1. Imunologia Prática 30 % da nota final É necessário aprovação para ir a exame teórico 2 trabalhos (Entregar final de Novembro): -Trabalho escrito (Grupos de 3 alunos max) -Apresentação oral (Método laboratorial) Teste prático (Dezembro)

2. Trabalhos teóricos Cancro Autoimunidade Transplantação Hipersensibilidade Imunodeficiências Primárias Vacinação Defesas às infecções Imunodeficiências Secundárias Diversidade de Anticorpos Anticorpos e Alergias Vacinas do cancro Terapia com anticorpos

3. Apresentações de métodos laboratoriais Reacções de precipitação Reacções de aglutinação Reacções do complemento ELISAs Radioimunoensaios Imunofluorescência Separação de populações celulares Citometria de fluxo Estimulação celular e citotoxicidade Estudos de funcionalidade celular

4. Ag-Ab reactions Tests for Ag-Ab reactions

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

6. Affinity =  attractive and repulsive forces Ab Ag High Affinity Ab Ag Low Affinity Affinity Strength of the reaction between a single antigenic determinant and a single Ab combining site

7. Calculation of Affinity Ag + Ab  Ag-Ab K eq = [Ag-Ab] [Ag] x [Ab] Applying the Law of Mass Action:

8. Avidity The overall strength of binding between an Ag with many determinants and multivalent Abs K eq = 10 4 Affinity 10 6 Avidity 10

9. 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.

10. 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 Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions

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

12. 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

13. Precipitation Tests Lattice Formation

14. Precipitation Reactions Involve soluble antigens with antibodies Figure 18.3

15. Radial Immunodiffusion (Mancini) Interpretation –Diameter of ring is proportional to the concentration Quantitative –Ig levels Method – Ab in gel – Ag in a well Ag Concentration Diameter 2 Ag Ab in gel

16. Factors Affecting precipitation Reactions pH variation Proportion Ag and Ab Ionic strength Temperature Ab excess Ag excess Equivalence – Lattice formation

17. Curve of precipitation

19. I mmunoelectrophoresis Method –Ags are separated by electrophoresis Interpretation – Precipitin arc represent individual antigens Ag - + Ab Ag Ab –Ab is placed in trough cut in the agar

20. Agglutination Tests Lattice Formation

21. Agglutination Reactions Involve particulate antigens and antibodies Antigens may be: On a cell (direct agglutination) Attached to latex spheres (indirect or passive agglutination) Figure 18.4

22. Hemagglutination Hemagglutination involves agglutination of RBCs. Viral hemagglutination inhibition tests for antibodies by the antibodies' ability to prevent viruses from agglutinating RBCs. Figure 18.7

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

24. Antibody Titer Is the concentration of antibodies against a particular antigen Figure 18.5

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

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

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

28. Neutralization Reactions Eliminate the harmful effect of a virus or exotoxin Figure 18.8b

29. Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test – Detects antibodies on erythrocytes +  Patient’s RBCs Coombs Reagent (Antiglobulin)

30. Coombs (Antiglobulin)Tests Indirect Coombs Test –Detects anti-erythrocyte antibodies in serum Patient’s Serum Target RBCs +  Step 1 +  Coombs Reagent (Antiglobulin) Step 2

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

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

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

34. I mmunoelectrophoresis Method Interpretation Qualitative –Relative concentration

35. Countercurrent electrophoresis Method –Ag and Ab migrate toward each other by electrophoresis –Used only when Ag and Ab have opposite charges Qualitative –Rapid Ag Ab - +

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

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

38. Competitive RIA/ELISA for Ag Method cont. –Determine amount of labeled Ag bound to Ab  NH 4 SO 4  anti-Ig Immobilize the Ab Quantitative – Most sensitive test +  Test + Patient’s sample Labeled Ag + –Concentration determined from a standard curve using known amounts of unlabeled Ag Solid Phase Solid Phase

39. 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 Solid Phase Ag Immobilized Ab in Patient’s sample Labeled Anti-Ig

40. 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 Solid Phase Ag Immobilized Ag in Patient’s sample Labeled Ab

41. Tests for Cell Associated Antigens Lattice formation not required

42. Immunofluorescence Direct – Ab to tissue Ag is labeled with fluorochrome Ag Fluorochrome Labeled Ab Tissue Section

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

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

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

46. Assays Based on Complement Lattice formation not required

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