Practical work

1. The Procedure of the Agglutination Test. This test is performed in the row of tubes. Patient’s serum with unknown antibodies is diluted in isotonic solution in simple numerical ratios 1:50, 1:100, 1:200, 1:400 and 1:800. Then 3 drops of diagnosticum are added into each tube with serum diluted and into the control tube with isotonic solution instead of serum (the antigen control). The serum control tube does not contain the diagnosticum (see Table 12-1).

Table 12-1

The Scheme of the Standard Agglutination Test with the Patient’s Serum

	Number of the test tube
Ingredients	1	2	3	4	5	serum control	antigen control
Isotonic solution (ml)	1	1	1	1	1	---	1
The patient’s serum in a 1:25 dilution (ml)	1	-->	--›	--›	--› pour out	1	---
The serum dilution	1:50	1:100	1:200	1:400	1:800	1:25	---
1-st row: O-diagnosticum (drops)	3	3	3	3	3	---	3
2-nd row: H-diagnosticum (drops)	3	3	3	3	3	---	3
Incubation at 37oC for 2 h., then at room temperature for 18-24 h.
Results: O-agglutination
Results: H-agglutination
Conclusion: the titer of antibodies in the patient’s serum to the O-antigen is __________; the titer of antibodies in the patient’s serum to the H-antigen is __________.

After 2 h. incubation at 37^oC the results are evaluated beginning with the controls. The absence agglutination in the control tubes must be pointed. In the positive result (denoted with "+"), on the bottom of the test tube there is a floccular sediment (antigen-antibody complexes), while the supernatant liquid is completely transparent. In the negative test ("-"), there is no sediment, and the suspension remains uniformly turbid, showing no difference between the test tube content and the antigen control. The fluid in the tube with the serum control must be quite transparent.

One should note the type of agglutination. If the large fluffy clumps appear on the bottom of the tube, leaving clear supernatant fluid in 2-4 h., the H-agglutination occur. If the small granular aggregates are detected, the O-agglutination is positive. The positive reaction is denoted with pluses. The last tube with agglutination is the titer of antibodies.

2. The Procedure of the Slide Agglutination Test. This test should be performed in two slide glasses: one -for non-absorbed antisera; and another one -for pre-absorbed antisera.

NOTE: This experiment shows the possible false-positive results with the same bacterial culture (the unknown antigen) and several non-absorbed antisera.

A. Take a clean grease-free slide glass. Divide the slide by wax marker into three cells.

B. Using a Pasteur pipette, apply a drop of diagnostic non-absorbed antiserum against Salmonella typhi in the first cell, a drop of diagnostic non-absorbed antiserum against Salmonella paratyphi A in the second cell, and a drop of isotonic solution as a control in the last one.

C. Flame the wire loop. Pick up the culture from the slant agar and place a loopful of tested bacterial culture («unknown pathogen») into the antiserum drop. Mix thoroughly the material.

D. Flame the wire loop again. Take the culture and inoculate another drop. Mix thoroughly the material.

E. Repeat all these steps to prepare the mixture of bacteria tested and the drop of isotonic solution.

NOTE: The reaction takes place at room temperature within 5-10 min. Positive test is indicated by the appearance of large or small flakes in the drop of antiserum. In negative test, the fluid remains uniformly turbid as well as in the control drop (saline).

F. Read the results.

NOTE: The test may be positive both with non-absorbed antiserum against S.typhi and S.paratyphi A non-absorbed antiserum. It is happened because S.typhi and S.paratyphi A possess related antigens that false interact with cross-reactive antibodies in the non-absorbed antisera. That is why pre-absorbed antisera are more preferable.

To perform Slide AT with pre-absorbed antisera repeat steps A-F.

3. The Procedure of the IHA-test. The test is usually employed to detect the specific antibodies in the patient’s serum. The IHA-test is performed in special plastic plates with a grate number of wells. One drop of erythrocytic diagnosticum (red blood cells coated with certain antigen) is added into each well with decreased amounts of unknown antibodies (two-fold dilutions of a patient’s serum) – see Table 12-2. As the erythrocytic diagnosticum control, the 1% suspension of non-sensitized erythrocytes is mixed with isotonic solution. The plates are thoroughly shaken and put in a 37^oC incubator for 30-40 min.

NOTE: Before performing the test, the patient’s serum is heated for 30 min at 56^oC to remove the non-specific hemagglutinins.

The results of the test are assessed by the presence of hemagglutination. Agglutinated erythrocytes form a granular film ("mat") over the bottom of the well. The non-agglutinated cells roll into a small, circular red button ("pellet"). It is necessary to check the antigen control for the absence of spontaneous agglutination or hemolysis.

The last well in row with hemagglutination is the titer of the reaction.

Table 12-2

The Scheme of the IHA-Test with the Patient’s Serum

The patient’s serum dilution (0,25 ml in the each well)	1:10	1:20	1:40	1:80	1:160	Antigen control	Serumcontrol
Results:
Conclusion: the titer of specific antibodies in the patient’s serum is ____________.

4. The Precipitin Ring Test. This test is used to determine the unknown antigen in a specimen (e.g., foodstuffs, leather, wool, blood stains or sperm, etc.) or to detect the antibodies in the patient’s serum. The precipitin ring test is performed in a narrow test tubes (0.5 cm diameter).

A. The procedure of the determining of unknown antigen (protein).

1. Take two narrow test tubes.

2. Add about 0.5 ml of unknown protein (or hapten) into each tube.

3. With a Pasteur pipette slowly add the same (equal) volume of precipitating antiserum against human protein in the first tube.

BE CAREFUL: The tube must be held in a titled position. The drops of antiserum must flow down the wall inside the tube.

4. Set up carefully the tube in a vertical position in order to avoid mixing of the fluids.

5. Pour carefully with Pasteur pipette about 0.5 ml of precipitating antiserum to the chicken protein into another tube. Set the tube up in a vertical position too.

6. Read the results of the test in 2-5 min. A zone of precipitation (white ring) promptly develops in the tube, where specific antibodies interfere with corresponding antigens (proteins).

7. Point out the results with (+) or (-) in Table 12-3.

Table 12-3

The Scheme of the Precipitin Ring Test to Identify the Unknown Protein

Ingredients	Unknown protein
Precipitating antiserum against human protein
Precipitating antiserum against chicken protein
Conclusion: the unknown protein is identified as the _________________ protein.