不同抗原对猪瘟病毒抗体ELISA检测效果的影响

Effect of ELISA Detection Result for the Classical Swine Fever Virus Antibody by Using different Antigents

关键词:  ELISA  检测效果  原核表达  E2蛋白

Key words:  enzyme linked immunosorbent assay  detection result  prokaryotic expression  E2 protein

摘要:

为研究不同抗原对猪瘟病毒抗体ELISA检测效果的影响，通过原核表达获取猪瘟病毒重组E2蛋白、E0蛋白、C蛋白和NS5B蛋白，大小分别约为35 kD、42 kD、16 kD和80 kD。经Ni-NTA亲和层析柱纯化并利用BandScan软件进行计算，纯度均在90%以上，满足ELISA检测包被用原料纯度的要求。将上述4种蛋白作为包被抗原进行ELISA，以10份企业阳性质控品血清和10份企业阴性质控品血清为检验指标比较不同抗原对猪瘟病毒抗体ELISA检测效果的影响。结果以E2蛋白为原料的包被板检测质控血清时，灵敏度和特异性均达到80%以上，能够满足猪瘟病毒抗体ELISA检测的要求，故选择E2蛋白作为包被抗原并进行相关检测试剂的研制。用研制的试剂与国际知名度高、产品质量好的美国IDEXX同类试剂对432份临床猪血清进行符合性检测，结果与美国IDEXX试剂的阳性符合率为95.53%，阴性符合率为86.56%，总符合率为91.67%，两种试剂检测结果具有较高的一致性。试验表明，用E2蛋白为包被抗原对猪瘟病毒抗体进行ELISA检测的效果最好，制备的检测试剂可用于临床猪瘟病毒血清抗体的检测，为今后猪瘟病毒抗体ELISA检测试剂的进一步研制奠定了基础。

Abstract:

To study the effect of detection result in ELISA for the Classical Swine Fever Virus (CSFV) antibody by using different antigents, the recombinant E2 protein, E0 protein, C protein and NS5B protein of CSFV were obtained by prokaryotic expression which were about 35 kD, 42 kD, 16 kD and 80 kD. These proteins were putiried by Ni-NTA affinity chromatographic column. The purity of the raw materials calculated by BandScan software was suitable for ELISA detection. The ELISA was carried on by using four proteins above as coating antigents. The effect of ELISA detection result for CSFV antibody by using different antigents was compared through ten samples of CSFV-positive sera and ten samples of CSFV-negetive sera from company. The result showed that the sensitivity and specificity of reagent can reach more than 80% by using E2 protein as the raw material which was coated to detecting the control sera. Therefore, E2 protein was satisfied for CSFV antibody ELISA detection. As the results, E2 protein was treated as the coating antigent to developing ELISA reagent. The positive coincidence rate, negetive coincidence rate and total coincidence rate of developed reagent were 95.53%, 86.56% and 91.67%, compared to IDEXX reagent with high popularity and good quality internationally from Untied States on 432 samples of pig clinical samples. In a word, the ability to detect the CSFV antibody from IDEXX and my company were almost the same. The results indicated that it''s the best effect of ELISA detection result for CSFV antibody by using E2 protein as coating antigen. This reagent can be used for the detection of pig clinical sera, which laid the foundation for the further study of the detection reagent in ELISA .