Laboratory of Veterinary Mycoplasmology, College of Veterinary Medicine, Southwest University, 2 Tiansheng Road, Beibei District, 400715, Chongqing, China.
Immunology Research Center, Medical Research Institute, Southwest University, 2 Tiansheng Road, Beibei District, 400715, Chongqing, China.
BMC Vet Res. 2021 Mar 16;17(1):123. doi: 10.1186/s12917-021-02828-7.
Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms.
In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, 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 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA.
The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.
用灭活的支原体肺炎疫苗（菌苗）对猪进行免疫会产生高浓度抗支原体肺炎 IgG 的超免疫血清。市售的 IgG-ELISA 试剂盒无法区分灭活菌苗诱导的超免疫血清与自然支原体肺炎感染后恢复期血清中的抗支原体肺炎 IgG。建立一种检测恢复期血清中抗支原体肺炎 IgG 的 ELISA 将有助于评估猪场的支原体肺炎状况。
本研究中，我们表达并纯化了含有支原体肺炎恢复期血清识别表位但不识别超免疫血清的重组 Mhp366-N 蛋白，将其用作包被抗原。对于支原体肺炎恢复期血清 IgG-ELISA，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 0.25μg/mL、2.5%脱脂乳、1 h、1:500、0.5 h、1:10,000、1 h 和 15 min。对支原体肺炎恢复期血清 IgG-ELISA 的验证表明，cut-off 值为 0.323，批内变异系数（CV）范围为 3.27%至 7.26%，批间 CV 范围为 3.46%至 5.93%，该检测方法能够区分恢复期血清和针对其他 7 种猪呼吸道病原体的抗体。该 ELISA 检测不出超免疫血清样品中的抗支原体肺炎 IgG，但商业 IgG-ELISA 鉴定出 145 份血清中的 95 份为阳性。支原体肺炎恢复期血清 IgG-ELISA 的准确性与 sIgA-ELISA 相当，但优于商业 IgG-ELISA。
该恢复期血清 IgG-ELISA 是一种重复性好、敏感且特异的间接 ELISA，可用于检测自然感染病原体诱导的恢复期血清中的抗支原体肺炎 IgG。该 ELISA 可用于对猪场的支原体肺炎感染进行大规模监测，而无需考虑疫苗接种状态。
