摘要
本试验旨在建立胶体金免疫层析法快速定量猪流行性腹泻病毒(PEDV)N蛋白的方法,评价其用于疫苗质量控制的可行性。通过大肠杆菌重组表达PEDVN蛋白,并进行Ni金属螯合层析纯化,制备N蛋白标准品;优化胶体金免疫层析法检测条件,包括检测体积、裂解温度、裂解时间、反应温度、反应时间;建立N蛋白标准品浓度与胶体金试纸条检测灰度的响应曲线,考察检测限、准确度、精密度、对不同生产阶段PEDV样品的检测适用性和应用于成品疫苗稳定性监测的可行性;比较胶体金免疫层析法定量结果与TCID_(50)测定结果的相关性。结果表明,制备的N蛋白标准品纯度为94.5%;检测最佳条件为将PEDV样品在室温下裂解10min后,取100μL于胶体金试纸条中,室温反应20min后检测;在最佳条件下,N蛋白浓度与胶体金试纸条检测灰度的四参数拟合曲线R2=0.99727,检测限为7.14ng/mL,不同浓度的N蛋白标准品回收率为92.2%~111.5%,高、低浓度的PEDV样品6次重复检测RSD<10%,可用于细胞培养液、灭活样、成品疫苗的检测和成品疫苗储存稳定性监测;N蛋白浓度与TCID_(50)结果具有相关性,线性拟合Rs^(2)=0.97566,Pearson相关系数为0.98776。综上所述,胶体金免疫层析法定量PEDVN蛋白检测速度快,具有良好的线性、准确性和重复性,可应用于PEDV样品的快速定量检测和疫苗质量快速评估。
rapid and quantitative method basing on colloidal gold immunochromatography assay was established for the detection of porcine epidemic diarrhea virus(PEDV)N protein.The utility of this method in the quality control of PEDV vaccines was evaluated.The PEDV N protein was recombinantly expressed in E.coli and purified by Ni metal chelate chromatography to produce the N protein standard.The assay conditions for the colloidal gold immunochromatography were optimized,including volume,lysis temperature,lysis time,reaction temperature,and reaction time.A response curve between the concentration of N protein standard and the gray level of the colloidal gold reagent test strip was established under the optimized conditions.The limit of detection,accuracy and precision were determined.Applicability of the method for detecting PEDV samples at different processing steps and for monitoring finished vaccines was evaluated.The correlation between the colloidal gold assay results and TCID_(50) assay was analyzed.The results showed that the purity of the prepared N protein standard was 94.5%.The optimal conditions for the colloidal gold immunochromatography assay were lysing the PEDV sample at room temperature for 10 min,adding 100μL to the colloidal gold test strip,and detecting after a 20-min reaction.Under these conditions,the four-parameter fit curve showed R=0.99727,and the limit of detection was 7.14 ng/mL.The recoveries of different concentrations of N protein standard were 92.2%~1l1.5%,and six replicates of PEDV samples with high and low concentrations showed an RSD of less than 10%.The method was proved suitable for detecting cell culture fluid,inactivated samples and finished products of vaccines,as well as for monitoring the storage stability of vaccines.The concentration of N protein showed a strong correlation with TCID_(50) results,with Rs^(2)=0.97566 and the Pearson's correlation coefficient of o.98776.In conclusion,the colloidal gold immunochromatography assay for quantitative detection of PEDV N protein is fast,exhibits good linearity,accuracy and reproducibility,and is applicable for the rapid quantification of PEDV samples and the rapid assessment of vaccine quality.
作者
宫萱
陈诗雅
杜久斌
陈晓洁
张承凤
黎明
杨延丽
贺笋
GONG Xuan;CHEN Shiya;DU Jiubin;CHEN Xiaojie;ZHANG Chengfeng;LI Ming;YANG Yanli;HE Sun(Tecon Pharmaceutical Co.,Ltd.,Suzhou 215000,China)
出处
《中国兽医科学》
北大核心
2025年第7期905-912,共8页
Chinese Veterinary Science
基金
新疆维吾尔自治区“天池英才”青年博士项目。
关键词
猪流行性腹泻病毒
胶体金免疫层析法
N蛋白
定量
疫苗
porcine epidemic diarrhea virus(PEDV)
colloidal gold immunochromatography assay
N protein
quantification
vaccine
