بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

3. Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com Antigen Antibody Reaction

4. TEACHING OBJECTIVES: 1. To discuss the factors affecting Ag-Ab reactions 2. To define Affinity & Avidity 3. To know different methods of Ag-Ab reactions 4. To describe the basics and types of precipitation reactions 6. To describe the basics and types of aggulination reactions 7. To describe the basics and types of immuno-flourescent techniques 8- To describe the basics and types of ELISA Clinical Immunology & Serology Practice (Code: MLIS 201)

5. Serology The science that deals with the properties and reactions of sera, especially blood serum. Usually refers to the diagnostic identification of Ag or Ab in the serum. Serological assays depends on Antigen-antibody interactions

6. Nature of antigen-antibody interaction i.e epitope-paratope binding forces

7. POOR FIT high repulsion high attraction low attraction low repulsion Binding of the epitopewith the paratope (antigen binding site) GOOD FIT Antigen binding site Antigen binding site epitope epitope antigen determinant

8. Factors Affecting binding of Ag/Ab Affinity Avidity Law of Mass Action Physical form of Ag Ag:Ab ratio

9. Antibody affinity is the strength of the binding between a single antigenic determinant and a single combining site on the antibody. • The higher the affinity of the antibody for the antigen, the more stable will be the interaction. 1-Affinity • 2-Avidity • Avidity is a measure of the overall strength of binding of multivalent Ag (i.e. antigen with many antigenic determinants) and multivalent antibodies. • Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.

10. Y Y Y Y Y Y Y 106 104 1010 Keq = Avidity Avidity Affinity 2-Avidity (cont.) The overall strength of binding between a multivalent Antigen and multivalent Antibodies

11. The ability of an individual Ab binding site to react with only one antigenic determinant. The ability of a population of antibody molecules to react with only one antigen. Specificity Cross Reactivity • The ability of an individual Abbinding site to react with more than one antigenic determinant. • The ability of a population of Abmolecules to react with more than one Ag

12. Anti-A Ab Anti-A Ab Anti-A Ab Ag B Ag C Shared epitope Similar epitope Ag A Cross reactions Specific Reaction

13. All tests based on Ag/Ab reactions can be used to detect either Ag or Ab Cannot be visualized, thus Detection of antigen/antibody reactions is difficult Various laboratory methods have been developed to make this reaction visible eg. Precipitation, agglutination, labeled reagents (ELISA, RIA, IF), …etc. Antigen antibody tests

14. Precipitation reactions (Ag is soluble) precipitin. • Agglutination reactions (Ag is particles) clumping. • Complement fixation reactions. • Labelling methods: a- Immuno-fluorescence reactions. b- ELISA. c- Flow cytometry Methods for detection of Ag-Ab Reactions

15. 1- Precipitation reactions Methods for detection of Ag-Ab Reactions

16. Precipitation Serological Tests

17. Precipitation reactions Involve soluble antigens with antibodies cross-linked and form lattice that Eventually develops into a visible precipitate. • In immunodiffusion, antigens and antibodies diffuse through a gel until they reach the zone of equivalence. • In immunoelectrophoresis, diffusion is combined with electrophoresis. Precipitation techniques

18. Polyclonal Abcan form lattices, or large aggregates with polyvalent Ag. • Monoclonal antibodycan link only two molecules (epitopes) of antigen andno precipitateis formed. Precipitation reactions ( Lattices or large aggregates ) ( no precipitate is formed)

19. The plot of the amount of antibody precipitated versus increasing antigen concentrations (at constant total Ab) reveals 3 zones: Precipitation curve • Zone of antibody excess: • Precipitation is inhibited. • Ab not bound to Ag can be detected in the supernatant; • 2. Zone equivalence: • Maximal precipitation in which antibody and antigen form large insoluble complexes. • No Ab or Ag can be detected in the supernatant • 3. Zone of antigen excess: • Precipitation is inhibited. • Ag not bound to Ab can be detected in the supernatant

20. Precipitation curve

21. 1. Tube precipitation test 2. Gel diffusion • Single radial • Double 3. Immuno-electrophoresis, immuno-fixation Precipitation techniques

22. Precipitation techniques • 1- Tube precipitation test • Ag & Ab molecules diffuse until they reach equivalence zone Visible Precipitation (In gel) PPT in Gel PPT in Fluid

23. Precipitation techniques: 2. Gel Immunodiffusion Techniques • Radial Immunodiffusion • A single diffusion technique where Ab is put into a gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel • Ouchterlony Double Diffusion • Both Ab and Ag diffuse from wells into a gel medium

24. Radial Immunodiffusion • A quantitative technique based upon the reaction between an Ag placed in a well diffuses into an agar containing the Ab. • During diffusion period the Ag & Ab • continue to react until the zone of • equivalence is reached with • formation of a well-defined ring of • precipitation around the Ag well which • isproportional to the Ag concentration.

25. Ouchterlony Double Diffusion • Both Ab and Ag diffuse independently from wells into a gel medium

26. Electrophoresis Techniques • Electrophoresis separates molecules according to differences in their electrical charge. • Rocket Immunoelectrophoresis • Countercurrent Immunoelectrophoresis • Immunoelectrophoresis • Immunofixation Electrophoresis

27. 3. Immuno-electrophoresis Ag molecultes are separated according to differences in their electrical charge by electrophoresis Ab is placed in a trough cut in the agar Ab diffuse towards Agsprecipitin arcs - + Ag Ag Ab Ag Ab • Interpretation- Precipitin arc represent individual antigens

29. Measurement of Precipitation • Turbidimetry • Passing light through a cloudy solution of Ag-Ab complexes. • Net decrease in light intensity correlates to the concentration of the Ag • Nephelometry • Measuring light scattered at a particular angle after being passed through a solution of Ag-Ab complexes. • Amount of light scattered correlates to the concentration of the Ag

30. 1- Precipitation Reactions 2- Agglutination Reactions Methods for detection of Ag-Ab Reactions

31. Agglutination Serological Tests

32. Agglutination • Principle • Particulate antigen + antibody –> clumping • The clumps will be called agglutinates • Lattice formation (antigen binds with Fab sites of 2 antibodies forming bridges between antigens) • Agglutination Curve follow the same roles as in precipitation curve • evaluation: • Qualitative (y/n), • Semi-quantitative (serum titration)

34. Agglutination Tests Types of Agglutination Reactions • Direct agglutination Tests • Indirect agglutination Tests • Agglutination Inhibition Tests

35. (I) Direct Agglutination Tests • Used to determine antibody titer against particulate antigens (Bacteria, Fungi or RBCs) • Particulate antigen reacts directly with antibodies. • Applications • Blood group typing • Direct Coomb’s Test • The diagnosis of certain diseases (Bacterial infection • Eg. Syphilis) • The identification of some viruses.

36. +  Y Y Y a) Blood Grouping Y Anti-RBC RBC Agglutinate

38. b) Direct Coombs (Antiglobulin)Test • detects the presence of antibodies on red blood cell • Simply add a second antibody ( Antiglobulinantibody, Coomb’ Reagent)directed against the immunoglobulin coating the red cells. • This anti-immunoglobulin antibodies cross link the red blood cells and result in agglutination.

39. Ag or Ab are not part of particulate matter but are attached to it (ANTIBODIES OR ANTIGENS ARE ATTACHED TO PLASTIC BEADS) EASY TO DO Eg. Latex test for RF & CRP C) Latex Test positive negative

40. (II) indirect agglutination (Coomb’s)test To detect anti- RBC’s antibodies in a serum sample. • This test is done by incubating RBC’s with the serum sample • washing out any unbound antibodies • then • adding a second anti-immunoglobulin reagent (Coomb’s Reagent) to cross link the cells resulting in agglutination.

41. Hemagglutination-Inhibition • Some viruses (eg. Measles virus) agglutinate RBCs in vitro. • Anti-virus Ab will bind the virus and inhibit hemagglutination.

42. Applications of agglutination tests in Serology • Determination of blood group antigens. • Diagnosis of cases with Rhincombatability (erythoblastosisfetalis) • Diagnostics of autoimmune hemolytic diseases • Latex test for detection of Ags or Abs (eg. RF, CRP) • To assess bacterial infections (e.g. Typhoid Fever, Syphilis)

43. Agglutination Insoluble or particulate Ag or Ab Ag must have at least two determinants Agxs results in postzone reaction Abxs results inprozonereactions Reaction time: minutes to hours Test results: qualitative or semi-quantitative Precipitation Soluble Ag & Ab Ag must have at least two determinants Agxs results in postzone reaction Abxs results inprozonereactions Reaction time: hours to days Test results: qualitative, semi-quantitative or quantitative Agglutination vs. Precipitation

44. 1- Precipitation Reactions 2- Agglutination Reactions Methods for detection of Ag-Ab Reactions 3- Labelling methods: a- Immuno-fluorescence b- ELISA. c- Flow cytometry

45. Immunofluorescence: Methods • Fix specimen on slide • Add Flourescein labeled antibody specific for the desired antigen • Look for fluorescence Direct Indirect • Fix specimen on slide • Add antibody specific for the desired antigen • Add second Flourescein labeled antibody • Look for fluorescence

49. Immunofluorescence:Interpretation Fluorescence = patient has the antigen

50. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA