PREPARATION OF EFFECTIVE DIAGNOSTIC REAGENTS SUITABLE FOR ELISA IN THE DIAGNOSIS OF BACILLUS ANTHRACIS
Main Article Content
Authors
B.K. Umuraliyev
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
O.N. Serikbayov
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
N.K. Orazymbetova
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
A.A. Issakhan
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
M.Zh. Kaukarbayeva
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
A.K. Adalbekova
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
R.Z. Nurgaziev
K.I. Skryabin Kyrgyz National Agrarian University, Kyrgyzstan
M.S. Seisenbayeva
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
Zh.K. Khoshemetov
Research Institute for Biological Safety Problems, National Holding “QazBioPharm”, Gvardeysky, Kazakhstan
Abstract
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.
Keywords
Bacillus anthracis, immunoperoxidase conjugate, gamma globulin, ammonium sulfate, Cohn alcohol fractionation method
Article Details
References
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