PRESS F1 FOR GUIDEANCE Immunology 3rd Practical MFSH 2003

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.

Antigen-Antibody reaction

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

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

Station 3

Station 4

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

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

Station 7

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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=1/1280. 1st titer=1/80. 1280/80=16 folds  the patient has the antigen (diseased). Station 8

Haemoglutination Test 1/10 1/20 1/40 1/80 1/160 1/320 1/640 1/1280 1/2560….etc Titer=1/80 Titer=1/1280 Reaction (Haemoglutination) No Reaction

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

Haemoglutination Inhibition Test

*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

Complement Fixation Test (C.F.T)

- 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

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

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

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

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 COORDINATOR1422@YAHOO.COM, It will mean too much to us, “even if it was just one word” THANX M. F. Shaheen 2003