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目的建立GⅡ.4诺如病毒(Norovirus,No V)抗原ELISA定量检测方法,用于重组GⅡ.4 No V病毒样颗粒疫苗研制中抗原含量检测。方法使用纯化的GⅡ.4 No V病毒样颗粒作为免疫原,分别免疫新西兰大白兔和豚鼠,制备抗血清。以豚鼠抗GⅡ.4病毒样颗粒多抗作为包被抗体,HRP标记的兔抗GⅡ.4病毒样颗粒多抗作为检测抗体,建立GⅡ.4 No V抗原ELISA定量检测方法,确定该方法的线性范围,并对该方法的准确度、精密度、稳定性、特异性进行验证;用建立的方法检测GⅡ.4病毒样颗粒制备过程中间品的抗原含量。结果 GⅡ.4病毒样颗粒内部参考品在15.6~250 ng/ml范围内,线性关系良好,R2>0.98,高、中、低3个浓度样品回收率在90.9%~110.2%之间,板内和板间精密度试验的CV值均不超过15%;包被微孔板37℃放置6 d,稳定性试验的CV值均小于15%;与GⅡ.4No V病毒样颗粒之外的抗原均无交叉反应;可用于定量检测重组No V抗原制备过程中不同阶段样品。结论建立了GⅡ.4 No V抗原ELISA定量检测方法,该方法准确度、精密度、稳定性、特异性、适用性均良好,为No V病毒样颗粒疫苗的研制提供了质控方法。
Objective To establish a method for the quantitative detection of GⅡ.4 Norovirus (No V) antigen, and to detect the antigenic content of recombinant GⅡ.4 No V virus-like particle vaccine. Methods Purified GⅡ.4 No V virus-like particles were used as immunogen to immunize New Zealand white rabbits and guinea pigs respectively to prepare antiserum. Using guinea pig anti-GⅡ.4 virus-like particle polyclonal antibody as coating antibody and HRP-labeled rabbit anti-GⅡ.4 virus-like particle polyclonal antibody as detection antibody, a GⅡ.4 No V antigen ELISA assay was established to determine the linearity of this method Range, and verify the accuracy, precision, stability and specificity of the method. The established method was used to detect the antigen content in the intermediate product of GⅡ.4 virus-like particles. Results The internal reference of GⅡ.4 virus-like particles showed a good linearity in the range of 15.6-250 ng / ml with R2> 0.98. The recoveries of three samples with high, medium and low concentrations ranged from 90.9% to 110.2% And CV between the plate precision test were not more than 15%; coated microplates placed at 37 ℃ for 6 d, the stability test CV values were less than 15%; and G Ⅱ .4No V virus-like particles were antigens No cross-reaction; can be used for quantitative detection of recombinant No V antigen in different stages of the sample preparation process. Conclusion The quantitative detection method of GⅡ.4 No V antigen ELISA was established. The accuracy, precision, stability, specificity and applicability of the method were satisfactory. The quality control methods for No V virus-like particle vaccine were established.