牛结核γ干扰素ELISPOT检测方法的建立

Development of an interferon-gamma ELISPOT for bovine tuberculosis

摘要:

本研究旨在建立一种牛结核γ干扰素ELISPOT检测方法，并评价该方法用于牛结核病检测的价值。通过筛选与天然牛γ干扰素特异性结合的单克隆抗体分别作为包被抗体和检测抗体，并探索不同的实验条件确定最佳包被抗体浓度、最佳细胞数量和最佳检测抗体浓度等，建立牛γ干扰素ELISPOT检测方法。采集30头奶牛尾静脉血并分离外周血单个核细胞，以结核菌素作为刺激原，使用建立的ELISPOT检测方法进行牛结核病检测，并与BOVIGAMTM ELISA试剂盒检测结果进行比较。筛选到两株与天然牛γ干扰素特异性结合的单抗2G5和5E11，确定ELISPOT检测方法的最佳实验条件为：包被抗体2G5浓度2.5 μg/mL，每孔细胞数量2.5×105个，检测抗体Bio-5E11浓度1 μg/mL。使用建立的ELISPOT检测方法与BOVIGAMTM ELISA试剂盒对30头奶牛进行同步检测，结果显示，以BOVIGAMTM ELISA试剂盒检测结果作为参考标准，14头BOVIGAMTM ELISA试剂盒检测阳性牛中，ELISPOT方法检出的阳性牛为11头，敏感性为78.6% (11/14)；16头BOVIGAMTM ELISA试剂盒检测阴性牛中，ELISPOT方法检出的阴性牛为12头，特异性为75% (12/16)。应用结核菌素作为刺激原的牛结核γ干扰素ELISPOT检测方法可用于牛结核病辅助检测，具有潜在的临床应用价值。

Abstract:

We established an ELISPOT for bovine interferon-gamma (BoIFN-γ), and applied it in the diagnosis of bovine tuberculosis (bTB). Monoclonal antibodies that can bind with native BoIFN-γ were screened as the coating antibody and detecting antibody. After optimization of detecting conditions including coating antibody concentration, cell number, and detecting antibody concentration, the ELISPOT assay was established. Peripheral mononuclear cells (PBMCs) isolated from 30 cows were co-cultured with PPD, and detected with the ELISPOT assay. The optimal conditions of ELISPOT assay were 2.5 μg/mL coating antibody 2G5, 2.5×105 cells/well, and 1 μg/mL detecting antibody Bio-5E11. In these 30 cows tested both with the ELISPOT assay and the BOVIGAMTM kit, 11 cows were proved to be positive in ELISOPT assay with the sensitivity of 78.6%, and 12 cows were proved to be negative in ELISOPT assay with the specificity of 75%. The ELISPOT assay for BoIFN-γ could be used to detect bTB efficiently and it might be an alternative method for the diagnosis of bTB.