抗人CA15-3单克隆抗体的制备、鉴定及化学发光检测体系的建立  

Preparation and characterization of the monoclonal antibodies against human CA15-3 and establishment of a sandwich CLIA system

作　　者：曹领改[1,2] 蓝兴国[1] 魏德强[1,2] 李玉花[1,3] 

机构地区：[1]东北林业大学生命科学学院,黑龙江哈尔滨150040 [2]大庆麦伯康生物技术有限公司,黑龙江大庆163316 [3]东北林业大学大庆生物技术研究院,黑龙江大庆163316

出　　处：《细胞与分子免疫学杂志》2014年第1期66-70,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：黑龙江省发展高新技术产业专项资金(FW11C201);黑龙江省应用技术研究与开发计划(GC13C122)

摘　　要：目的获得抗人CA15-3单克隆抗体（mAb），建立CAl5-3双抗体夹心化学发光检测体系。方法人CAl5-3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CAl5-3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CAl5—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10qg／mL，轻链均为K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0．59U／mL；线性相关系数为0．9978；低高值的变异系数（cy）均小于10％；与肿瘤标志物AFP、CEA、CAS0、CAl9-9和CA72-4均不交叉。结论成功制备了抗人CAl5-3mAb，建立了双抗体夹心检测CAl5-3化学发光体系。Objective To prepare the monoclonal antibodies （mAbs） against human carbohydrate antigen 15-3 （CA15- 3） and establish a double-antibody sandwich chemilurninescent immunoassay （CLIA） system for detecting CA15-3 in the human serum. Methods BALB/c mice were immunized with human CA15-3 antigen. Spleen cells of the immunized mice were fused with Sp2/0 cells and the positive hybridoma cells were selected and subcloned. The supernatant was taken for purify mAbs using protein A chromatography. The purity, titer, epitope and subtype of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity, repetitiveness and specificity. Results Five hybridoma cell lines named #3-1-3, #5-2-2, #11-2-2, #12-1-3 and #16-1-3 were obtained respectively, which are of strong positive signal and high secretion. The titers of the mAbs secreted by these hydridomas were above 10-8 g/mE All of the mAbs expressed K light chains, and their heavy chains were as follows： #3-1-3 mAb had IgG2a, #5-2-2 mAb and #12-1-3 mAb had IgG2b, #11-2-2 mAb and #16-1-3 mAb had IgG3. The double-antibody sandwich CLIA system had a good linear relationship between 0.59 U/mL and 300 U/mL. The recovery rate for accuracy was 97.45% and the limit of detection was 0.59 U/mL. The assay was highly linear （correlation coefficient 0.9978） and highly repeatable [ coefficient of variation （CV） 〈 10% ]. This CLIA system was also highly specific without cross-reactivity to the tumor marker AFP, CEA, CA50, CAI9-9 and CA72-4. Conclusion The mAbs against human CA15-3 have been prepared and a sandwich CLIA system for detecting CAI5-3 in the human serum has been established successfully, which provides a basis for CA15-3 quantification and clinical application.

关 键 词：肿瘤标志物 糖抗原CA15-3 单克隆抗体 化学发光免疫分析 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]