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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)×106(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 high titer and high specificity anti-myoglobin monoclonal antibody (m Ab) and establish ELISA double antibody sandwich method to detect MYO in human serum. Methods Immunized with natural MYO, hybridoma technology was used to obtain a stable cell line secreting anti-human MYO m Ab. Ascites m Ab was prepared and characterized by affinity purification. The best matched pair and the establishment of double antibody sandwich ELISA one-step, serum samples were tested and compared with the imported kit. Results Nine strains of cells that could stably secrete m Ab were screened. Among them, the titer of 2M1, 3M4, 5M7 and 10M4 reached to (1.0 ~ 2.6) × 106 after affinity purification (dilution of antibody when A450 was about 1.0). Three pairs of antibodies (2M1 / HRP-3M4, 5M7 / HRP-3M4 and 10M4 / HRP-5M7) were screened by sandwich ELISA. The pairings with 5M7 / HRP-3M4 had higher sensitivity and larger size The linear range was 25-1000 ng / mL, which was better than that of the imported kit (25-500 ng / mL). The standard curve was established by 5M7 / HRP-3M4. The sample test results showed that the positive rate of self-made kit was 95% (19/20), the negative detection rate was 100% (40/40). Conclusions Two highly specific and high affinity anti-human MYO m Ab were obtained and a one-step sandwich ELISA was established, which laid the foundation for the development of ELISA kit.