Patent RU-2599035-C1 (RU)
Inventor: IVANOV ARKADIJ VASILEVICH
IPC: C12N1/20
Filed: 2015-07-13
Granted: 2016-10-10
Abstract: FIELD: medicine. 
     SUBSTANCE: group of inventions relates to medicine, namely to immunology, and can be used to obtain erythrocyte anthrax antigen for kit for detection of antibodies in the blood serum of animals vaccinated against anthrax. To do this method comprises a preliminary preparation of the filtrate, placing it in a dialysis bag, storage in an inverted position in a refrigerator at +4 °C to concentration of up to ¼ of the original volume, preparation of 2.5 %-th suspension of formalinized erythrocytes. Pre-selection is carried out in blood of intact sheep in bottles with glass balls, stirring for 15 minutes is performed, dilution of 1:1 phosphate buffer solution pH of 6.4, the addition of a mixture of 100 ml of diluted blood per 20 ml of neutral formalin and 20 ml of phosphate buffer solution with pH of 7.2, placement in the thermostat on 2 hours at a temperature of +37 °C with continuous stirring. Subsequent centrifugation at 1,500 rpm for 10 minutes, 4-fold laundering of settled erythrocytes and suspending in 400-500 ml of phosphate-buffered solution, pH 7.2, placement of erythrocyte suspension in a refrigerator at a temperature of +4 °C. Withdrawal of the supernatant is carried out after 48 hours, 4-fold laundering of sediment with phosphate buffered solution pH 7.2 is conducted, then perform dilution of obtained sediment with phosphate buffered solution pH 6.4, 2.5 % suspension preparation, preserving by formalin to 1 % of its concentration. Then perform 3-fold laundering of 2.5 % resulting suspension with phosphate buffered solution pH 7.2 in a centrifuge at 1,500 rpm for 10 minutes each, followed by mixing in equal volumes with tannin, 20-minute exposure in water bath at +37 °C and 3-fold laundering in a centrifuge at a rate of 1,500 rpm for 10 minutes each. Further removal of supernatant and sediment filling with phosphate-buffer solution pH 7.2, and after laundering perform dilution of settled erythrocytes with phosphate buffer solution pH 6.4 based on receipt of a 2.5% suspension of erythrocytes, preserving by formalin to 1 % of its concentration and further 3-fold laundering in a centrifuge at 1,500 rpm for 10 minutes each with phosphate-buffered solution, pH 7.2. Add to the received based on 100 ml of washed formalinized erythrocytes with 5 ml filtrate, sensitization is carried out for 2 hours on a water bath at a temperature of +37 °C in the presence of 0.2 % glutaraldehyde with laundering and further adding of 0.5 % inactivated normal horse blood serum and preparation from sediment of 2.5 % erythrocyte suspension. Then the obtained erythrocyte anthrax antigen is conserved by 1 % formalin, pack in bottles and store in the refrigerator at a temperature of +4 °C. Group of inventions also relates to kit for detection of antibodies in the blood serum of animals vaccinated against anthrax. 
     EFFECT: use of this group of inventions allows to obtain a sensitive and reliable method for detection of antibodies in the blood blood serum of animals, vaccinated against anthrax in RIHA to control the tension of post-vaccination immunity, identify animals with low antibody titers and tolerant, to take timely measures for the prevention of disease. 
     3 cl, 5 ex, 1 tbl