Application of A29L Protein Specific Monoclonal Antibodies A-A29L_MPoxV for Monkeypox Diagnosis

Abstract

The spread of the disease caused by monkeypox virus (MPox) since 2022 has shown the urgency of developing countermeasures. The development of modern methods of clinical laboratory diagnostics of MPox contributes to this. Enzyme-linked immunosorbent assay (ELISA) is an accessible and sensitive platform for developing diagnostic tools. Detection of MPox antigens using ELISA kits based on monoclonal antibodies (MAbs) is promising due to the quick time of analysis and minimal requirements for sample preparation. We have developed and deposited two strains of Escherichia coli that produce recombinant proteins. Mice were immunized with the AgPOX protein, which contains unique antigenic sequences of MPox. The Trx + A29 protein for selecting MAb producers includes the original amino acid sequence A29L. The absence of antibody crossover to Trx protein and native preparations of variola virus and vaccinia virus tested by ELISA. As a result of hybridization of splenocytes from immunized mice, MAb producers were obtained. Fifteen MAb-producing hybridomas were selected based on ELISA results with three specific MPox antigens and three nonspecific ones. Three hybridomas were selected for deposit according to the productivity criteria. The possibility of detection by means of its MAbs of the native MPox antigen at various concentrations was tested and method sensitivity was determined. The MAbs a-A29L_MPoxV of three hybridomas detected the native antigen MPox at a concentration of 10² PFU/mL. It is likely that the method is even more sensitive when selecting analysis conditions. Based on labeled MAbs a-A29L_MPoxV, it is possible to develop a sensitive and specific indirect two-step ELISA kit for immunodiagnostics of MPox.