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

文献类型： 外文期刊

作者： Wei, Yanna 1 ; Khoza, Thandeka 3 ; Yu, Yanfei 2 ; Wang, Li 2 ; Liu, Beibei 2 ; Wang, Jia 2 ; Gan, Lanxi 2 ; Hao, Fei 2 ; Shao, Guoqing 2 ; Feng, Zhixin 2 ; Xiong, Qiyan 2 ;

作者机构： 1.Minist Sci & Technol, Jiangsu Key Lab Food Qual & Safety, State Key Lab Cultivat Base, Nanjing, Peoples R China

2.Jiangsu Acad Agr Sci, Inst Vet Med, Minist Agr & Rural Affairs, Key Lab Vet Biol Engn & Technol, Nanjing, Peoples R China

3.Univ KwaZulu Natal, Coll Agr Engn & Sci, Discipline Biochem, Sch Life Sci, Private Bag X54001, Durban, South Africa

4.Univ KwaZulu Natal, Coll Agr Engn & Sci, Discipline Microbiol, Sch Life Sci, Private Bag X54001, Durban, South Africa

5.Hunan Agr Univ, Coll Vet Med, Changsha, Hunan, Peoples R China

6.Nanjing Agr Univ, Coll Vet Med, Nanjing, Peoples R China

7.Jiangsu Univ, Sch Food & Biol Engn, Zhenjiang, Jiangsu, Peoples R China

8.Jiangsu Univ, Coll Life Sci, Zhenjiang, Jiangsu, Peoples R China

关键词： Mycoplasma hyopneumoniae; Indirect competitive-ELISA; Quantitative detection; Vaccine; CCU

期刊名称：JOURNAL OF IMMUNOLOGICAL METHODS （ 影响因子：2.287； 五年影响因子：2.475 ）

ISSN： 0022-1759

年卷期： 2022 年 500 卷

页码：

收录情况： SCI

摘要： 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.

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