环鸟苷酸兔单克隆抗体的制备和竞争酶联免疫吸附测定方法的建立  

Preparation of rabbit monoclonal antibody against cGMP and development of competitive ELISA for cGMP

作　　者：谭兴梅[1,2] 胡海峰 陈枢青[1] 

机构地区：[1]浙江大学药学院药理毒理与生化药学研究所,浙江杭州310058 [2]宜康(杭州)生物技术有限公司,浙江杭州310015

出　　处：《浙江大学学报（医学版）》2015年第4期423-428,共6页Journal of Zhejiang University(Medical Sciences)

摘　　要：目的：研制环鸟苷酸（cGMP）兔单克隆抗体，并建立测定cGMP浓度的竞争ELISA方法。方法：用人工合成的环鸟苷酸一钥孔血蓝蛋白（cGMP-KLH）免疫新西兰大白兔，采用Epitomics公司的兔单克隆抗体技术制备针对cGMP的兔单克隆抗体；应用cGMP兔单克隆抗体建立竞争ELISA方法，并对该方法的特异性、精密度和回收率进行测定。结果：所制备的cGMP兔单克隆抗体的效价为3．1．g／mL，50％抑制浓度（IC50）为12．57ng／mL。与环磷酸肌苷的交叉反应率为33．1％，与其他结构类似的化合物无交叉反应。所建立的cGMP竞争ELISA方法的检测范围为0～120ng／mL，最低检测限为1．95ng／mL，回收率为89％～103％，批次内和批次间的精密度分别小于11．68％和13．85％。结论：成功制备高效价、高特异性的cGMP兔单克隆抗体，并建立了测定cGMP浓度的竞争ELISA方法。Objective： To prepare rabbit monoclonal antibody （RabMab） against guanosine 3＇ , 5＇-cyclic monophosphate （cGMP） and to develop a competitive ELISA for the detection of cGMP. Methods： New Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin （cGMP-KLH） to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined. Results： The RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration （IC50） of 12. 57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3＇, 5＇-cyclic monophosphate （cIMP） and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0 - 120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89% 103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively. Conclusion： The RabMab against cGMP with high affinity and high specificity has been generated successfully, of cGMP has been developed with the prepared and a competitive ELISA for detection cGMP RabMAb

关 键 词：环GMP 抗体 单克隆 酶联免疫吸附测定 兔 

分 类 号：R392.11[医药卫生—免疫学]