Antigen-Antibody Reaction Serial Dilution Technique

1. Laboratory2 Antigen-Antibody Reaction Serial Dilution Technique Assist. Prof. Suwin Wongwajana, M.D., M.Sc. Dept.of Microbiology Fac.of Medicine, KKU For 2nd Year Pharmaceutical Science Student

2. Serial dilution 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml Color water 1 2 3 4 5 6 D.W.0.5 ml Dilution 1:2 1:4 1:8 1:16 1:32 control

3. Calculation of Dilution N1V1 = N2V2 N1 = Dilution (concentration) of the 1st tube V1 =Volume that pipette from the 1st tube N2 = Dilution (concentration) of the 2nd tube V2 = Total volume of the 2nd tube (V2 = V1 + initial volume of the 2nd tube)

4. N2 = N1 x V1 V2 N2 = 1/4 x 1/2 =1/8 1 2 3 V1 0.5 ml V2 = 0.5+0.5 =1 D.W. 0.5 ml 1:4 ? N1 N2

5. Experiment 1. Tube precipitation test Demonstration - On the bench near the windows Answer:Ab titer= ?

6. Serum 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 1 2 3 4 5 6 Diluent0.5 ml Dilution 1:2 1:4 1:8 1:16 1:32 control Antigen 1 1 1 1 1 1 drop

7. Dilution 1:2 1:4 1:8 1:16 1:32 control Ab Titer = 1:8 or 8 precipitate

8. Experiment 2.Immunodiffusion 1 Ag1 6 Ag3 Ag2 2 7 Ab 5 3 Ag3 Ag2 4 Ag1 -Practice applying the Ag and Ab into the wells -Observe the precipitin band (from your Gr. tutor)

11. Experiment 3. Blood grouping - Perform the finger stick on your friend’s finger. - Use sterile technique – to prevent the infection - Wipe the finger with 70% Alcohol, stick the finger withlancet, drop the blood onto the glass slide. - Discard the lancet, cotton, etc. to the appropriate receptacles.

12. Anti-B Anti-A Agglutination Blood drop from the finger A What is the blood gr. ?

13. Agglutination NoAgglutination

14. Demonstration 1. Single radial immunodiffusion (SRID) 2. Immunoelectrophoresis (IEP) 3. Immunodiffusion (ID) 4. Tube precipitation 5. Enzyme-linked immunosorbent assay (ELISA)

17. Enzyme-linked immunosorbent assay (ELISA) By... Assistance Professor Suwin Wongwajana,MD.

18. Microtiter Plate (ELISA Plate) 96 wells plate (8 rows, 12 columns)

20. Indirect ELISA (For detection of antibody) 1. Coat plate with Antigen

21. 2. Wash

23. (Y) with specific Ab No specific Ab Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 3. Add specimens (with Ab, or No Ab)

24. (Y) with specific Ab Y 4. Wash No specific Ab

25. Y Y Y Y Y Y E E Y E E E E E E E E E E E E 4. Add conjugate (anti-human IgG=Enzyme) Y

26. Y Y E E E E E E Y 5. Wash (Y) with specific Ab No specific Ab

27. Y = Ag Human-IgG immunized Y Y Y Rabbit anti-human IgG

28. Y Y Y Y E E E E E E E E E E Y 6. Add substrate (Chromogenic) E E Y

29. Y Y E E E E E E Y

30. Absorbance (O.D.)Measurement Intensity of the color developed, Correlated with the concentration of the unknown Ab in the specimen

32. ELISA READER

33. Immunofluorescence(IF) Cell Tissue

34. CD4 CD3 Direct Immunofluorescence (For detection of antigen)

35. CD4 F* F* F* F* F* F* F* F* F* F* CD3 F* F* F* F* F* Anti-CD4=Fluorescein dye + F*

36. CD4+ve Fluorescent Microscope

37. CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD8 CD8 CD8 CD8 CD8 CD8 CD8 CD8 CD8 CD8 CD8 CD8

38. F* F* F* F* F* F* F* F* F* Conjugate Specific Ab Indirect Immunofluorescence (For detection of antigen)

39. F* F* F* F* F* F* F* F* F* Mouse anti-rabbit = F* Rabbit anti-CA Ag CA Ag

42. What should you do, today 1. Practiceserial dilution by sterile technique. 2. Perform blood grouping. 3. Practice applying the well inslide immunodiffusion. 4. Observe all the demonstration points. 5. Conference today start at3.30 pm . - Quizand discussion The time given for the lab hour until3.30 pm