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

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

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

4 1:4 ? ml N1N1 N2N2 V1V1 V2 = =1 N2 = 1/4 x 1/2 =1/8 1 D.W. 0.5 ml N2 = N1 x V1 V2

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

Dilu ent0.5 ml Serum 0.5 ml 0.5 ml Antigen drop Dilution 1:2 1:4 1:8 1:16 1:32 control 0.5 ml

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

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

9

11 - 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 with lancet, drop the blood onto the glass slide. - Discard the lancet, cotton, etc. to the appropriate receptacles. Experiment 3. Blood grouping Experiment 3. Blood grouping

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

13 Agglutinatio n No Agglutination

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

15

16

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

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

19

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

21 2. Wash

22

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

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

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

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

27 Human- IgG Y immuniz ed Rabbit anti-human IgG Y = Ag Y Y

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

29 Y Y E E Y E E E E

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

31

32 ELISA READER

33 Cell Tissue Immunofluorescence(IF)

34 Direct Immunofluorescence (For detection of antigen) CD4 CD3

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

36 CD4+ve Fluorescent Microscope

37 CD 4 CD 8 CD 4 CD 8 CD 4 CD 8 CD 4 CD 8 CD 4

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

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

40

41

42 What should you do, today 2. Perform blood grouping. 3. Practice applying the well in slide immunodiffusion. 4. Observe all the demonstration points. 1. Practice serial dilution by sterile technique. 5. Conference today start at 3.30 pm. - Quiz and discussion The time given for the lab hour until 3.30 pm