Experimental Peroxidase Conjugate for Detection of Specific Antibodies to Anthrax Agent in Enzyme Immunoassay

Abstract

Anthrax poses a pressing issue for veterinary medicine and public health in many countries, including the Russian Federation, which necessitates the improvement and development of new, sensitive and specific diagnostic tools.The aim of the work was to create an experimental peroxidase conjugate for the detection of specific antibodies to the anthrax pathogen and to optimize the conditions for performing enzyme immunoassay (ELISA).Materials and methods. The peroxidase conjugate was constructed using horseradish peroxidase and Staphylococcus aureus protein A (Sigma-Aldrich, USA). Bacterial antigens isolated from strains of Bacillus anthracis 55ΔTPA-1Spo, B. anthracis Sterne 34 F2 were used as sensitizing agents. The developed experimental batches of the conjugate were tested in ELISA for the ability to bind antibodies in the blood sera of anthrax patients and vaccinated individuals. The sensitivity, specificity, and accuracy of the method were calculated using the built-in functions of the ROCR software package.Results and discussion. The peroxidase conjugate to detect specific antibodies to the anthrax pathogen in the study of clinical material has been developed; conditions for the ELISA performance have been optimized. To interpret the results of the study, a threshold value of the positivity coefficient was used, below which the result was considered negative, and at an equal or higher value, positive. The test demonstrated significant differences in the “positivity coefficient” indicator for the “Healthy”/“Sick” and “Healthy”/“Vaccinated” groups, while the differences between the “Sick”/“Vaccinated” groups were statistically insignificant. The maximum accuracy of the method was observed at blood serum dilutions of 1:250 and 1:500. 100 % intra-run, run-to-run and series-to-series reproducibility has been established for all positive samples. The sensitivity and specificity of the experimental peroxidase conjugates were 100 and 95.8 %, respectively, and the accuracy was 97.6 %.