草鱼肠型点状产气单胞菌双抗夹心ELISA试剂盒的研制

Development of Double-antibody Sandwich ELISA Kit for Aeromonas punctata fintestinalis Ctenopharyngodon Idellus

通过对包被单抗和酶标单抗以及抗原最佳工作浓度的优化,建立草鱼肠型点状产气单胞菌双抗夹心ELISA试剂盒检测体系。用分光光度法对检测体系的特异性、稳定性和灵敏度进行测定;用脱脂奶平板法对试剂盒的临床应用进行检测。结果表明,单抗最适稀释液为pH9.4的碳酸盐缓冲液,洗涤液为含φ=0.05%Tween-20的PBS,包被单抗最适体积稀释倍数为1∶800,酶标单抗最适体积稀释倍数为1∶6 000,抗原最佳工作浓度为3×104cfu/mL。试剂盒与肠型点状产气单胞菌有特异性反应,脱脂奶平板法与试剂盒法对草鱼患病检出率基本一致,符合率达90%;试剂盒有很好的储存稳定性,检测灵敏度为7×103cfu/mL。

Double-antibody sandwich ELISA kit for Aeromonas punctata fintestinalis Ctenopharyngodon idellus was developed on the base of establishing double-antibody sandwich ELISA method,determining working concentration of coating monoclonal antibody and enzyme-labeling monoclonal antibody,as well as the best working concentration of antigen;measuring specificity,stability and sensitivity of the kit by spectrophotometry,and detecting the clinical application of the kit by skim milk plates.We used PBS（pH=7.4） as diluents for kit coating monoclonal antibody,and PBS（pH=9.4） with 0.05% Tween-20 as washing solution;the dilution multiple of coating monoclonal antibody was 1∶800,and t the enzyme-labeling antibody was 1∶6 000,the best working concentration of antigen was 3×104cfu/mL.The kit could responded to Aeromonas punctata fintestinalis,however,the kit did not have cross reaction with other strains;there was no significant difference of the kit by OD detection after 1 week storage at 4 ℃ and 37 ℃,respectively,as well as after 1,2 and 3 months storage at 4 ℃,respectively;kit sensitivity was 7×103cfu/mL;the kit and skim milk plate method showed a similar detection rate,the coincidence rate was more than 90%.The kit took a good performance in stabilization specification and detection rate,it was suitable for fast field detection.