FIELD: microbiology; biotechnology.
SUBSTANCE: method implies using monoclonal peroxidase conjugate as a diagnostic reagent. The method includes the following technological steps: activation of peroxidase, conjugation of activated peroxidase with specific immunoglobulins, purification of unbound proteins, storage and control. The difference of the invention is as follows: the activation is carried out in the following sequence: the enzyme horseradish peroxidase is dissolved in 0.3 M carbonate-bicarbonate buffer with pH 9.5 at the rate of 5 mg per 1 ml. At certain intervals — 1 h, 30 min, 1 h, respectively, 100 μl of 1% 2,4-dinitro1-fluoro-benzene, 1.0 ml of 0.08 M sodium periodate, 1.0 ml of 0.16 M ethylene glycol, then the mixture of reagents is incubated for 3 hours in the darkness at room temperature with constant stirring on a magnetic stirrer at a speed of 80–100 rpm. After that, for conjugation, the activated peroxidase solution is combined with monoclonal antibodies dissolved in 0.1 M phosphate-buffered saline with pH 7.2–7.4 in a ratio of 1:3, namely: 5 mg of peroxidase and 15 mg of MCA, the mixture is adjusted to pH 9.5 by adding 10 μl of 0.5 M carbonate-bicarbonate buffer. The conjugation is continued for 3 hours in the darkness at room temperature on a magnetic stirrer at a speed of 80–100 rpm, periodically monitoring the pH to be kept at 9.5. After the reaction is stopped after 3 hours by adding 5 mg of dry sodium bromide and left overnight at a temperature of 4°C. Purification of the conjugate from unbound components is carried out by dialysis in 0.1 M phosphate-buffered saline with pH 7.2–7.4 at a temperature of 4°C, changing the buffer during the day. The final purification of the conjugate is carried out using gel filtration on Sephadex G-50, obtaining a conjugate with a titer of 1:64–1:128.
EFFECT: invention can be used to identify strains of vibrio cholerae O1 serogroup and R-variants isolated in the process of cholera monitoring studies on the territory of the Russian Federation.
1 cl, 1 dwg, 1 tbl, 2 ex
