免疫小鼠血清中猪圆环病毒2型抗体ELISA检测试剂盒的研究

Study on ELISA Kit for Detection of Porcine Circovirus Type 2 Antibody in Serum of Immunized Mouse

关键词:  猪圆环病毒2型  抗体  ELISA试剂盒

Key words:  porcine circovirus type 2  antibody  ELISA kit

摘要:

为研制一种小鼠血清中猪圆环病毒2型（Porcine circovirus type 2,PCV2）抗体检测试剂盒，以PCV2 Cap蛋白兔多抗作为包被用抗体，捕获纯化的PCV2 Cap蛋白，制备抗原检测板，辣根过氧化物酶(HRP)标记羊抗小鼠IgG作为二抗，建立检测小鼠血清中PCV2抗体ELISA检测方法并制备试剂盒。采用所制备的ELISA试剂盒对50份已知阴性血清样本进行检测，临界OD450nm值为0335；采用ELISA试剂盒与IFA分别对100份血清检测，总符合率为96%，检测小鼠血清敏感性效价达1∶16000，与其他常见的猪病毒小鼠阳性血清无交叉反应，2～8 ℃保存15个月稳定，批内变异系数为2.28%～4.17%，批间变异系数为4.27%～6.02%，具有良好的敏感性、特异性、稳定性及重复性；采用ELISA试剂盒对不同来源疫苗免疫组小鼠血清进行检测，均显示良好效果。研究表明，本研究制备的ELISA试剂盒可用于小鼠血清中PCV2抗体效价检测。

Abstract:

To develop a kit for detecting porcine circovirus type 2 (PCV2)antibody in mouse serum， PCV2 Cap protein rabbit polyclonal antibody was used as the coating antibody, the purified PCV2 Cap protein was captured to prepare an antigen detection plate, and horseradish peroxidase (HRP) labeled goat anti-mouse IgG was used as a secondary antibody, an ELISA method for detecting PCV2 antibody in mouse serum was established and a kit was prepared. The ELISA kit was used to detect 50 known negative serum samples, and the critical OD450 nm value was 0.335. This ELISA kit and IFA were used to parallel detection of 100 serum samples, the total coincidence rate was 96%, and the sensitivity of mouse serum was detected. The titer was 1∶16000, and there was no cross-reaction with other common pig virus mouse positive serum. It was stable for 15 months at 2～8 ℃. The intra-assay coefficient of variation was 2.28% to 4.17%, and the inter-assay coefficient of variation was 4.27% to 6.02%, with good sensitivity, specificity, stability and reproducibility. The serum of mice immunized with vaccines from different sources were detected by the ELISA kit, and all showed good results. In conclusion that the ELISA kit prepared in this study could be used for the detection of PCV2 antibody titer in mouse serum.