摘要
本试验旨在建立一种准确、灵敏的定量检测猪流行性腹泻病毒(PEDV)S蛋白的夹心ELISA方法,用于PED疫苗的质量控制。采用重组PEDV全长S蛋白作为标准品,测定家兔源单克隆抗体GC44F3-1和GC32C11-5与PEDV S蛋白标准品的亲和力;优化夹心ELISA条件,验证方法的检测线性、检测限、准确性、重复性、稀释平行性及特异性;监测PEDV S蛋白标准品与预包被的ELISA酶标板保存稳定性,验证方法作为质控方法的可行性。制备的PEDV S蛋白纯度>90%,尺寸均一性较好;GC44F3-1和GC32C11-5的结合速率常数k_(on)分别为4.294×10^(5)和9.445×10^(4)(mol/L)^(-1)·s^(-1),二者解离速率常数k_(off)<1×10^(-7)s^(-1),解离常数Kd<10^(-12)mol/L。ELISA最优检测条件为:捕获抗体2μg/m L,检测抗体0.125μg/m L,捕获抗体4℃过夜包被,抗原37℃孵育1 h,检测抗体37℃孵育1 h,Streptavidin-HRP 37℃孵育25 min,37℃显色15 min。方法学验证表明,该方法具有良好的检测线性(R^(2)=0.9993,n=7),检测限为0.098 ng/m L;高、中、低3个浓度的PEDV S蛋白标准品回收率为94.5%~100.2%,加标回收率为84.3%~98.1%;批内和批间检测CV<5%,重复性良好;不同稀释倍数下检测值CV<5%,稀释平行性好;仅对PEDV检测时结果呈阳性,特异性良好。PEDV S蛋白标准品和预包被的ELISA酶标板稳定保存12个月。该方法能够用于不同生产过程样品的检测和不同批次乳化样品的保存稳定性监测。本研究建立的PEDV S蛋白夹心ELISA定量检测方法具有灵敏、准确、稳定等优点,对PED疫苗质量控制具有良好的应用前景。
This study aimed to develop and validate a double-antibody sandwich ELISA for the quantitative detection of the spike(S)protein of porcine epidemic diarrhea virus(PEDV),with the goal of establishing a sensitive and reliable method for quality control of PED vaccines.Recombinant full-length S protein was used as the standard.The antibody affinities of the screened rabbit monoclonal antibodies GC44F3-1 and GC32C11-5 were determined.Conditions for sandwich ELISA were optimized and the ELISA method was validated for linearity,limits of detection,accuracy,repeatability,dilutional parallelism,and specificity.Storage stability of the PEDv S protein standard and pre-coated ELISA plates was monitored.The feasibility of the established method as a quality control tool was validated.The purity of the prepared PEDV S protein was>90% and good size homogeneity.The association rate constants(k_(on))of GC44F3-1 and GC32C11-5 were 4.294×10^(5) and 9.445×10^(4)(mol/L)^(-1)·s^(-1),respectively.The dissociation rate constants(k_(off))of both antibodies were<1×10^(-7) s^(-1),and their dissociation constants(K_(d))were<10^(-12) mol/L.Optimal assay conditions included capture antibody of 2μg/mL and detection antibody of 0.125μg/mL,capture antibody overnight coating at 4 C,antigen incubation at 37 C for 1 h,detection antibody incubation at 37 C for 1 h,streptavidin-HRP incubation at 37 C for 25 min,and chromogenic reaction at 37 C for 15 min.The method showed good linearity(R^(2)=0.9993,n=7)and a detection limit of O.O98 ng/mL.The recovery rates for high,medium,and low concentrations of PEDV S protein standards were 94.5% to 100.2%,with spiked recovery of 84.3% to 98.1%.Both intra-assay precision and inter-assay precision coefficients of variation(CV)were less than 5%,indicating good repeatability.The CV of detected values under different dilution factors were<5%,demonstrating good dilution parallelism.The method showed a positive result only for PEDV,confirming excellent specificity.The PEDV S protein standard and pre-coated ELISA plates remained stable for up to 12 months.Furthermore,it demonstrated broad applicability for detecting PEDV in various production process and for monitoring the storage stability of emulsified samples across different batches.The established sandwich ELISA method for quantitative detection of PEDv S protein offers high sensitivity,accuracy,and stability.It is well-suited for the quality control of PED vaccine,demonstrating promising application prospects.
作者
张承凤
杜久斌
曹光辉
陈晓洁
黎明
贺笋
杨延丽
ZHANG Chengfeng;DU Jiubin;CAO Guanghui;CHEN Xiaojie;LI Ming;HE Sun;YANG Yanli(Tecon Pharmaceutical Co.,Ltd.,Suzhou 215000,China;Tecon Biology Co.,Ltd.,Urumqi 830010,China)
出处
《中国兽医科学》
北大核心
2026年第2期216-224,共9页
Chinese Veterinary Science
基金
新疆维吾尔自治区“天池英才”青年博士项目(ZH-03)。
关键词
猪流行性腹泻病毒
S蛋白
双抗体夹心ELISA
疫苗
质量控制
porcine epidemic diarrhea virus(PEDV)
S protein
double-antibody sandwich ELISA
vaccine
quality control
