19-去甲睾酮异源性ciELISA试剂盒的研制及应用

Development and Application of Heterologous ciELISA Kit for Rapid Detection of 19-Nortestosterone Residues

筛选抗19-去甲睾酮(NT)单克隆抗体,建立异源性ciELISA试剂盒。用人工抗原NT-17-BSA免疫Balb/C小鼠,细胞融合技术建立1株抗NT单克隆杂交瘤细胞NT1-B7,同种型为IgG1/k,腹水效价达0.64×105,亲和常数(Ka)为4.31×109L/mol,半数抑制浓度(IC50)为0.55ng/ml,与群勃龙和雌二醇的交叉反应率(CR)分别为2.2%和1.6%,与其它化合物无CR。异源性ciELISA试剂盒在猪肉、猪尿、牛肉和牛尿四种基质中的检测限分别为0.24ng/ml、0.23ng/ml、0.22ng/ml和0.17ng/ml,批内和批间相对标准差(RSD)在4.4%～21.8%,添加回收率在85%～110%,4℃下保存期至少6个月。NT-Kit的基质pH适用范围为6.5～7.5,温育过程优化后可节约检测时间30～90min。试剂盒灵敏度高、特异性强,可应用于样品基质中NT残留检测。

To screen anti-Nortestosterone monoclonal antibody （NT mAb） and develo Pheterologous ciELISA kit.Balb/C mice were immunized with artificial antigen of NT-17-BSA and a strain of hybridoma cell named NT1-B7 was produced,which can stably secret NT mAb.The isotype of mAb was IgG 1 /k,ascites titer was 0.64×105,affinity constant （Ka） was 4.31×109 L/mol and IC 50 value determined by ciELISA was 0.55ng/ml.Of all the analogous competitors,2.2% and 1.6% cross-reactivity （CR） were observed for trenbolone and estradiol,respectively,while no CR was obtained for other compounds.With coating antigen of NT-3-OVA,heterologous ciELISA kit was developed,which limits of detection in pork,swine urine,beef and cattle urine were 0.24ng/ml,0.23ng/ml,0.22ng/ml and 0.17ng/ml,respectively.The recoveries of NT spiked in the four matrix were in the range of 85%~110%,while intra-and inter-relative standard deviation （RSD） were in the range of 4.4%~21.8%.when stored at 4℃,the NT-Kit could preserve 6 months or more.The pH of validation detection in matrix ranged from 6.5 to 7.5,and the program could save 30~90 min after the incubation process was optimized.With high sensitivity and specificity,this ciELISA kit could be advantageously utilized for the primary screening of sample matrix for presence of 19-NT residue.