抗黄曲霉毒素B1重链IgG2b亚型单克隆抗体的制备及应用  

Preparation and Application of Heavy Chain IgG2b Isotype McAb against Aflatoxin B1

作　　者：裴世春[1] 李玉花[2] 张园园[1] 才琳[1] 

机构地区：[1]东北林业大学,哈尔滨150040 [2]黑龙江八一农垦大学,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2009年第6期602-604,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30771799);黑龙江省教育厅项目(1151hz025);中国博士后科学基金资助项目(20070410882)

摘　　要：目的制备抗黄曲霉毒素B1(AFB1)的重链IgG2b亚型单克隆抗体,并建立黄曲霉毒素B1的间接竞争ELISA检测方法。方法采用AFB1-BSA偶联物为免疫抗原,AFB1-STI偶联物为检测抗原,利用间接竞争ELISA筛选分泌抗AFB1单抗的细胞株,并对抗体进行鉴定。结果筛选到1株能分泌特异性抗AFB1单克隆抗体的杂交瘤细胞株,其染色体数目为(90±10)条;所分泌的单抗亚类重链为IgG2b,轻链为λ;间接ELISA检测该株杂交瘤细胞分泌上清效价在1∶12800～1∶25600,纯化腹水效价为1∶105～1∶106。传30代及液氮中保存6个月,抗体效价稳定;建立的间接竞争ELISA检测方法的最低检出限为0.001ng/ml,校正曲线的线性范围为0.005～5ng/ml,IC50为0.01ng/ml;与AFB1的结构类似物AFM1和化学结构有差异的脱氧雪腐镰刀菌烯醇的交叉反应率分别为1.4%和<1%。结论所制备的抗黄曲霉毒素B1单克隆抗体具有高度特异性和稳定性,可应用于间接竞争ELISA检测方法。Objective To prepare the heavy chain IgG2b isotype McAb against aflatoxin B1 （AFB1） and develop an indirect competitive ELISA. Methods The cell strains secreting MeAb against AFB1 were screened by indirect competitive ELISA using AFB1-BSA conjugate as an antigen for immunization and AFB1-STI conjugate as an antigen for detection, and the secreted MeAbs were identified. Results A hybridoma cell strain secreting McAb against AFB1 specifically, with a chromosome number of 90 ±10, was screened, and the heavy and light chains of secreted McAb were IgG2b and k respectively. The titers of MeAb in euhure supernatant of hybridoma cells and purified murine aseites determined by indirect ELISA were 1 ： 12 800 - 1 ： 25 600 and 1 ： 105- 1 ： 106 respectively. After subculture of hybridoma cells for 30 passages or storage at liquid nitrogen for 6 months, the titer of secreted McAb was stable. The minimum detection limit, linear range of correction curve and IC50 of the developed indirect ELISA was 0. 001, 0. 005 - 5 and 0. 01 ng/ml respectively. The cross reaction rates of AFB1 with AFM1 and deoxynivalenol were 1.4% and less than 1% respectively. Conclusion The prepared McAb against AFBI showed high specificity and stability and might be used for development of indirect competitive ELISA.

关 键 词：黄曲霉毒素B1 重链IgG2b亚型 单克隆抗体 酶联免疫试验 

分 类 号：R446.61[医药卫生—诊断学]