Immunology (elective) MLIL-101

1. Immunology (elective)MLIL-101 Prepared by: Dr. Mohamed S. Abdel-Latif

2. Ag-Ab reactionsTests for Ag-Ab reactions

3. Learning Outcome: At this time you should know the following: 1. To describe the nature of Ag-Ab reactions 2. To compare and contrast antibody affinity and avidity. 3. To delineate the basis for antibody specificity and cross reactivity. 4. To discuss the principles of commonly used tests for antigen/antibody Reactions.

4. http://www.med.sc.edu:85/chime2/lyso-abfr.htm Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 Nature of Ag/Ab Reactions • Lock and Key Concept • Non-covalent Bonds • Hydrogen bonds • Electrostatic bonds • Van der Waal forces • Hydrophobic bonds • Multiple Bonds • Reversible

5. 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

6. [Ag-Ab] Keq = [Ag] x [Ab] Calculation of Affinity Ag + Ab  Ag-Ab Applying the Law of Mass Action:

7. 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

8. 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.

9. 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

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

11. 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

12. Agglutination Tests Lattice Formation

13. Qualitative agglutination test • Ag or Ab Y +  Y Y Agglutination/Hemagglutination • Definition - tests that have as their endpoint the agglutination of a particulate antigen • Agglutinin/hemagglutinin

14. 1/1024 1/256 1/512 1/128 1/16 1/64 1/32 Pos. 1/8 Neg. 1/4 1/2 Titer Patient 64 1 8 2 512 3 <2 4 32 5 128 6 32 7 4 8 Agglutination/Hemagglutination • Quantitative agglutination test • Titer • Prozone

15. 1/256 1/512 1/128 1/16 1/64 1/32 1/8 1/4 1/2 Agglutination/Hemagglutination • Definition • Qualitative test • Quantitative test • Applications • Blood typing • Bacterial infections • Fourfold rise in titer • Practical considerations • Easy • Semi-quantitative

16. Y Y +  Y Passive Agglutination/Hemagglutination • Definition - agglutination test done with a soluble antigen coated onto a particle • Applications • Measurement of antibodies to soluble antigens

17. 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

18. 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

19. Coombs (Antiglobulin)Tests • Applications • Detection of anti-Rh Ab • Autoimmune hemolytic anemia

20. Prior to Test Y Y +  Y Y Test Y + +  Y Patient’s sample Agglutination/Hemagglutination Inhibition • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

21. Agglutination/Hemagglutination Inhibition • Definition • Applications • Measurement of soluble Ag • Practical considerations • Same as agglutination test

22. Precipitation Tests Lattice Formation

23. 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 • Ig levels

24. - + 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

25. Immunoelectrophoresis • Method • Interpretation • Qualitative • Relative concentration

26. - + 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

27. Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required

28. Prior to Test Y Y +  Labeled Ag Test Y Y + + +  Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method • Determine amount of Ab needed to bind to a known amount of labeled Ag • Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

29. Solid Phase Solid Phase Test Y Y + + +  Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method cont. • Determine amount of labeled Ag bound to Ab • NH4SO4 •  anti-Ig • Immobilize the Ab • Concentration determined from a standard curve using known amounts of unlabeled Ag • Quantitative • Most sensitive test

30. Labeled Anti-Ig Ab in Patient’s sample Y Y Ag Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • 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

31. Labeled Ab Ag in Patient’s sample Y Ag Y Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • 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. Tests for Cell Associated Antigens Lattice formation not required

33. Fluorochrome Labeled Ab Y Ag Tissue Section Immunofluorescence • Direct • Ab to tissue Ag is labeled with fluorochrome

34. Fluorochrome Labeled Anti-Ig Unlabeled Ab Y Y Ag Tissue Section Immunofluorescence • Indirect • Ab to tissue Ag is unlabeled • Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. • Qualitative to Semi-Quantitative

35. Flow Tip FL Detector Light Scatter Detector Laser Immunofluorescence • Flow Cytometry • Cells in suspension are labeld with fluorescent tag • Direct or Indirect Fluorescence • Cells analyzed on a flow cytometer

36. Two Parameter Histogram Green Fluorescence Intensity Red Fluorescence Intensity Immunofluorescence • Flow Cytometry cont. • Data displayed One Parameter Histogram Unstained cells FITC-labeled cells Number of Cells Green Fluorescence Intensity

37. Assays Based on Complement Lattice formation not required

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

39. Assignment: As a part of the semester activity, Group of students are selected every week to prepare a short seminar about his/her point of interest in one of the lecture topics. That to be discussed and evaluated during the next lecture.