METHOD OF DETERMINING GENERAL REDOX ACTIVITY OF PHAGOCYTES IN NITROBLUE TETRAZOLIUM REDUCTION TEST FOR LEUKAEMIA OF CATTLE

摘要

FIELD: veterinary medicine.SUBSTANCE: method comprises isolation of neutrophilic granulocytes from venous blood of cattle by centrifugation in a density gradient of ficoll-urografin (density 1.077 g/cm), application of 100 mcl of the cell suspension in two parallel wells of 96-well flat-bottomed plate for immunoassay. In one well of the plate 50 mcl of buffered saline or saline (spontaneous NBT-test) are added, and in another - 50 mcl of stimulator (BCG) at optimal dilution in buffered saline, or saline (induced NBT-test), then in both wells of the plate 50 mcl of 0.15% solution of nitroblue tetrazolium is added, stirred, incubated for 20 min at 37°C, centrifuged at 500 g for 10 minutes. Then the supernatant is separated, 200 mcl of absolute ethanol is added. The solution is centrifuged for 5 minutes at 500 g, the supernatant is separated. The packed cells is added to 200 mcl of saline. Centrifuged for 5 minutes at 500 g. 130 mcl dimexide is added to each well and incubated at 60°C for 20 minutes, 70 mcl of 2M KOH solution is added, mixed thoroughly. The results of the reaction are fixed through immunochemical analyser and are expressed in arbitrary units of optical density. After registering the reaction the stimulation coefficient is determined, and in determining the level of induced activity 50 mcl of levamisole is additionally added into the well at a final concentration of 0.5 mcg/ml. Results of the reaction are determined by the difference of the extinction at a wavelength of 630 and 490 nm. The stimulation coefficient (SC) is determined by the ratio of the induced level of cell activity to the spontaneous level. At SC?0.95 the animal is considered sensitive to leukaemia infection of cattle.EFFECT: method enables to assess quickly and accurately the susceptibility to leukaemia infection of cattle and gives an opportunity to diagnose the development of infection-inflammatory processes in animals with leukaemia.4 tbl, 3 ex