抗人肌红蛋白单克隆抗体的制备及应用

Preparation and application of monoclonal antibody against human myoglobin

目的制备高效价、高特异性的抗人肌红蛋白（MYO）单克隆抗体（m Ab）,建立ELISA双抗体夹心法检测人血清中MYO。方法用天然的MYO进行免疫,用杂交瘤技术获得稳定分泌抗人MYO m Ab的细胞株;制备腹水型m Ab,经亲和纯化后进行抗体特性鉴定;确定最佳配对组合并建立双抗体夹心ELISA一步法,对血清样本进行检测,并与进口试剂盒进行比较。结果共筛选出9株能稳定分泌m Ab的细胞株,其中2M1、3M4、5M7、10M4亲和纯化后效价达到（1.0-2.6）×10^6（A450约为1.0时的抗体稀释倍数）。抗体配对共筛选出3对能进行夹心配对的抗体（2M1/HRP-3M4、5M7/HRP-3M4、10M4/HRP-5M7）,其中5M7/HRP-3M4这个配对有较高的灵敏度和较大的线性范围;利用最佳配对组合5M7/HRP-3M4建立标准曲线,其线性范围为（25-1000）ng/m L,优于进口试剂盒的线性范围（25-500）ng/m L;样本检测结果显示,自制试剂盒的阳性检出率为95%（19/20）,阴性检出率为100%（40/40）。结论获得了2株高特异性,高亲和力的抗人MYO m Ab,建立了双抗体夹心ELISA一步法,为ELISA试剂盒的研制奠定了基础。

Objective To prepare the monoclonal antibody（m Ab） against human myoglobin（MYO） of high titer and specificity and develop double-antibody sandwich ELISA for detecting MYO in human serum samples. Methods The BALB / c mice were immunized with natural human MYO,and the hybridoma cell lines secreting anti-MYO m Ab were established using cell fusion and hybridoma screening techniques. The characteristics of the m Ab were identified after affinity purification from ascites. Then the best antibody pair was selected from m Ab to establish a one-step sandwich ELISA method. Sixty human serum samples were detected by the homemade ELISA kit and the imported one,respectively. Results Nine strains of hybridoma cell lines stably secreted anti-MYO m Ab. Four strains named 2M1,3M4,5M7 and 10M4 could secrete high-quality m Ab and the titers of them were in the range of 1. 0 × 10^6 to 2. 6 × 10^6（A450value was about 1. 0）. Three antibody pairs（2M1 /HRP-3M4,5M7 /HRP-3M4,10M4 /HRP-5M7） were selected by double-antibody sandwich ELISA. Among them,the5M7 / HRP-3M4 had higher sensitivity and larger linear range. The homemade ELISA kit had a larger linear range（ 25-1000 ng / m L） than the imported one（ 25- 500 ng / m L） and showed high accuracies in detecting human serum samples,being 95%（19 /20） in positive samples and 100%（ 40 /40） in negative samples. Conclusion With the anti-human MYO m Abs of high specificity and affinity,a one-step sandwich ELISA for detecting human MYO has been established successfully,which provides a basis for the development of domestic ELISA kit.