MFSH MFSH 20032003

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Antigen-Antibody reactionAntigen-Antibody reaction

Station1:Precipitation reaction

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

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Be careful, this was the practical that we couldn’t answer its questions well in the exam. That 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 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 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.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 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.upon your bacterial knowledge on the first place. As you will see.

Positive negative

clear

Magnifying lens

TurbidPrecipitation

Antigen-

Antibody

reaction

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

The line indicates antigen-antibody reaction between the circle containing the antigen and the circle containing the antibodies.

Counter-current Immunodiffusion

Station 5

Dila

tion

1/10 1/20 1/40 1/80 1/160 1/320 1/640 1/1280

Tube AgglutinationThe 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.

1 2 3 4 5 6 7 8

Tube 3 Tube 4

Positive control

Negative control

No Antibodies

With Antibodies

Station 6

Station 7

Page 1

Page 2

*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).

Dilution 1/10

1/20

1/40

1/80

1/640

1/1280

1/160

1/320

Station 8

Haemoglutination Test

Reaction (Haemoglutination)

No Reaction

Titer=1/1280Titer=1/1280

1/10 1/20 1/40 1/80 1/160 1/320 1/640 1/1280 1/2560….etc

Titer=1/80Titer=1/80

*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.

Dilution 1/10

1/20

1/40

1/80

1/640

1/1280

1/160

1/320

Station 9

Haemoglutination Inhibition Test

*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.

Dilution 1/10

1/20

1/40

1/80

1/640

1/1280

1/160

1/320

No Lysis (Positive) Lysis

(Negative)

Station 10

Complement Fixation Test (C.F.T)

*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)

Dilution

Station 11

1/400

1/100

No antibodies

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

يا يا دعواتكم دعواتكم

شبابشباب

A word can change the outcome of A word can change the outcome of

many things. So, if you either liked or many things. So, if you either liked or

didn’t like this project, why keep your didn’t like this project, why keep your

thoughts to yourself ?thoughts to yourself ?

Send us your comments and ideas Send us your comments and ideas

to to COORDINATOR1422@YAHOO.COM, It will

mean too much to us, “even if it was just “even if it was just

one word”one word”

THANXTHANX

M. F. ShaheenM. F. Shaheen 2003 2003