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 i: 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.%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.
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