石房蛤毒素时间分辨荧光免疫检测试剂盒的研制

Development of time-resolved fluoroimmunoassay kit for detection of saxitoxin

目的制备石房蛤毒素（STX）的单克隆抗体,利用时间分辨荧光免疫分析技术建立石房蛤毒素超微量的检测方法。方法利用STX-KLH偶联抗原免疫小鼠的脾细胞与骨髓瘤细胞Sp2/0融合,筛选后获得稳定分泌抗STX抗体的杂交瘤细胞株,腹水诱生法制备大量单抗,采用间接竞争法建立STX-TRFIA时间分辨荧光免疫检测试剂盒,对试剂盒各项性能进行评价。结果该试剂盒最低检出浓度为1.0 ng/ml,校准曲线线性范围为2.0~512 ng/ml,线性方程Y=-0.455 7X＋22.872（｜r｜=0.998 3）,与腹泻性贝类毒素（DSP）、神经毒性贝类毒素（NSP）、失忆性贝类毒素（ASP）均无交叉反应,平均回收率为94.66%,与进口试剂盒同时检测广州市水产品中贝类毒素的含量,其相关系数为0.9764,P〈0.001。结论试剂盒各项指标均接近进口试剂盒水平,达到检验要求,可满足实际工作需要。

Objective To make monoclonal antibodies against Saxitoxin（STX）and develop a rapid, sensitive and quantitative ELISA kit for STX based on time-resolved fluoroimmunoassay technology. Methods Spleen cells from 6-8 weeks old female BALB/c mice immued with FB1-KLH were fused with the Sp2/0 myeloma cells. Hybridoma cell linesstably secreting McAb against FB1 were selected.Ascites in mice was produced by intraperitoneal injection and antibodies were purified with protein A affinity chromatography. The STX-TRFIA detection kit was established based on indirect competitive ELISA method and evaluated. Results The limited concentration of detection was 2 ng/ml, linear range was 2-300 ng/ ml; the linear equation was Y=-0.455 7X＋22.872 （r=0.998 3）. There was no cross reaction with ASP, NSP, and DSP. The mean recovery rate was 94.66%. Compared with another kit from USA, the correlation coefficient of results from shellfish toxin levels detection in aquatic products in Guangzhou city with the kit was 0.957 4,P〈0.001. Conclusion The ELISA-kit deteetition ability meet the needs of practical work.