本研究旨在建立一种特异性高的猪肺炎支原体抗体检测方法。首先,我们构建了重组菌株大肠杆菌BL21(DE3)-pET-32a()-mhp336来表达重组蛋白Mhp336,并使用纯化的Mhp336作为包被抗原。然后,我们优化了ELISA参数,包括抗原浓度,阻塞缓冲区,阻塞时间,血清稀释,血清孵育时间,二级抗体稀释,二次抗体孵育时间,比色反应时间,和截止值。之后,进行了重复性实验,并测定了Mhp366与猪主要病原体其他抗血清的交叉反应性和血清的最大稀释比。最后,采用本研究建立的方法对226份猪血清进行检测,用于猪肺炎支原体抗体检测的商业ELISA试剂盒,和用于猪肺炎支原体抗体检测的恢复期血清ELISA试剂盒。比较三种方法的检测结果,评价本研究建立的ELISA方法的敏感性和特异性。对于此方法,最佳抗原浓度,阻塞缓冲区,阻塞时间,血清稀释,血清孵育时间,二级抗体稀释,二次抗体孵育时间,比色反应时间为0.05μg/mL,含有2.5%脱脂牛奶的PBS,1h,1:500,0.5h,1:10000,1小时,5分钟,分别。基于Mhp336的ELISA方法的验证显示截断值为0.332。批次内和批次间试剂盒的变异系数均低于7%。猪血清样品的检测结果表明,本研究建立的猪肺炎支原体抗体检测方法在灵敏度和特异性方面均优于商业ELISA试剂盒和恢复期血清ELISA试剂盒。我们成功建立了基于Mhp336蛋白的猪肺炎支原体抗体的ELISA检测方法。该方法具有较高的灵敏度和特异性,作为预防猪场猪支原体肺炎的工具。
This study aims to establish an ELISA method with high
specificity for the detection of antibodies against Mycoplasma hyopneumoniae. Firstly, we constructed a recombinant strain Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 to express the recombinant protein Mhp336 and used the purified Mhp336 as the coating antigen. Then, we optimized the ELISA parameters, including antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, colorimetric reaction time, and cut-off value. Afterwards, reproducibility experiments were conducted, and the cross reactivity of Mhp366 with other antisera of porcine major pathogens and the maximum dilution ratios of the sera were determined. Finally, 226 porcine serum samples were detected using the method established in this study, a commercial ELISA kit for M. hyopneumoniae antibody detection, and a convalescent serum ELISA kit for M. hyopneumoniae antibody detection. The detection results of the three methods were compared to evaluate the sensitivity and
specificity of the ELISA method established in this study. For this method, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, and colorimetric reaction time were 0.05 μg/mL, PBS containing 2.5% skim milk, 1 h, 1:500, 0.5 h, 1:10 000, 1 h, and 5 min, respectively. Validation of the ELISA method based on Mhp336 showed a cut-off value of 0.332. The coefficients of variation of both intra-batch and inter-batch kits were below 7%. The detection results of porcine serum samples indicated that the method established in this study outperformed the commercial ELISA kit and the convalescent serum ELISA kit for M. hyopneumoniae antibody detection in terms of sensitivity and
specificity. We successfully established an ELISA method for detecting the antibodies against M. hyopneumoniae based on Mhp336 protein. This method demonstrated high sensitivity and
specificity, serving as a tool for the prevention of mycoplasmal pneumonia of swine in pig farms.