Development of a McAb-based indirect competitive ELISA for the detection of CD58 in goat serum | 山羊血清中CD58间接竞争ELISA检测方法的建立

中国人. 制备抗CD58单克隆抗体，在此基础上建立可用于定量检测山羊血清中CD58含量的间接竞争ELISA 方法。以纯化的CD58蛋白为抗原免疫BALB/c 小鼠，通过杂交瘤细胞融合技术制备抗CD58单抗，并对其特异性进行鉴定；在此基础上，建立CD58间接竞争ELISA 检测方法，对不同生理时期山羊血清中CD58的含量进行检测。筛选出1 株稳定分泌抗CD58单克隆抗体的杂交瘤细胞株G6；玫瑰花环抑制试验和Western-blotting 试验表明，制备的细胞株能分泌针对CD58的特异性单克隆抗体；对间接竞争ELISA 方法的反应条件进行优化后，建立了检测CD58的间接竞争ELISA 方法，该方法最适可测范围为10～1 000 000 ng/mL，理论最低检测限为3.087 ng/mL，批内和批间平均变异系数分别为3.50%和5.22%；用建立的方法检测未发情和发情山羊血清中CD58含量分别为（32.160±3.69）和（35.929±3.08）μg/mL，怀孕山羊血清中CD58含量为（61.564±5.10）μg /mL。建立了山羊血清中CD58 间接竞争 ELISA 检测方法，该方法敏感、特异，具有良好的重复性；对不同生理时期山羊血清中CD58含量的检测表明，怀孕山羊血清中CD58 含量显著高于未发情和发情山羊。

英语. The study was done to develop an indirect competitive ELISA method for the quantitative determination of CD58 in goat serum by preparation of the anti-CD58 monoclonal antibody. The CD58 protein was immunized to BALB/c mice to prepare the specific monoclonal antibody against CD58 by hybridoma technique. On this basis, goat serum contents of CD58 at different physiological periods were detected through the establishment of an indirect competitive ELISA method. One clone of the hybridoma cell stably secreting anti-CD58 antibody was obtained and named G6. This monoclonal antibody could react to CD58 protein in Et-rosseting inhibition test, as well as with CD58 in western-blotting. After optimization of the reactive condition, the method of indirect competitive ELISA for detection of CD58 was established. The optimal detection range was 10C1 000 000 ng/mL, providing the detection limit of 3.087 ng/mL in theory. Meanwhile, the coefficient of variation of intra and inter assays was 3.50% and 5.22% respectively. The results of samples showed that the content of CD58 in not estrus and estrus goat serum was (32.160±3.69) and (35.929±3.08) μg/mL respectively, and the content of CD58 in pregnant goat serum was (61.564 ±5.10) μg/mL. The indirect competitive ELISA detection method of CD58 of goat serum was established. This method is sensitive, specific and has high consistency with CD58 detection. Testing CD58 of goat serum during different physiological periods showed that the CD58 of goat serum of pregnant goat were significantly higher than those of non-estrus and estrus goat.