| Number of the test tube | |||||||
| Ingredients | serum control | antigen control | |||||
| Isotonic solution (ml) | --- | ||||||
| The patient’s serum in a 1:25 dilution (ml) | --> | --› | --› | --› pour out | --- | ||
| The serum dilution | 1:50 | 1:100 | 1:200 | 1:400 | 1:800 | 1:25 | --- |
| 1-st row: O-diagnosticum (drops) | --- | ||||||
| 2-nd row: H-diagnosticum (drops) | --- | ||||||
| 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 37oC 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 l arge 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 thefirst 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.
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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 plateswith 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 37oC incubator for 30-40 min.
NOTE: Before performing the test, t he patient’s serum is heated for 30 min at 56oC 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