Preparation of Effective Diagnostic Reagents Suitable for Elisa in the Diagnosis of Bacillus Anthracis

The aim of this study was to optimize methods for precipitating gamma-globulins from hyperimmune sera obtained from animals immunized with Bacillus anthracis strains, followed by the preparation of immunoperoxidase conjugates for use in enzyme-linked immunosorbent assays (ELISA). Two methods for gamma-globulin isolation were compared: ammonium sulfate precipitation and multi-step ethanol fractionation according to a modified Kon method. Protein yield, purity, and precipitating activity in the radial diffusion in gel (RDG) assay were evaluated. The results showed that the ethanol method provided higher gamma-globulin yield (up to 85 %) and protein concentration (17.4 mg/mL) compared to ammonium sulfate precipitation. Maximum precipitating activity (1:16) was achieved using the optimized ethanol precipitation, whereas the activity obtained with ammonium sulfate was 1:8. The isolated fractions were conjugated with horseradish peroxidase using the periodate oxidation method. Spectrophotometric analysis at 280 nm and 403 nm demonstrated that conjugates prepared from ethanol-precipitated gamma-globulins exhibited more uniform enzymatic activity and stable optical properties. These results indicate that ethanol fractionation is an effective approach for obtaining immunoperoxidase conjugates suitable for ELISA-based detection of Bacillus anthracis.