脑心肌炎病毒抗体双抗原夹心ELISA检测方法的建立及初步应用

Development and preliminary application of double antigen sandwich ELISA method for encephalomyocarditis virus

目的建立脑心肌炎病毒(Encephalomyocarditis virus,EMCV)抗体双抗原夹心ELISA检测方法,用于EMCV血清学调查及临床检测。方法采用EMCV基因重组蛋白VP1、VP2和2C作为包被抗原,HRP标记EMCV作为酶标抗原,建立EMCV抗体双抗原夹心ELISA检测方法;用内部参考血清对建立的方法进行评价,并与间接ELISA法进行比较;采用所建立的方法对1 475份人血清和1 309份猪血清进行检测。结果建立的双抗原夹心ELISA法的最佳抗原包被浓度为7.5μg/ml,封闭液为10%马血清或2%鱼蛋白胨,酶标抗原稀释度为1∶400。该方法的批内和批间变异系数均小于10%;热稳定性较好;与间接ELISA法检测结果的符合率为93.5%。应用建立的方法检测人血清和猪血清中的EMCV抗体阳性率分别为16.8%和63.4%。结论已建立了EMCV抗体双抗原夹心ELISA检测方法,该方法可靠、稳定、特异、敏感、操作简便,为EMCV抗体的临床检测提供了有效的技术手段。

Objeelive To develop a double antigen sandwich ELISA （Ds-ELISA） method and apply to serological investigation and clinical determination of encephalomyocarditis virus （EMCV）. Methods A Ds-ELISA method was developed by using recombinant EMCV proteins VP1, VP2 and 2C as coating antigen, and HRP-labeled EMCV as enzyme-labeled antigen, then evaluated with internal reference serum, and the result was compared with that of indirect ELISA. A total of 1 475 human serum samples and 1 309 porcine serum samples were determined by the developed method. Results The optimal antigen concentration for coating of the developed Ds-ELISA method was 7. 5 I^g / ml, while the optimal blocking solution was 10% horse serum or 2% peptone, and the optimal dilution of enzyme-labeled antigen was 1 ： 400. Both intra- and inter-coefficients of determination results by the developed method were less than 10%. The method showed high heat stability, of which the coincidence rate of determination result to that of indirect ELISA was 93. 5%. The EMCV antibody positive rates in human and porcine serum samples determined by the developed method were 16. 8% and 63. 4% respectively. Conclusion A Ds-ELISA method for EMCV antibody was developed, which was reliable, stable, specific, sensitive and easy to handle. It provided an effective technique for clinical determination of EMCV antibody.