三聚氰胺单克隆抗体的制备及鉴定  

Development And Characterization of Hybridoma Cell Line H2C6 Secreting Monoclonal Antibody Ggainst Melamine

作　　者：蔡家利[1] 陈昕[1] 徐社会 

机构地区：[1]重庆理工大学,重庆400050 [2]广州瑞森生物科技有限公司,广州511400

出　　处：《重庆理工大学学报（自然科学）》2010年第4期30-34,48,共6页Journal of Chongqing University of Technology：Natural Science

摘　　要：制备了抗三聚氰胺(melamine)单克隆抗体,鉴定了其免疫学特性并建立三聚氰胺标准抑制曲线。以人工合成抗原Melamine-BSA免疫BALB/c小鼠,应用杂交瘤技术建立分泌抗三聚氰胺单克隆抗体的细胞株,以体内诱生腹水法生产三聚氰胺单抗,用硫酸钠盐析法提纯,以直接竞争ELISA法建立三聚氰胺标准抑制曲线。获得了1株能稳定分泌抗三聚氰胺单克隆抗体的杂交瘤细胞株H2C6。用间接ELISA方法测定腹水单抗效价为2×10-7。经金标免疫层析实验鉴定单抗与三聚氰胺特异性结合,H2C6杂交瘤细胞株单抗对三聚氰胺的半数抑制浓度(IC50)为4.15μg/mL。除与三聚氰酸交叉反应性为72%外,与三嗪及各类抗生素等交叉反应性都小于5%。体外多次传代培养和反复冻存复苏后抗体分泌稳定。Monoclonal antibody（mAb） against melamine was prepared,its immunity characters were appraised,and a melamine calibration restraining curve was made.The spleen cells of BALB/ C mouse immunized by Melamine-BSA were fused with SP2/0 plasmacytoma cells using PEG3 000 to artificially produce antigen,cells which excrete antibody against melamine were obtained by hybridoma technique.monoclonal antibody against melamine was prepared by an inside body induced ascites method,the sodium sulfate salt extracting method was applied for purification,and a melamine calibration restraining curve was made by a direct ELISA method.One hybridoma cell line of H2C6 was screened for specificity to Melamine,which secrete high affinity monoclonal antibodies against Melamine with indirect ELISA titers of 2×10-7.The mAb had specificity integrated with Melamine conjugated antigen that determined by gold-immuno chromatography assay（GICA）.The mAb of H2C6 showed good sensitivity with an IC50 of 4.15 μg/mL to Melamine.And it generally had 72% cross-reactivity to Cyanuric acid,but little or no cross-reactivity to triazine and antibiotics.which secreted stable monoclonal antibodies against Melamine after it was repeated subculture in vitro and cryopreservation many times.Conclusion Results indicated that the mAb would be very useful in the development of analytical reagent kit to detect Melamine and laid a well foundation for the detection and control of Melamine.

关 键 词：三聚氰胺 单克隆抗体 免疫学特性鉴定 

分 类 号：R392[医药卫生—免疫学]