检测瞬时受体电位通道6的ELISA双抗体夹心法的建立  

Construction of a double antibody sandwich ELISA for determination of transient receptor potential channel 6

作　　者：张萃[1] 梅俪凡[2] 卢文菊[3] 刘晓波[1] 黄超贤[1] 李红枝[1] 

机构地区：[1]广东药学院微生物学与免疫学教研室,广州510006 [2]中山市博爱医院肾内科,528403 [3]广州医科大学第一附属医院呼吸疾病国家重点实验室,510230

出　　处：《免疫学杂志》2013年第12期1084-1087,共4页Immunological Journal

基　　金：中山市科技计划项目(20102A021);华南中医药城项目(20101H014)

摘　　要：目的建立ELISA双抗夹心法以用于TRPC6蛋白的测定。方法采用mAb相加法和配对实验,选择最佳配对组合;并通过cELISA法测定mAb阻断率,以选亲和力较高的mAb进行HRP标记,最终建立ELISA双抗体夹心法以用于大鼠和小鼠脑组织TRPC6蛋白的测定。结果 mAb-A2与mAb-F11的AI>50%,配对较佳;mAb-F11亲和力较强,IC50为0.6μg/ml;HRP标记mAbF11的最低工作浓度为1∶1 600;建立ELISA双抗夹心法可检测到大鼠和小鼠脑组织TRPC6蛋白分别为(1.42±0.06)μg/ml和(0.88±0.04)μg/ml。检测线性范围0.625～10μg/ml,R2=0.992,组间变异系数小于10%。结论所建立的ELISA双抗夹心法灵敏高,特异性强,重复性好,可用于脑组织中TRPC6蛋白的测定。This study aimed to establish a double antibody sandwich ELISA method for determination of transient receptor potential canonical （TRPC） 6 protein. The best matching double antibody combination was determined by ELISA combined method and antibody pair matching test. The blocking rate of monoclonal antibodies （mAb） was measured by competitive ELISA. Then the mAb with high affinity were marked with HRP and used in double antibody sandwich ELISA for detecting TRPC6 protein in brain-tissue of rat or mouse. Results demonstrated that mAb-A2 and -Fll posses high AI-value （AI〉50%）, which were then selected as matching antibody pairs for ELISA. Clone-F11 had higher affinity （IC50=0.6 txg/ml） and was taken as HRP-linked detection antibody in ELISA with maximal working dilution of 1：1 600. The minimal detection levels of the ELISA on brain TRPC6 of rats and mice were （1.42±0.06） μg/ml and （0.88±0.04） μg/ml respectively, with a liner detection range of （0.625-10） μg/ml （R2=0.992）, and inter variation coefficient of less than 10%. In conclusion, the developed sandwich ELISA is sensitive, specific and reproducible, thus might be used in testing TRPC6 protein in brain.

关 键 词：TRPC6 MAB ELISA双抗体夹心法 

分 类 号：R446.6[医药卫生—诊断学]