Gan Yujie, Dong Qingshuang, Zhang Yang, He Jiawei, Wang Dongjing, Feng Zhixin, Wang Jia, Zhou Kexin, Ding Honglei
Laboratory of Veterinary Mycoplasmology, College of Veterinary Medicine, Southwest University, Chongqing 400715, China.
Institute of Animal Husbandry and Veterinary, Xizang Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850009, Xizang, China.
Sheng Wu Gong Cheng Xue Bao. 2024 Jul 25;40(7):2322-2332. doi: 10.13345/j.cjb.230699.
This study aims to establish an ELISA method with high specificity for the detection of antibodies against . Firstly, we constructed a recombinant strain 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 . antibody detection, and a convalescent serum ELISA kit for . 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 . antibody detection in terms of sensitivity and specificity. We successfully established an ELISA method for detecting the antibodies against . 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.
本研究旨在建立一种具有高特异性的酶联免疫吸附测定（ELISA）方法，用于检测抗……的抗体。首先，我们构建了重组菌株BL21(DE3)-pET-32a(+)-mhp336以表达重组蛋白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、封闭缓冲液、封闭时间、血清稀释度、与血清孵育时间、二抗稀释度、二抗孵育时间、比色反应时间和临界值。之后，进行了重复性实验，并测定了Mhp366与猪主要病原体的其他抗血清的交叉反应性以及血清的最大稀释倍数。最后，使用本研究建立的方法、用于……抗体检测的商业ELISA试剂盒以及用于……抗体检测的恢复期血清ELISA试剂盒检测了226份猪血清样本。比较这三种方法的检测结果，以评估本研究建立的ELISA方法的敏感性和特异性。对于该方法，最佳抗原浓度、封闭缓冲液、封闭时间、血清稀释度、与血清孵育时间、二抗稀释度、二抗孵育时间和比色反应时间分别为0.05μg/mL、含2.5%脱脂牛奶的磷酸盐缓冲液（PBS）、1小时、1:500、0.5小时、1:10000、1小时和5分钟。基于Mhp336的ELISA方法验证显示临界值为0.332。批内和批间试剂盒的变异系数均低于7%。猪血清样本的检测结果表明，本研究建立的方法在敏感性和特异性方面优于用于……抗体检测的商业ELISA试剂盒和恢复期血清ELISA试剂盒。我们成功建立了一种基于Mhp336蛋白检测抗……抗体的ELISA方法。该方法具有高敏感性和特异性，可作为猪场预防猪支原体肺炎的工具。
