鸭疫里氏杆菌双抗体夹心ELISA检测方法的建立

Establishment of a double antibody sandwich ELISA for the detection of Riemerella anatipestifer

为快速检测鸭疫里氏杆菌,以纯化的兔抗1型鸭疫里氏杆菌抗体作为捕获抗体,以纯化的鸭疫里氏杆菌单克隆抗体作为检测抗体,建立了双抗体夹心ELISA方法。最佳的方案为用184ng/孔纯化的多抗包被过夜,100mL/L小牛血清37℃封闭2h,检测抗原、单抗、酶标抗体均为37℃作用1h,显色20min后终止反应,D450nm值高于0.330且P/N>2.1判定为阳性。该方法特异性强,与禽源巴氏杆菌、大肠杆菌、沙门菌无交叉反应,最低检测剂量为104 CFU/mL。与传统细菌分离方法相比,该方法的敏感性为100%,符合率为85.2%。本研究首次建立了检测鸭疫里氏杆菌的双抗体夹心ELISA方法,且特异、敏感。

The aim of the present study was to develop a rapid and accurate method for the detection of Riemerella anatipestifer based on a double antibody sandwich ELISA. Purified polyclonal antibody against serotype 1 R. anatipestifer was used as a capture antibody, while purified monoclonal antibody 5G7 was used as a detection antibody. The optimal conditions for the ELISA were as follows：polyclonal antibody with 184 ng per well was added to coat overnight,the plate was blocked by 100 mL/L neonatal calf serum for 2 hours at 37℃ ,the incubation times for detection antigen,monoclonal antibody and second labeled an- tibody were 1 hour at 37℃for each,the value of D450nm was obtained after 20 min coloration. This method has no cross-reaction with poultry-origin Pasteurella multocida, Escherichia coli and Salmonella, and its minimum detectable limit was 104 CFU/mL. The sensitivity of the method was 100% ,and the coincidence between bacterial isolation method and the double antibody sandwich ELISA was 85.2M. Therefore,this study provides a rapid, specific and sensitive detection method for R. anatipestifer.