1. PRESS F1 FOR GUIDEANCE
Immunology
3rd Practical
MFSH 2003

2. Contents Antigen-Antibody reaction Station1:Precipitation reaction 4
Station2: Controlled reaction
Station3:Serum electrophoresis
Station4:Single radial immunodiffusion
Station5:Counter current immunodiffusion
Station6:Tube agglutination
Station7:Florescent antibody test
Station8:Heamagglutination test
Station9:Heamagglutination inhibition test
Station10:Complement fixation test
Station11:Anti-Streptolysin test
Station12:Important notes 4 5 6 7 8 9 10 13 15 18 21 22
Be careful, this was the practical that we couldn’t answer its questions well in the exam. That is because we got surprised by the questions. But don’t worry, the way will be illustrated in this version. You may not find all the information needed here, but through the guide lines present here you can start your search for the information in your hand-outs.
The most important point you should know is that, the immunity questions are depending upon your bacterial knowledge on the first place. As you will see.

3. Antigen-Antibody
reaction

4. Positive negative Magnifying lens Antigen- clear Antibody Turbid
reaction
clear
Turbid
Precipitation
Station 1

5. Antigen
The dark line indicates antigen-antibody reaction between the circle containing the antigen and the circle with patient serum (containing the antibodies)
Station 2

6. Station 3

7. Station 4

8. Counter-current Immunodiffusion
The line indicates antigen-antibody reaction between the circle containing the antigen and the circle containing the antibodies.
Station 5

9. 1 2 3 4 5 6 7 8
Positive control
Negative control
With Antibodies
No Antibodies
1/10
1/20
1/40
1/80
1/160
1/320
1/640
1/1280
Dilation
Tube Agglutination
The patient’s serum is diluted in a series of tubes (see above), and then a drop of known bacterial (antigen) suspension is added to each tube. If the concentration of antibodies in a tube is high enough, agglutination will occur and the color will disappear, if not the blue color persists. The last dilution at which agglutination occurs is called Titer. In this example, Titer is 1/40 (as the last clear tube is number 3). Note that each tube is diluted twice as much as the tube before it.
Tube 3
Tube 4
Station 6

10. Station 7

11. Page 1

12. Page 2

13. Reaction  Agglutination (positive) No reaction  red dot (negative)
Dilution
1/10
1/40
1/160
1/640
1/20
1/80
1/320
1/1280
*Concept: Serum (containing antibodies) is added to antigen+RBCs. If there is an antibody-antigen reaction, haemaglutination occurs. If there is no reaction, the RBCs will fall and form a small dot in the center.
Reaction  Agglutination (positive) No reaction  red dot (negative)
The antigen+RBCs are inserted into wells. Then, diluted serum (see above) is added to wells. The (1st patient serum) is taken from a patient that we suspect has the antigen (disease). We take another sample (2nd Serum) from the patient after 14 days, if the titer is more than 4 folds, then he has acute infection (disease). In this example, 2nd titer=1/ st titer=1/ /80=16 folds  the patient has the antigen (diseased).
Station 8

14. Haemoglutination Test
1/ / / / / / /640 1/ /2560….etc
Titer=1/80
Titer=1/1280
Reaction (Haemoglutination)
No Reaction

15. Dilution
1/10
1/40
1/160
1/640
1/20
1/80
1/320
1/1280
*Concept: Some antigens will react with RBCs without the presence of antibodies. Therefore the serum (antibodies) are added with the antigen to the wells, after this the RBCs are added. Therefore, if there is an antibody-antigen reaction, RBCs will fall and form a dot in the center. However if there is no reaction, the antigen will act on the RBCs and haemagglutination will occur. Note that calculations are made like the previous slide.
Station 9

17. Haemoglutination Inhibition Test

18. *Calculations are the same.
Dilution
1/10
1/40
1/160
1/640
1/20
1/80
1/320
1/1280
No Lysis (Positive)
Lysis (Negative)
*Concept: If a specific antibody meets a specific antigen they form a complex (positive test). These complexes make a further complex with “complement”. Free complement (not in complexes) has the ability to lyse RBCs. So:
Antigen-antibody reaction + complement + RBC’s  no lysis of RBCs. (positive)
Antigen (no reaction with antibody) + complement + RBC’s  lysis of RBCs. (negative)
*Calculations are the same.
Station 10

20. Complement Fixation Test (C.F.T)

21. - Answer: Serum 3 (Highest titer)
Dilution
1/100
No antibodies
1/400
*Concept: Streptolysin is a bacterial product (streptococci) that has the ability to lyse RBCs.
We mix the serum with the bacteria (antigen), and then add RBCs. If lysis occurs it means there was no antibody-antigen reaction (negative). However, If lysis doesn’t occur it means there was an antibody-antigen reaction (positive).
- Question: From the 3 serum samples above, which has the most antibodies?
- Answer: Serum 3 (Highest titer)
Station 11

22. Important notes
1- Be sure that you know the names of all the tests and how to differentiate between them
2- You should which kind of test goes with which kind of bacteria.
E.g. Syphilis  haemagglutination test
3- Some titers are diagnostic, you should know them.
E.g. a titer of 1:100 for liptospira is diagnostic.
4- Be careful, the bacteria you studied in the practical classes are not the only ones you should know. In other words, you should know all the bacteria related to the immunity tests.
Station 12

23. my dear friend who helped
With many thanks to
my dear friend who helped
me in this
A.A.Yami

24. اللهم انك سلطت علينا عدوا عليما بعيوبنا
يرانا هو وقبيله من حيث لا نراهم
اللهم آيسه منا كما آيستـه من رحمتك
وقنطه منا كما قنطـته من عـفوك
وباعــد بيننا وبينه
كما باعـدت بينه وبين رحمتك وجنتك
دعواتكم يا شباب

25. I hope it was useful.
A word can change the outcome of many things. So, if you either liked or didn’t like this project, why keep your thoughts to yourself ?
Send us your comments and ideas to It will mean too much to us, “even if it was just one word”
THANX
M. F. Shaheen 2003