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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 etiological, specificity and stability of recombinant e antigen of Japanese encephalitis cloned for the development of ELISA diagnostic kit for IgM antibody against Japanese encephalitis B virus. Methods The antigens of JEV SA14-14-2 strain were cloned and expressed in vitro. The antigens were purified by inactivation and purification and used as coating antigen. The optimal coating concentration, stability, reactivity and specificity were determined by ELISA A series of comments. Results The dilution of the recombinant antigen at a dilution of 1: 500 gave a maximum positive serum / negative serum value of 6.647 with optimal reactivity and was therefore optimal for dilution; at a dilution of 1: 500 it accurately reflected Changes of Serum IgG in JE Patients. The activity of recombinant antigens remained above 75% at 37 ℃ for 6 days, with good stability. Compared with the same type of commercially available kit, the coincidence rate reached 95%. There was no cross-reaction of recombinant JE against 27 human normal sera and 31 dengue virus IgG positive and JE virus IgG negative sera, indicating good specificity. Conclusion The recombinant JE antigen protein has good stability and specificity, and has good serological detection value.