METHOD OF PREPARING PURIFIED CHOLERAIC 0-ANTIGEN Russian patent published in 1999 - IPC

FIELD: microbiology, biochemistry. SUBSTANCE: strains of three serovara (Inaba, Ogawa, 0139) are grown under conditions of submerged culturing and additional feeding with glucose and ammonia. The native preparation of 0-antigen is obtained that is excreted in cultural medium spontaneously. Inert proteins are removed by their precipitation at pH 4.4 ± 0.2 and nonspecific impurities (molecular mass is less 100 kDa) are concentrated and separated by ultrafiltration. Nonspecific impurities (molecular mass is less 300 kDa) are separated by column chromatorgaphy being at all stages of purification 0-antigen is retained in the monophase dissolved state. The concentrated solution of 0-antigen is lyophilized in ampoules. One ampoule has 5 ± 0.5 mg of dry preparation, 250-500 conventional antigen doses equal to reverse titer in RPGA with 0-serum (choleraic or against 0139 strain) depending on serovarum. Protein content is 7-8% (by Lowry). Toxicity in white mice by LD₅₀ is 2 ± 0.5 mg. The preparation shows homogeneity by data of high-performance liquid chromatography. Invention ensures to obtain highly purified, good soluble 0-antigen of cholera vibrio 01 serovara (Inaba, Ogawa) and showing epidemically significance nonagglutinating (NAG) vibrio 0139 serovara. Invention can be used for specific identification and diagnosis of cholera caused by pathogens of three serovara: Inaba, Ogawa and 0139. EFFECT: improved method of 0-antigen preparing. 3 dwg