Method for obtaining anthrax erythrocyte antigenic diagnosticum

Abstract

This utility model relates to biotechnology and microbiology, specifically to the production of anthrax erythrocyte antigen diagnostic solution for the indirect hemagglutination assay (IHA). The technical result achieved by this utility model is increased specificity, sensitivity, and standardization of the erythrocyte antigen diagnostic solution, and reduced conjugating agent content, all while maintaining a long shelf life. A method for obtaining an anthrax erythrocyte antigen diagnosticum, including obtaining an antigen from the culture filtrate of the vaccine strain Bacillus anthracis 55, treating formalized ram erythrocytes with a conjugating preparation, followed by sensitization of the erythrocytes with anthrax antigen, to 100.0 mg of acetone-dried bacterial mass of the anthrax vaccine strain Bacillus anthracis STI-1 add 10.0 ml of a 70% phenol solution, shake and leave in the cold at 4°C for 3 hours, then the mixture is centrifuged at 6000 rpm for 90 minutes, the supernatant liquid is drained and neutralized by adding a saturated solution of sodium carbonate until a pH of 7.1±0.1 is obtained, and to remove insoluble particles the mixture is centrifuged at 6000 rpm for 30 minutes, the resulting yellowish opalescent supernatant is placed in a laboratory dialysis bag and dialyzed against running water for 2 days and then for 24 hours against distilled water, changing the water 4-5 times; the lipopolysaccharide antigen is extracted from the dialyzed solution by adding 5 volumes of 96° ethyl alcohol and acetone 1:1; the resulting mixture is shaken and left at a temperature of 4°C for 18-20 hours, After the specified period, the mixture is centrifuged at 5000 rpm for 90 min, then the supernatant is drained, and the sediment is washed twice with 3 volumes of 96° ethyl alcohol by centrifugation at 5000 rpm for 30 min, then the sediment is dissolved in distilled water, the resulting liquid lipopolysaccharide antigen in a volume of 3.0 ml is poured into glass vials with a capacity of 20.0 ml, after which the frozen antigen in the vials is transferred to a drying chamber, pre-cooled to a temperature of minus 30°C, hermetically close the lid and turn on the vacuum pump, after creating a vacuum in the chamber, after 2 hours turn on the heating of all the plates, while the temperature of the plates after 1 hour is raised to + 28°C, this heating temperature is maintained until the end of lyophilization, while the temperature in the vial with the antigen after 13 hours is also brought to a temperature of + 28°C, when this temperature is reached, lyophilization is completed after 6 hours, then the sensitized erythrocytes are washed and preserved, the residue of atkisencitin that has not bound to the erythrocyte receptors is removed, they are washed four times for 10 minutes at 3000 rpm with a solution of Tween-80 in a dilution of 1:5000 at the rate of 25-fold volume to the erythrocyte sediment, the sediment of sensitized erythrocytes after washing is diluted with a 0.9% solution of sodium chloride pH (6.8 ± 0.1) and sodium azide to a 10% concentration of erythrocytes.