罗丹明B单克隆抗体的制备及初步鉴定

Preparation and preliminary characterization of monoclonal antibody against Rhodamine B

采用N-羟基琥珀酰亚胺活性脂（NHS）法将罗丹明B（Rhodamine B,RB）与牛血清白蛋白（BSA）和卵清蛋白（OVA）分别偶联形成免疫原和检测原,经紫外分光光度计扫描鉴定初步判断偶联成功。以人工合成免疫原免疫BALB/c小鼠,利用杂交瘤技术获得了3株稳定分泌针对RB抗体的杂交瘤细胞株,分别命名为2D1、4C3和7F11,将7F11注入BALB/c小鼠腹腔产生腹水,用所得到的腹水建立了间接竞争ELISA（ic ELISA）标准曲线。对ic ELISA的工作条件进行了优化,方阵试验确定了包被抗原的工作浓度为1∶2 000,腹水抗体的工作浓度为1∶64 000。利用RB作药物抑制标准曲线,其线性方程为y=0.2848x-0.2847（R2=0.9924）,线性范围为1-1 000 ng/m L,最低检测浓度为1 ng/m L。交叉试验表明,该单抗与RB的抑制率为100%,与罗丹明6G、罗丹明123、苯酚红、副品红、中性红、曙红Y、橙黄Ⅱ、甲基橙、结晶紫均无交叉反应。

Bovine serum albumin and ovalbumin were used as two protein carriers respectively to couple with semiantigen RBby NHS method.The artificial antigen of RB was examined by Ultraviolet absorbance.Mice（BALB/c）were immunized with the com-pelete antigen.Three hybridoma cell lines secreting monoclonal anti-bodies against RB,named as 2D1,4C3 and 7F11,respective-ly,were obtained by using the hybridoma technique.After cloning,the positive hybridoma（7F11）was injected intraperiton-eally in-to mice to obtain ascitic fluid.Indirect competitive ELISA was established with this ascites.The most appropriate concentration ofcoating antigen was 1∶2 000,and the monoclonal antibody in the abdominal dropsy was attenuated by 1∶64 000.The regressionequations of the detection method was y=0.2848x-0.2847（R2=0.9924）. The detectable limit was in the range of 1-1 000 ng/m L.The curve indicated that the lowest detection limit was 1 ng/m L.The cross showed that the monoclonal antibody had 100% inhibi-tion rate for RB,and it had no cross reaction with Rhodamine 6G,Rhodamine 123,Phenol red,Pararosaniline,Neutral red,EosinY,Orange Ⅱ,Methyl orange and gentian violet.