拉沙里菌素单克隆抗体的研制及间接竞争ELISA检测方法的建立

摘要

To prepare monoclonal antibodies (MAb) against lasalocid (LAS) and establish an indirect competitive ELISA (Ci-ELISA) detection method,conjuaction of LAS-BSA and LAS-OVA were synthetized as the immunogen and coating antigen by using active ester method in this experiment.After 6 times of immunization,the spleen cells of mice and myeloma cells were fused.Finally,a hybridoma cell line that could stably secrete specific MAb against LAS was screened.The immunological subtype of the MAb was identified as IgG1,and its light chain was κ type.The titer of the ascites was 1 ∶ 16 000,which showed no cross activity with monensin sodium,salinomycin sodium,maduramycin,cefalotin sodium and streptomycin sulfate.Ci-ELISA method was established based on the MAb against LAS with the linear equation was y=0.376x-0.2374(R2 =0.9914),and the linear range was 5 to 1 000 ng/mL and the IC50 was 90.22 ng/mL.The method was used to detect LAS in spiked samples and the recovery rate was 81.76％ to 102.41％within the detection range,indicating that the method was successfully established with good accuracy and high sensitivity.The preparation of MAb against LAS and the establishment of Ci-ELISA method laid the foundation for the development of LAS detection kit.%为制备拉沙里菌素(LAS)单克隆抗体,建立针对LAS的间接竞争ELISA (Ci-ELISA)方法,试验采用了活性酯法将LAS分别与BSA和OVA偶联成LAS-BSA和LAS-OVA作为免疫原和检测原,6次免疫后,取小鼠脾细胞与骨髓瘤细胞融合,最终筛选出一株能稳定分泌抗LAS单克隆抗体的杂交瘤细胞株C11.经鉴定,C11的抗体类型为IgG1,轻链为κ链;所制腹水效价为1∶16 000,与莫能菌素钠、盐霉素钠、马杜霉素胺、头孢噻吩钠和硫酸链霉素均无交叉反应.用此单克隆抗体建立LAS Ci-ELISA检测方法显示,Ci-ELISA标准工作曲线为y=0.376x-0.2374(R2=0.9914),LAS在5～1 000 ng/mL时,线性关系良好,IC50为90.22 ng/mL,加标回收率为81.76％～102.41％,表明方法精确度好、灵敏度高.LAS单克隆抗体的制备和Ci-ELISA方法的建立为下一步试剂盒的研发奠定了基础.