抗人甲胎蛋白单克隆抗体的制备及双抗体夹心ELISA检测技术的建立

目的制备、筛选多株具有商用价值的可配对的高特异性、高亲和力的抗人甲胎蛋白(hAFP)单克隆抗体,初步建立双抗体夹心ELISA检测方法。方法通过经典的淋巴细胞杂交瘤技术筛选分泌抗hAFP单抗的杂交瘤细胞株;对筛选所得单抗的特性进行鉴定分析;双抗体夹心ELISA法筛选最佳配对抗体;初步建立DAS-ELISA检测方法并检测血样,绘制其标准检测曲线并与进口试剂盒比较。结果共获得12株稳定分泌抗hAFP单抗的杂交瘤细胞株,其中Ab 1~Ab 5 5株单抗的腹水效价均高于600万,Ab 5的效价高达3000万;特异性鉴定结果表明Ab 1~Ab 5均能够特异识别天然hAFP分子,但Ab 5与人血清白蛋白(HSA)存在较强交叉反应;抗体配对实验共筛选出5对(Ab 1/HRP-Ab 2、Ab 1/HRP-Ab 4、Ab 3/HRP-Ab 2、Ab 3/HRP-Ab 4和Ab 4/HRP-Ab 1)能够满足hAFP检测要求且无交叉反应的配对抗体,最终确定Ab 1+Ab 3/HRP-Ab 2和Ab 1+Ab 3/HRP-Ab 4为最佳配对组合;利用最佳配对抗体组合建立标准检测曲线,其线性检测范围为5~250 ng/ml,最低检测限2 ng/ml,检测上限400 ng/ml,优于进口试剂盒的线性范围5~200 ng/ml和最低检测限5 ng/ml;样检结果显示自制试剂盒的阳性血清检测准确率99.2%(119/120例),阴性血清准确率100%(40/40例)。结论筛选获得的4株高亲和力、高特异性抗hAFP单抗均可应用于DAS-ELISA试剂盒的研制,Ab 1和Ab 3适合作为捕获抗体,Ab 2和Ab 4适合作检测抗体;自制试剂盒的线性检测范围和最低检测限均优于进口试剂盒,表明具有商用价值。

Preparation of monoclonal antibodies against hAFP and development of mAb-based sandwich ELISA

Objective To prepare several hAFP-specific monoclonal antibodies with high specificity and high affinity. To screen antibody pairs with potential commercial value from these mAbs and to develop a sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for the detection of hAFP. Methods mAbs were prepared using the classic hybridoma fusion method, and the specificity of mAb was identified and analyzed. Using DAS-ELISA, the best antibody pair was selected from the mAbs. DAS-ELISA was optimized and compared to an ABCam ELISA kit. Then the initially self-made kit was used to detect a total of 160 samples （including 120 positive and 40 negative serums）. Results A total of 12 mAbs was prepared and 5 of them had very high affinities. The titers of ascites of both Ab 1-Ab 5 were less than 1：6 million and Ab 5 had the best titer：1：30 million. Both Ab 1-Ab 5 could recognize the natural hAFP specifically, but Ab 5 had cross-reaction with HSA. In the selected antibody pairs, Ab 1/HRP-Ab 2, Ab 1/HRP-Ab 4, Ab 3/HRP-Ab 2, Ab 3/HRP-Ab 4 and Ab 4/HRP-Ab 1 were identified to be capable of application. Ab 1＋Ab 3/HRP-Ab 2 and Ab 1＋Ab 3/HRP-Ab 4 were identified as the best antibody pairs to establish the DAS-ELISA kit. The self-made kit had better linear scale of detection （5-250 ng/ml） than that of ABCam kit （5-200 ng/ml）, and better limitation of detection （2 ng/ml） than that of ABCam （5 ng/ml）. The self-made kit also showed good results in detecting the samples with accuracies of 99.2%（119/120） in positive serums and 100%（40/40） in negative serums. Conclusion 4 mAbs （ including capture mAbs Ab 1 and Ab 3 and detection mAbs Ab 2 and Ab 4） with high affinity and specificity are used in developing the DAS-ELISA kit. The better linear scale and limitation of detection also suggest that this kit would have good commercial value.