Antigen and antibody reactions

1. Antigen and antibody reactions • Antigen reacts with a specific antibody in an observable manner • Ag-Ab reaction in vitro - serology

2. 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

3. 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

4. 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.

5. 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

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

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

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

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. 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

11. 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

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

13. 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

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

15. Ring test

16. VDRL

17. Immunodiffusion test • Precipitation in 1% agar gel • Single diffusion in one dimension – Oudin • Double diffusion in one dimension – Oklaey-Fulthrope • Single diffusion in two dimensions - Radial Immunodiffusion

18. Ab in gel Ag Ag Ag Ag Diameter2 Ag Concentration Radial Immunodiffusion (Mancini) • Method • Ab in gel • Ag in a well • Interpretation • Diameter of ring is proportional to the concentration • Quantitative • To estimate Immunoglobulin levels

19. In this example, Anti-dog IgG is Mixed in agar so only what is Placed in wells (Ag) diffuses out

20. Double diffusion in two dimensions – Ouchterlony – Elek’s test

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

22. 22

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 – Rapid • Hepatitis B Ag, Cryptococcus antigen in CSF

24. Rocket electrophoresis

25. Applications of precipitation reactions • Identification of microbial antigens - Anthrax bacilli, Streptococcus, Cryptococcus • Blood grouping • VDRL • Forensic applications • Estimation & Identification of Immunoglobulins

26. NEUTRALIZATION TESTS • Detection of bacterial toxins by using antibodies • Toxigenicity tests – detection of toxins produced by C.diptheriae, Cl.welchi etc • Antistreptolysin ‘O’ test • Viral neutralisation test

27. Neutralization tests • To measure virus neutralizing antibodie in patient’s serum • Serum( in dilutions) + Viral suspension inoculated into test cell culture • Viral suspension inoculated into control cell culture • Failure to develop CPE if Antibodies present • CPE in control cell culture • Four fold rise in Ab titer is required to establish diagnosis

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

29. RadioImmunoassay • The radioactivity of the specific labeled antibody or antigen is used to quantify the antigen or antibody in patient’s serum

30. Radioimmunoassays (RIAs) utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.

31. Labeled Ab Ag in Patient’s sample Y Ag Y Immobilized Solid Phase Solid Phase RIA for antigen/direct • Ag detection • Immobilize Ab • Incubate with sample • Add labeled antibody • Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative

32. Labeled Anti-Ig Ab in Patient’s sample Y Y Ag Immobilized Solid Phase Solid Phase RIA for antibody/indirect • Ab detection • Immobilize Ag • Incubate with sample • Add labeled anti-Ig • Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative

33. ELISA technique Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The technique is divided into 1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA

34. Competitive ELISA The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal).

36. Sandwich ELISA The ELISA plate is coated with Antibody to detect specific antigen

37. Indirect ELISA

38. Cassette ELISA Few sample can be tetsed Specific antigens are coated on nitroclellulose membrane These strips are incubated with patients serum positive samples develop a colred spot or band

39. Enzyme Immunoassay • Enzymes used include: • Horseradish peroxidase • Glucose-6-phosphate dehydrogenase • Alkaline phosphatase • Β-D-galactosidase

40. Immunochromatography

41. Chemiluminescent Immunoassays • The process of chemiluminescence occurs when energy in the form of light is released from matter during a chemical reaction. 

42. Chemiluminescent Immunoassays • Can be used for heterogeneous or homogeneous assays. • Can attach label to antigen or antibody. • Heterogeneous assays use competitive and sandwich assay. • Competitive assays used to measure smaller analytes. • Sandwich assays are used to measure larger analytes.

43. Chemiluminescent Immunoassay • Many applications. • Can measure antigen or antibody. • Add chemiluminescently tagged analyte. • Measure light which is emitted which is directly related to concentration although competitive binding assays are available.

44. Immunoblotting

45. Western Blot HIV-1 Western Blot • Lane1: Positive Control • Lane 2: Negative Control • Sample A: Negative • Sample B: Indeterminate • Sample C: Positive