重组乙脑抗原对乙型脑炎病毒IgG抗体反应活性的研究

Study on reactivity of recombinant encephalitis B antigen to IgG antibody of Japanese encephalitis virus

目的对克隆表达的乙脑重组抗原反应原性、特异性以及稳定性等进行评价优化,用于乙脑血清Ig G抗体ELISA诊断试剂盒的研制。方法基于乙脑病毒(JEV)SA14-14-2株的抗原蛋白经克隆表达,灭活纯化后作为包被抗原,利用ELISA方法对其最佳包被浓度,稳定性,反应活性以及特异性进行一系列的评价。结果重组抗原在1∶500的稀释条件下,阳性血清/阴性血清值最大为6.647,具有最佳反应原性,因此此稀释度为最佳;在1∶500稀释条件下,能准确地反映出乙脑病人血清IgG的变化。重组抗原37℃保存6 d活性仍保持75%以上,具有较好的稳定性;与市售同类型试剂盒比较,符合率达到95%。重组乙脑抗原与27份人正常血清和31份登革热病毒IgG抗体阳性而乙脑病毒IgG抗体阴性的血清均无交叉反应,表明具有良好的特异性。结论重组乙脑抗原蛋白稳定性好,特异性强,具有良好的血清学检测价值。

Objective To evaluate and optimize the reactionogenicity, specificity and stability of recombinant Japanese encephalitis antigen, which was used in the development of lgG antibody ELISA kit for Japanese encephalitis. Methods The recombinant antigen of Japanese encephalitis virus （JEV） from SA14-14-2 strain was inactivated and purified, used as a coating antigen, and then to evaluate the optimal concentration, stability, reactionogenicity and specificity by ELISA method. Results The optimal concentration of recombinant antigen is 1 ： 500, which has the maximum value of positive serum/negtive serum, 6.647; and can accurately reflect the change of serum IgG in Japanese encephalitis patients. The recombinant antigenactivity remained above 75%, which stored at 37℃ for 6 days. With the commercialization of the same kind of imported kits, the conformity rate was 95% and there was no cross reactivity. There was no cross reaction between the recombinant JEV antigen and the 27 human normal serums and 31 dengue virus IgG antibody positive serums. Conclusion The recombinant antigen had good stability and strong specificity and could be used for clinical labortory and detection of serum antibody.