions depending on the nature of the Ag & conditions of reaction. Both views fallacious. True that single Ab can cause precipitation & agglutination & other serological reactions, but an Ag can stimulate different classes of immunoglobulins which differ in their reaction capabilities,

9. Role of Immunoglobulin Classes in different Serological Reactions. Negative Strong Weak Lysis Negative Weak Strong Complement fixation Moderate Strong Weak Agglutination Variable Weak Strong Precipitation IgA IgM IgG Immunoglobulin Class Serological Reactions

10. Antigen – Antibody reactions Some Ag - Ab reactions initiate chain reactions in vivo. Lead to neutralisation, destruction of injurious Ag or to tissue damage. Tertiary Reactions -Humoral immunity against infectious diseases , Clinical allergy , other immunological diseases.

11. 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. Only surface Ag participate in reaction. Ag – Ab combination firm but reversible. Firmness of combination depends on - Affinity & Avidity

12. Affinity Strength of the reaction between a single antigenic determinant and a single Ab combining site. Intensity of attraction between Ag & Ab molecules. Function of closeness of fit between an epitope & Ag combining region of Ab. Affinity = ∑ attractive and repulsive forces

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

14. Affinity & Avidity

15. Measurement of Ag & Ab Measurement in terms of mass ( eg mg nitrogen) or commonly units or titre. Antibody titre of serum = Highest dilution of serum showing an observable reaction with antigen in the test. Two parameters of serological tests :- Sensitivity - Ability of test to detect even minute quantities of antigen or antibody. Specificity – Ability of the test to detect reactions between homologous antigens & with no other.

16. 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

17. Factors Affecting Measurement of Ag/Ab Reactions Affinity. Avidity. Ag : Ab ratio. Physical form of Ag.

18. Tests Based on Ag/Ab Reactions All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes. All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

19. Precipitation Tests Lattice Formation Equivalence – Lattice formation

20. 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. When a colloidal antigen combines with its antibody in presence of electrolytes ( NaCl) at a suitable temperature & pH, the Ag – Ab complex forms an insoluble precipitate which remains suspended instead of suspended ‘ Flocculation’.

21. Applications Qualitative & Quantitative test. Very sensitive in detecting antigens & as little as 1  g of protein. Less sensitive to detect antibody. Ring test - Ascoli’s thermoprecipitin 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 & toxoides.

22. 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. Each Ag – Ab reaction forms a line of ppt. & the number of different Ag in the mixture readily observed. Immunodiffusion indicates identity, cross reaction & nonidentity bet n different Ag. Performed in soft (1%) agar or agarose gel.

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

24. Radial Immunodiffusion (Mancini) 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

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

26. Immunoelectrophoresis

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

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

29. Laurell’s two dimensional electrophoresis Variant of Rocket electrophoresis Several Ag in mixture quantitated. Antigen mixture electrophoretically separated first. Secondly electrophoresis done perpendicular to that of first stage to get rocket like precipitation.

30. Agglutination Reactions Lattice Formation Equivalence – Lattice formation

31. Agglutination Reactions Particulate antigen combining with its antibody in presence of electrolyte at optimal temp. & pH, resulting in visible clumping of particles. Lattice formation. Zonal phenomenon. Incomplete or monovalent Ab do not cause agglutination, though combine with Ag. They act as blocking Ab inhibiting agglutination. More sensitive than precipitation for detection of Ab. Better with IgM than IgG.

32. Applications Slide Agglutination : - 1. Routine procedure to identify bacterial strains from clinical specimens e.g. Salmonella sp. 2. Blood grouping & cross matching. Y Y Y + ↔

33. 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 .

34. Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes + ↔ 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)

35. Coombs (Antiglobulin)Tests Indirect Coombs Test Detects anti-erythrocyte antibodies in serum Detection of Anti Rh Ab & Autoimmune hemolytic anemia. Y Y Y Y Y Patient’s Serum Target RBCs + ↔ Step 1 + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Antiglobulin ) Step 2

36. 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. 1. Latex Agglutination test : # For detection of Hepatitis B Ag. # ASO, CRP, # RA factor ( Rose Waller test) # HCG # N. meningitidis typing

37. Passive Agglutination Test 2. Hemagglutination Test : Erythrocytes sensitised with antigen used for detection of antibodies. In rheumatoid arthritis, an auto Ab ( RA factor) appears in the serum which acts as Ab to gammaglobulin. RA factor can agglutinate red cells coated with gammaglobulins. Ag used is sheep red blood cells sensitised with rabbit antisheep erythrocyte Ab (amboreceptor). = Principle of Rose Waller test for RA factor detection.

38. Passive Agglutination Test Based on the presence of protein A on the surface of some strains of Staph. Aureus ( Cowan I strain) . Specific IgG coated on Cowan I strains of Staph. Aureus . Fc portion of IgG binds to protein A while antigen combining Fab portion remains free. When the corresponding Ag is mixed with these coated cells , Fab terminal binds to Ag resulting in agglutination. = coagglutination. Used for detection of bacterial antigens in blood, urine, CSF. N.gonorrhoeae, Strepto. Pyogenes, H. infuenzae Ag can be detected.

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