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.

中文翻译：

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A29L蛋白特异性单克隆抗体A-A29L_MPoxV在猴痘诊断中的应用

摘要

2022年以来猴痘病毒（MPox）引起的疾病的传播表明制定对策的紧迫性。MPox 临床实验室诊断现代方法的发展对此做出了贡献。酶联免疫吸附测定 (ELISA) 是用于开发诊断工具的易于使用且灵敏的平台。使用基于单克隆抗体 (MAb) 的 ELISA 试剂盒检测 MPox 抗原具有广阔的前景，因为分析速度快且样品制备要求极低。我们开发并保藏了两种能够产生重组蛋白的大肠杆菌菌株。使用 AgPOX 蛋白对小鼠进行免疫，该蛋白含有 MPox 的独特抗原序列。用于选择MAb生产者的Trx+A29蛋白包括原始氨基酸序列A29L。通过 ELISA 测试，不存在与 Trx 蛋白以及天花病毒和牛痘病毒天然制剂的抗体交叉。作为来自免疫小鼠的脾细胞杂交的结果，获得了MAb生产者。根据 ELISA 结果，使用三种特异性 MPox 抗原和三种非特异性抗原选择 15 个产生 MAb 的杂交瘤。根据生产力标准选择三个杂交瘤进行保藏。测试了通过其单克隆抗体检测不同浓度的天然 MPox 抗原的可能性，并确定了方法的灵敏度。三个杂交瘤的单克隆抗体a-A29L_MPoxV检测到浓度为10 ² PFU/mL的天然抗原MPox。在选择分析条件时，该方法可能更加灵敏。基于标记的单克隆抗体a-A29L_MPoxV，可以开发用于MPox免疫诊断的灵敏且特异的间接两步ELISA试剂盒。