Assessment of methods of purification of corynebacterium  allergen with definition of concentration and its   experimental application

Abstract

Aim: Search for an effective method for obtaining bacterial protein and determining the optimal concentration for identification of specific types. Material and Methods. Using the example of a C. xerosis N 1911 reference strain grown on a nutrient medium improved by us, methods were investigated of protein precipitation with sodium chloride, ammonium sulphate, sodium hexametaphosphate, trichloroacetic acid and polyethylene glycol. The threshold sensitivity of the allergen in six different cultures was determined in tests on 24 guinea pigs infected with corynebaсteria. Biological activity was studied in cultures from 36 guinea pigs infected with M. BCG (Bacillus Calmette–Guérin vaccine), M. avium, C. xerosis N 1911, C. ulcerans N 675 and C. bovis, as well as 3 rabbits infected with Corynebacterium xerosis. Results. Comparative testing of five protein precipitation methods was carried out. When using ammonium sulphate as a precipitant relatively high results were obtained at a salt concentration of 30% and a pH of at least 5.8. More significant protein precipitation occurred at the isoelectric point of sodium chloride at pH 3.9. It was noted that trichloroacetic acid and sodium hexametaphosphate had insignificant precipitating properties while there was none with polyethylene glycol. The threshold sensitivity (0.00005 mg in 0.1 ml) and allergen unit of action (0.0003 mg) were established. Intensity of skin reaction to sensitin homologous to infection was detected. Conclusions. The data obtained revealed the optimal method of protein precipitation, the unit of action of the corynebacterium allergens, and expanded the understanding of the mechanisms of the sensitization of the macro‐organism to tuberculin.

Keywords

• ppd‐tuberculin
• corynebacterium sensitin
• allergen
• precipitation
• sensitivity
• specificity
• concentration