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Influenza Virus Agglutination Inhibition Test (IVAIT)
Guinea pig erythrocytes (GPE) : add 5-6 volume blood from guinea pig to 1 volume of 3.8% sodium citrate solution for to prevent coagulation. The resulting suspension is then thoroughly washed with 10 volume of cold (3 °C) physiological saline solution to remove the sodium citrate, the plasma and soluble salts of the blood. After several washing 10 volume of animal erythrocytes pellet is suspended in 90 volume of cold (3 °C) suspension solution (dissolve in 2000 ml of dH2O, 60 g boric acid, 80 ml of 1.0 N sodium hydroxide, 4 g of NaN3 and 18 g of NaCl and adjust at pH 7.0 using boric acid or sodium hydroxide solution and strict storing at 2-4 °C) and if the pH of the resulting mixture changes from about seven, it should be adjusted to seven adding more boric acid or sodium hydroxide. The resulting mixture is incubated at about 2-4 °C, with stirring either continuously or at intervals (e.g. three times a day) to prevent settling of the cells. If continuous agitation is employed the incubation time will tend to be reduced. Continue incubation until the formation of a white-grayish layer (the antigen) is observed. This usually occurs within 8 to 20 days. Allow the antigen formation to continue for about 3 additional days. Then stir the mixture for 10 min. and centrifuge for 30 minutes at 4500 rpm to separate the antigen from the remaining materials such as the hemoglobin, plasma, cell proteins and sodium citrate. Centrifugation produces three layers, a bottom layer of heavy cellular material, middle layer of the white-grayish material which contains the antigen, and a top layer of supernatant liquid. Discard both the supernatant liquid (top layer) and the heavy cellular material (bottom layer). Wash the middle layer, containing the antigen, with saline solution by centrifugation, once more discarding top and bottom layers and retaining the middle antigen containing layer. Repeat the washing until the supernatant is clear, indicating that the hemoglobin and other separated materials have been removed. Usually three washings suffice. The white-grayish sediment constitutes the GPE that is taken up in 20 ml of saline solution, and preserved by adding 0.2% (w/v) NaN3.
Virus target : noise mucus secretion from influenza affected patients is treated with acetylcisteine and formalin or phenol for to fluidify mucus and destroy virus infectivity while preserving its antigencity. Dilute redoubling the viral solution in 0.15 M NaCl from 1:2 to 1:512 in MTP with 50 µl as final volume and then add 50 µl of GPE. Shaker at 200 rpm for 10 sec. and after at 50 rpm for 2-3 min. The last dilution which shows distinct agglutination (i.e. when the next higher dilution produces no agglutination) represents one agglutinative unit of viral antigen.
Treat serum patient with kaolin to remove non-specific agglutination inhibitors and place one drop of this on a glass plate, on same glass, separately, dispense one drop of standardized virus target and both reagents (drops) are mixed with a wooden applicator and allowed to stand for about 2 minutes. Then a drop of GPE is added to the mixture, and mixing is carried out as before. Finally the glass plate is rotatively manipulated gently over an indirect light for not more than about 2 minutes, and the presence or absence of agglutination reactions is visually observed. In a positive test, the viral antigen will have been neutralized by the specific antibodies in the patient's serum, and no agglutination occurs with the antigen erythrocytes.