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目的建立及验证西尼罗病毒(West Nile virus,WNV)抗体间接ELISA检测方法,并用于评价疫苗的免疫效果。方法以纯化后的WNV E蛋白第三结构域(EDⅢ)蛋白作为包被抗原,建立WNV抗体间接ELISA检测方法,对抗原包被浓度(2.5、5、10、20、40、80μg/ml)、血清稀释度(1:10、1:20、1:40、1:80、1:160、1:320)、抗原封闭时间(0.5、1、1.5、2 h)、血清孵育时间(0.5、1、1.5、2 h)、HRP标记的山羊抗马Ig G稀释倍数(1:5 000、1:10 000、1:20 000、1:40 000、1:80 000稀释)及其孵育时间(0.5、1、1.5、2 h)进行优化,并验证该方法的特异性、敏感性及精密性。采用建立的方法检测WNV阴性马血清和病毒样颗粒(WNV-VLPs)疫苗免疫的马血清,与ALPHA公司试剂盒的检测结果进行比较。结果间接ELISA法最佳检测条件为:抗原包被浓度为20μg/ml,4℃包被过夜;Blocking ACE封闭液于37℃封闭1.5 h;加入血清(1:80稀释),37℃孵育1.5 h;加入HRP标记的山羊抗马Ig G(1:20 000稀释),37℃孵育1 h。该方法可特异性检测WNV阳性血清中的WNVE-Ig G抗体;阳性血清稀释至1:2 560时,检测结果仍为阳性;批内和批间的变异系数(CV)均小于7%。25份WNV阴性马血清检测结果均为WNVE-Ig G抗体阴性,经WNV-VLPs疫苗免疫的马血清中的WNVE-Ig G抗体效价水平随免疫次数的增加呈递增趋势,间接ELISA法与ALPHA公司试剂盒的检测结果完全一致。结论成功建立了WNV抗体间接ELISA检测方法,可用于疫苗免疫效果的评价,为开展WNV流行病学调查、诊断及新型疫苗的研发奠定了基础。
Objective To establish and validate an indirect ELISA for the detection of West Nile virus (WNV) antibody and to evaluate the immunogenicity of the vaccine. Methods The purified WNV E domain protein (EDⅢ) was used as coating antigen to establish an indirect ELISA for detection of WNV antibody. The antigen concentration (2.5, 5, 10, 20, 40 and 80μg / ml) Serum dilutions (1:10, 1:20, 1:40, 1:80, 1:16, 1:20), antigen blocking time (0.5,1,1.5,2 h), serum incubation time (0.5,1 , 1.5, 2 h), the HRP-labeled goat anti-horse Ig G dilution (1: 5000, 1:10 000, 1:20 000, 1:40 000, 1:80 000 dilution) and incubation time , 1, 1.5, 2 h), and verify the specificity, sensitivity and precision of this method. The established method was used to detect the serum of horse serum immunized with WNV-negative horse serum and virus-like particles (WNV-VLPs) vaccine and compared with the results of ALPHA kit. Results The optimal conditions of indirect ELISA were as follows: the antigen was coated at a concentration of 20μg / ml overnight at 4 ℃; Blocking ACE blocking solution was blocked at 37 ℃ for 1.5 h; serum (1:80 dilution) HRP-labeled goat anti-horse IgG (1:20 000 dilution) was added and incubated at 37 ° C for 1 h. The method can specifically detect WNVE-Ig G antibody in WNV positive sera. The positive serum is still positive when diluted to 1: 2 560; the coefficient of variation (CV) within and between batches is less than 7%. The results of 25 WNV negative horse serums were negative for WNVE-Ig G antibody. The titer of WNVE-Ig G antibody in horse serum immunized with WNV-VLPs vaccine increased with the increase of the number of immunizations. Indirect ELISA and ALPHA Test results of the company kit exactly the same. Conclusion The ELISA method for indirect ELISA of WNV antibody was successfully established and could be used to evaluate the vaccine immunization efficacy. It laid the foundation for the investigation and diagnosis of WNV epidemiology and the development of novel vaccine.