Immunology

Lab 10-Immunology د.زينب عادل چـابك
م.جوان احمد علي الهماوندي
Serology: is the science dealing with the study of antigen–antibody reaction.
Uses:
1- Diagnosis of infections.
2- Evaluation of the immunological status.
Antigen – Antibody Reaction:
Reaction of antigens and antibodies are highly specific. An antigen will react only with antibodies elicited by itself or by a closely related antigen.
Because of the great specificity, reactions between antigens and antibodies are suitable for identifying one by using the other. This is the basis of serologic reactions.
Antigens + Antibodies = product

Types of diagnostic tests:
1. Agglutination test: This reaction can be done in a small cup or tube or with a drop on a slide.
One very commonly used agglutination test is the test that determines a persons’ ABO blood group and the bacterial agglutination tests including Widal and Brucella test (Rose Bengal Test).
Widal test:
Is a presumptive serological test for enteric fever. In case of salmonella infections, it is a demonstration of agglutinating antibodies against antigen O-somatic and H-flagellar in the blood. The test is not
a very specific test, since patients are often exposed to other bacteria (ex. Salmonella enteritidis, S. typhimurium and some types of E. coli).

Procedure:
Patient’s serum is diluted with normal saline by tenth-double dilution by mixing and transferring from 1:10 to 1:640 (serum:normal saline) in three-four rows. Somatic S. typhi O antigen is added to the first row of tubes. For all the remaining rows, different flagellar H antigens are added. Each tube must contain (0.5 ml) of diluted serum. A test tube with only saline is kept in each row as a negative control. All the tubes (including control) in a row are mixed with (0.5 ml) of antigen suspension.
The first row is treated with S. typhi O antigen, the second row with S. typhi H antigen, the third row with S. paratyphi AH antigen and the fourth row with S. paratyphi BH antigen. Since infections by S. paratyphi B are rare, this antigen is usually omitted in the test.
After all the tubes have been treated with specific antigen suspensions, the Widal rack of tubes is placed in a thermostatically controlled water bath maintained at 37°C for overnight incubation. Another approach is to incubate the tubes at 50-55 °C.
Reading the results:
The control tubes must be examined first, where they should give no agglutination. The agglutination of O antigen appears as a “math” or “carpet” at the bottom. Agglutination of H antigens appears loose, wooly or cottony. The highest dilution of serum that produces a positive agglutination is taken as titre. The titres for all the antigens are noted.

Brucella test (Rose Bengal Test):
Brucella antigens are bacterial suspensions for use in slide agglutination test to detect the presence of bacterial agglutinins associated with bacterial infection or previous exposure to a related organism. This slide test is recommended as a screening procedure only to establish the presence or absence of antibody.
Reagents:
1. Rose Bengal-Brucella antigen.
2. patient s serum
Specimen collection and preparation:
1. Collect 5 ml whole blood samples aseptically from the patient.
2. Allow blood to clot and remove serum.
3. Store serum at 2-8°C until testing can be preformed.
Procedure:
1. Allow reagents and serum samples to reach room temperature for testing.
2. Shake the antigen bottle gently to insure a uniform suspension.
3. Place (40 ?l) sample serum onto the selected ring of the slide.
4. Place one drop of the Rose Bengal antigen onto serum sample.
5. Mix serum sample with Rose Bengal antigen using a stick.
6. Repeat these steps using the positive (known infected person) and negative (healthy person) controls instead of serum sample.
7. Gently rock the slide for 2 minutes.
8. Observe for agglutination after 2 minutes from beginning of shaking.
Results:
- Negative: No agglutination.
- Positive: (presence of specific antibodies) agglutination.

2. Precipitation test:
This test consist of the following:
A) Precipitation is solution.
B) Precipitation in agar.
C) Precipitation in agar with an electric field.



Precipitation in agar:
This is done as either single or double diffusion.
1- Single diffusion:
Antibody is incorporated into agar and antigen is added into
a well. As the antigen diffuses with time, precipitation rings will be formed depending on the antigen concentration. Ex. Radial immunodiffusion is used to measure IgG, IgM, complements and other substances in serum.
2- Double diffusion:
Antigen and antibody are placed in different wells in agar and allowed to diffuse and form concentration gradients. Where optimal proportions occur, lines of precipitate form. This method indicates whether antigens are identical, related but not identical, or not related.