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目的获得抗人CA15-3单克隆抗体(mAb),建立CA15-3双抗体夹心化学发光检测体系。方法人CA15-3糖抗原免疫小鼠后,通过细胞融合,筛选到阳性杂交瘤细胞。杂交瘤细胞扩大培养后,纯化获得抗CA15-3的mAb。对抗体进行纯度、效价、表位和亚型的鉴定并建立双抗体夹心检测CA15-3化学发光体系,对该体系进行准确度、最低检测限、线性、重复性与特异性评估。结果获得5株阳性信号较强的杂交瘤细胞(分别为#3-1-3﹑#5-2-2、#11-2-2、#12-1-3和#16-1-3)。其分泌的抗体效价均大于10-8g/mL,轻链均为κ链,重链#3-1-3为IgG2a、#5-2-2与#12-1-3为IgG2b、#11-2-2与#16-1-3为IgG3。双抗体夹心体系在(0~300)U/mL范围内线性关系良好,其准确度的回收率为97.45%;最低检测限为0.59 U/mL;线性相关系数为0.9978;低高值的变异系数(CV)均小于10%;与肿瘤标志物AFP、CEA、CA50、CA19-9和CA72-4均不交叉。结论成功制备了抗人CA15-3 mAb,建立了双抗体夹心检测CA15-3化学发光体系。
Objective To obtain anti-human CA15-3 monoclonal antibody (mAb) and establish the sandwich ELISA system of CA15-3 double antibody. Methods Human CA15-3 carbohydrate antigen was used to immunize mice and positive hybridoma cells were screened by cell fusion. After hybridoma cells were expanded and cultured, mAbs against CA15-3 were obtained. The purity, potency, epitopes and subtypes of the antibodies were identified and the double antibody sandwich assay was used to detect the CA15-3 chemiluminescence system. The accuracy, the lowest detection limit, the linearity, the repeatability and the specificity of the system were evaluated. Results Five hybridoma cells with strong positive signal were obtained (# 3-1-3, # 5-2-2, # 11-2-2, # 12-1-3 and # 16-1-3, respectively) . The secreted antibody titers were all greater than 10-8g / mL, the light chains were both κ chain, heavy chain # 3-1-3 was IgG2a, # 5-2-2 and # 12-1-3 were IgG2b, # 11 -2-2 and # 16-1-3 are IgG3. The linearity of the double antibody sandwich system was good in the range of (0 ~ 300) U / mL with the accuracy of 97.45%, the lowest detection limit of 0.59 U / mL, the linear correlation coefficient of 0.9978, the coefficient of variation of low value (CV) were less than 10%; and tumor markers AFP, CEA, CA50, CA19-9 and CA72-4 do not cross. Conclusion Anti-human CA15-3 mAb was successfully prepared and double antibody sandwich assay was developed for the detection of CA15-3 chemiluminescence.