凝血酶-抗凝血酶Ⅲ复合物单克隆抗体的制备及其免疫检测方法的建立  

Preparation of Monoclonal Antibodies to Thrombin-Antithrombin III Complex and Establishment of an Immunoassay Method

作　　者：周泓圣 刘萌[1,2] 郭超林 白仲虎 ZHOU Hongsheng;LIU Meng;GUO Chaolin;BAI Zhonghu(The Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China;National Engineering Laboratory of Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,无锡214122 [2]江南大学粮食发酵工艺与技术国家工程实验室,无锡214122

出　　处：《中国生物工程杂志》2025年第2期44-53,共10页China Biotechnology

基　　金：国家重点研发计划(2018YFA0900804);国家轻工技术与工程一级学科计划(LITE2018-24)资助项目。

摘　　要：为了量化凝血酶-抗凝血酶Ⅲ复合物(TAT)的浓度,创建并评估了一种用于检测TAT的化学发光免疫分析法(CLIA)。首先在血浆中提取凝血酶(T)和抗凝血酶(AT),在体外反应生成TAT作为免疫原制备单抗,通过筛选获得1E9、16G4和1D7三株可特异性识别TAT复合物的杂交瘤细胞株。进一步筛选配对后,以单抗16G4偶联生物素作为捕获抗体,1D7标记辣根过氧化物酶作为检测抗体,建立了双抗夹心的检测方法。最佳反应流程为两步法,每步孵育时间为10 min,检测限为0.6 ng/mL,小于25 min即可获得检测结果。测试结果与希森美康试剂盒测试结果之间的强相关性(R^(2)=0.9769),表明试剂对TAT的检测具有等同性。进一步研究中将所有试剂优化并集中于一个单人份的卡条上,结合全自动化学发光分析仪进行定量检测,方便快捷。To quantify the concentration of thrombin-antithrombin III complexes(TAT),we developed and evaluated a chemiluminescent immunoassay(CLIA)for TAT detection.Initially,thrombin(T)and antithrombin(AT)were extracted from plasma and reacted in vitro to form TAT,which served as the immunogen.Through screening,we identified three hybridoma cell lines,1E9,16G4,and 1D7,that specifically recognize TAT.After further screening and pairing,biotin-conjugated monoclonal antibody 16G4 was used as the capture antibody and horseradish peroxidase-labled 1D7 was used as the detection antibody,thus establishing a double-antibody sandwich detection system.The optimal procedure for this method is a two-step process with an incubation time of 10 mins per step and a detection limit of 0.6 ng/mL,yielding results in less than 25 mins.The strong correlation between our measurements and those obtained using the Sysmex kit(R^(2)=0.9769)showed that the reagents were equivalent for the detection of TAT.In this study,all reagent components were integrated into a single cartridge,and quantitative detection was performed using a fully automated chemiluminescence analyzer for convenience and speed.

关 键 词：凝血酶-抗凝血酶Ⅲ复合物 化学发光检测法 单克隆抗体 辣根过氧化物酶 定量 

分 类 号：Q5-33[生物学—生物化学]