Development of an indirect competitive enzyme linked immunosorbent assay for the quantitative detection of Mycoplasma hyopneumoniae during the vaccine production process

• Published in: Journal of immunological methods Vol. 500; p. 113196
• Main Authors: Wei, Yanna, Khoza, Thandeka, Yu, Yanfei, Wang, Li, Liu, Beibei, Wang, Jia, Gan, Lanxi, Hao, Fei, Shao, Guoqing, Feng, Zhixin, Xiong, Qiyan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2022
• Subjects: Animals; Antibodies, Bacterial - metabolism; Antigens, Bacterial - metabolism; Bacterial Vaccines - immunology; Binding, Competitive; CCU; Enzyme Assays; Enzyme-Linked Immunosorbent Assay; Indirect competitive-ELISA; Mycoplasma hyopneumoniae; Mycoplasma hyopneumoniae - physiology; Pneumonia of Swine, Mycoplasmal - immunology; Quality Control; Quantitative detection; Rabbits; Reproducibility of Results; Sensitivity and Specificity; Swine - immunology; Swine - microbiology; Vaccine; Vaccine; Mycoplasma hyopneumoniae; CCU; Quantitative detection; Indirect competitive-ELISA
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2021.113196

Inactivated Mycoplasma hyopneumoniae vaccine is used extensively to control M. hyopneumoniae infection worldwide. Quantification techniques are essential in the process of standardizing and validating vaccines. In this study, we developed and optimized an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) for the rapid quantification of M. hyopneumoniae antigen during vaccine production. Briefly, whole M. hyopneumoniae antigen was coated onto microtiter plates, and a polyclonal antibody against M. hyopneumoniae recombinant elongation factor thermo unstable (EF-Tu) protein was prepared and added with the samples to be tested. The methods were optimized and showed significant reproducibility, with coefficients of variation of 4.01% and 6.14% for the intra-and inter-assays, respectively. Quantification of M. hyopneumoniae cultures at different growth stages using the ic-ELISA test showed a similar curve to that of the traditional color changing units (CCU) assay, with a delay in the time when the amount reached the peak and started to fall. In the inactivated vaccine production process, the cultures could be harvested later than that for the live vaccine, at about 12 h after the end of the logarithmic growth phase. Different batches of cultures were measured for their relative potency value compared with the in-house reference vaccine, which was used to determine whether the cultures met the antigen amount requirements for vaccine preparation. The curves of the CCU titer and ic-ELISA titer in the logarithmic phase correlated strongly and a linear regression equation was established to calculate the CCU values rapidly using the ic-ELISA results. In conclusion, an ic-ELISA method was established to rapidly assess the amount of antigen in an M. hyopneumoniae culture during the vaccine production process.