CN116410270A - 一种猫杯状病毒orf1重组非结构蛋白及其在制备间接elisa抗体检测试剂盒中的应用 - Google Patents
一种猫杯状病毒orf1重组非结构蛋白及其在制备间接elisa抗体检测试剂盒中的应用 Download PDFInfo
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- CN116410270A CN116410270A CN202310509792.4A CN202310509792A CN116410270A CN 116410270 A CN116410270 A CN 116410270A CN 202310509792 A CN202310509792 A CN 202310509792A CN 116410270 A CN116410270 A CN 116410270A
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Abstract
本发明涉及一种猫杯状病毒ORF1重组非结构蛋白及其在制备间接ELISA抗体检测试剂盒中的应用。本发明提供的间接ELISA抗体检测试剂盒包含以猫杯状病毒ORF1重组非结构蛋白制备的包被抗原、酶标一抗、酶标二抗、标准阳性血清、标准阴性血清、洗涤液、显色液、终止液。该间接ELISA抗体检测试剂盒可从血清学上区分FCV野毒感染抗体和FCV商品化灭活疫苗免疫抗体的临床样本,提升了FCV鉴别诊断的能力,为该病的防控工作提供技术支撑。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种猫杯状病毒ORF1重组非结构蛋白及其在制备间接ELISA抗体检测试剂盒中的应用。
背景技术
猫杯状病毒是一种引起猫科动物传染性疾病的主要病原体之一,该病毒的传染性极强,目前已呈世界性分布。目前我国针对该病主要通过免疫FCV商品化灭活疫苗来进行预防,由于FCV的高度变异,导致现有疫苗对不同毒株的交叉保护力不足,为了更好地在临床上区分FCV野毒感染和疫苗免疫情况,加快对FCV的鉴别诊断方法研究已经迫在眉睫。
目前对FCV的临床诊断主要根据FCV感染猫后引起的主要临床症状来进行初步判断,大部分猫科动物感染FCV后主要表现为流涎、鼻塞、咳嗽、发热等典型临床症状,但FCV与猫常见病原体如猫细小病毒(Feline panleukopenia virus,FPV)、猫疱疹病毒(Felineherpesvirus type 1,FHV-1)等病毒感染所引起的临床症状非常相似,仅通过临床症状判断难以确诊,且FCV极易发生变异,这给临床检测带来了极大挑战。如需要确诊是否为FCV感染,还需根据FCV核酸检测结果、猫杯状病毒抗体检测结果或其他分子生物学检测结果等实验室诊断结果进行确诊。
用于检测猫杯状病毒的实验室检测方法有很多,主要包括胶体金试纸条、聚合酶链式反应、病毒分离等。病毒分离鉴定是最古老的一种检测病原体的方法。RT-PCR法作为一种分子生物学检测方法因价格低廉,被许多学者作为实验室检测方法检测临床样本的FCV感染情况,该方法不仅用于FCV病原检测,还可分析FCV的变异情况,为FCV的流行病学调查提供了技术支撑,随着猫杯状病毒的不断变异,原有的RT-PCR检测方法检出率持续下降。近年来,随着病原体检测技术的迅速发展,胶体金检测技术越来越成熟,因其价格低廉,可快速出结果,目前国内在临床上对FCV病原体的检测主要采取胶体金拭子条检测技术。
由于猫杯状病毒的基因极易变异且猫感染后终身带毒,患猫康复后仍会持续排毒,增加了FCV的防控难度,然而上述检测技术并不能准确区分出野毒感染猫和疫苗感染猫,为此建立一种准确、快速、便捷的FCV野毒感染抗体检测方法已显得刻不容缓。
发明内容
猫杯状病毒是一种RNA病毒,基因结构为单股正链,没有囊膜。全基因约7700bp,全基因组分别编码ORF1、ORF2和ORF3 3个开放阅读框。编码非结构蛋白的ORF1氨基酸全长约1763aa,该部位位于全基因组的上游(5’端),此区域的蛋白比较保守,主要由RNA病毒解旋酶、RNA聚合酶和一个半胱氨酸蛋白酶构成,多聚蛋白前体被半胱氨酸蛋白酶切成p5.6、p32、p39、p30、p13和p76等多种非结构蛋白,基因组的5’端共价连接有病毒连接蛋白(Viralgenome-linekedprotein,VPg),VPg主要进行病毒蛋白的翻译工作,并决定猫杯状病毒基因组的感染性;ORF2基因组全长约2000bp,分为A-F六个区,ORF2最初编码衣壳蛋白的前体蛋白,经蛋白酶处理后转化为前导衣壳蛋白LC和成熟衣壳蛋白VP1。ORF3全基因大小约为320bp,位于全基因组的下游(3’末端),参与结构蛋白VP2的形成,VP2与猫杯状病毒Viruslike particles,VLPs的形成和复制有关。
国内外许多学者针对猫杯状病毒抗体检测方法进行了大量研究,目前国内主要通过VP1结构蛋白或者纯化的FCV作为包被抗原建立检测FCV抗体的ELISA方法,利用病毒作为抗原建立方法时,需要对病毒进行大量培养,纯化,此方法工作量大且容易造成病毒传播。以结构蛋白作为抗原建立的ELISA方法,无论是对疫苗产生的抗体还是野毒株产生的抗体都能与之结合,在检测时难以区分是疫苗免疫产生的抗体还是野毒感染产生的抗体。针对猫杯状病毒血清抗体的检测,国外研发出了商品化的猫杯状病毒抗体检测试剂盒,但进口的试剂盒价格昂贵,不适合作为FCV抗体检测的常规诊断方法。
基于现有技术中存在的技术问题,本发明将FCV ORF1非结构蛋白保守部位进行截短表达和纯化,以纯化的重组非结构蛋白作为抗原包被96孔酶标板,建立一种检测猫杯状病毒野毒感染抗体的间接ELISA方法。
为了解决上述技术问题,本发明采用以下技术方案:
一种猫杯状病毒ORF1重组非结构蛋白,其核苷酸序列为SEQ ID NO:1所示。
本发明还提供了一种猫杯状病毒的间接ELISA抗体检测试剂盒,包括前述的以猫杯状病毒ORF1重组非结构蛋白制备的包被抗原。
在本发明中,所述抗原的包被量为1~8μg/mL。
进一步的,所述抗原的包被量为2μg/mL。
在本发明中,所述间接ELISA抗体检测试剂盒还包括酶标一抗、酶标二抗、标准阳性血清、标准阴性血清、洗涤液、显色液、终止液中的至少一种。
在本发明中,所述酶标一抗为FCV商品化灭活疫苗高免血清;酶标二抗为HRP标记的兔抗猫IgG。
在本发明中,所述酶标一抗的稀释度为1:100~1600;优选地,所述酶标一抗的稀释度为1:800。
在本发明中,所述酶标二抗的稀释度为1:2000~10000;优选地,所述酶标二抗的稀释度为1:4000。
在本发明中,所述标准阳性血清是由如下方法制备得到的:
(1)将猫杯状病毒ORF1重组非结构蛋白与完全佐剂混合后,对猫进行皮下注射150~300μg,进行首次免疫;
(2)将猫杯状病毒ORF1重组非结构蛋白与不完全佐剂混合后进行第二、三次免疫,5~10d后收集高免血清,即得。
在本发明中,所述封闭液选自明胶、脱脂奶粉、PBST、BSA、PEG8000中的至少一种;优选为脱脂奶粉;进一步为1~5%脱脂奶粉。
本发明具有如下优点:本发明建立的间接ELISA抗体检测试剂盒对猫血清样本进行抗体检测,结果判定方便、特异性好,稳定性强,可用于从血清学上鉴别猫杯状病毒的野毒感染和FCV商品化灭活疫苗免疫感染情况,具有很强的实用性和推广性,非常适合作为FCV抗体检测的常规诊断方法。
附图说明
图1为FCV ORF1重组蛋白可溶性分析结果;
图2为FCV ORF1重组蛋白Western Blot鉴定结果;
图3为重组蛋白与FCV商品化灭活疫苗高免血清Western Blot鉴定结果;
图4为猫高免血清Western Blot鉴定结果;
图5为封闭时间、封闭液筛选结果;
图6为一抗孵育时间和抗体稀释液筛选结果;
图7为酶标二抗最适工作浓度、最佳孵育时间筛选结果;
图8为显色时间筛选结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
应当明确的是,下述实施例中所使用的实验方法如无特殊说明,均为常规方法,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本文中所用的术语“包含”、“包括”、“具有”、“含有”或其任何其它变形,意在覆盖非排它性的包括。例如,包含所列要素的组合物、步骤、方法或制品不必仅限于那些要素,而是可以包括未明确列出的其它要素或此种组合物、步骤、方法或制品所固有的要素。
实施例1ORF1重组非结构蛋白的表达、鉴定
1材料
临床样本与细胞系
临床样本采集自2021-2022年成都地区2家动物医院患有相应临床症状的猫眼鼻咽拭子,共96份。中华田园猫购买于成都市小动物交易市场,PET32a(+)载体、DH5a和Rosetta(DE3)感受态细胞均由实验室保存。
2.方法与结果
2.1氨基酸的生物信息学分析
2.1.1FCV ORF1氨基酸同源性分析
下载GenBank数据库中国内外上传的所有完整FCV ORF1非结构蛋白的氨基酸序列,利用电脑软件(MEGA7.0和MegAlign)对下载的氨基酸序列进行比对分析。截取非结构蛋白中的几段氨基酸序列进行串联。
2.1.2B细胞抗原表位预测
利用在线资源(Bcepred、ABCpred)和DNAstar软件对串联的ORF1氨基酸序列进行抗原表位预测,同时对亲水性、柔韧性和表面可及性等进行综合分析以确定本研究所需要合成的基因序列。
2.2原核表达载体的构建
2.2.1基因序列的优化与合成
由上海生工生物工程股份有限公司对FCV ORF1非结构重组蛋白的保守基因序列进行序列优化和合成,串联的氨基酸序列见表1。利用EcoRⅠ限制性内切酶和Xho1限制性内切酶进行双酶切后连接到PET32a(+)构建原核表达载体。合成的目的基因大小为954bp,序列见SEQ ID NO:1。
表1 FCV ORF1基因串联表达
2.2.2重组质粒的验证
重组质粒用PET32a(+)载体通用引物(T7、T7t)进行PCR鉴定和双酶切鉴定,将经PCR和双酶切鉴定结果均为阳性的重组质粒进行测序再次鉴定基因序列有无缺失,替换等异常情况。
2.2.3重组质粒的转化
通过热激法将连接至PET32a(+)载体的FCV阳性重组质粒转化至DH5a感受态细胞,以5000rpm/min离心5min后弃掉部分上清液,吸取100μL菌液涂布于加有氨苄青霉素的LB选择培养皿,37℃恒温培养箱培养24h~36h左右,用无菌白枪头挑取菌落用10μL ddH2O稀释混匀,进行PCR和双酶切鉴定,吸取10μL经PCR和双酶切鉴定均为阳性的细菌加入到3mL LB选择培养基进行细菌增菌,12h后收集菌液送测序鉴定。
将上述转化成功的重组质粒转化至Rosetta(DE3)感受态细胞中获得表达工程菌。转化步骤:从-80℃冰箱中取出50μL Rosetta(DE3)感受态细胞,放冰上静置10min左右使其充分融化,无菌吸取2μL重组质粒加入到装有Rosetta(DE3)感受态细胞的EP管内,混匀后放入4℃冰箱静置30min,迅速取出放入42℃恒温水浴锅内,90s后取出放冰上静置5min,用LB液体培养基补足至1mL,以190r/min的恒温摇床培养2h,2h后取出菌液以5000rpm/min离心15min,弃掉800μL上清液,无菌吸取100μL涂布于LB选择培养皿,37℃培养12-36h,挑取菌落进行双酶切和PCR鉴定,将鉴定成功的菌落增菌后送测序确认。
2.3 ORF1重组非结构蛋白的表达、鉴定
2.3.1 ORF1重组蛋白表达
吸取500μL种子菌放入50mLLB选择培养基培养,OD450nm值达到0.7左右时分组加入IPTG,使其终浓度分别为0.3mM,0.6mM,0.9mM,1.2mM,1.5mM,对IPTG诱导浓度进行筛选;其余条件不变,将诱导温度分别设为37℃、25℃、19℃和16℃4个梯度,筛选出蛋白表达最佳诱导温度;采取单一变量法将诱导时间分别设为:0h,2h,4h,6h,8h和过夜。最后收集不同条件表达的产物进行SDS-PAGE电泳检测,根据电泳检测结果确定蛋白最佳表达条件。如图1所示,当IPTG终浓度为0.6mM、0.9mM、1.2mM和1.5mM时对蛋白表达量无明显差异。诱导温度为19℃时蛋白表达量最高。诱导时间为2h、4h、6h和8h时,诱导时间对蛋白表达量无显著差异,过夜诱导时蛋白表达量最低。
利用优化的蛋白表达条件对ORF1重组非结构蛋白进行大量表达,通过10%的分离胶对表达的蛋白进行SDS-PAGE电泳检测。
2.3.2蛋白可溶性分析
将诱导表达的菌液在低温条件下用超声波破碎仪破碎30min后以12000rpm/min低温离心30min,将离心后的沉淀和上清进行分装。细菌沉淀用PBS稀释后和上清分别进行SDS-PAGE电泳检测,对表达的FCV ORF1重组非结构蛋白进行可溶性分析。结果显示,FCVORF1重组蛋白主要为包涵体蛋白,结果如图1所示。
2.3.3 FCV ORF1重组非结构蛋白的纯化
将诱导表达的菌液在4℃低温离心机中以12000rpm/min离心10min,弃上清液,收集菌体用PBS稀释重悬后在低温条件下超声破碎30min,离心弃上清,沉淀用BindingBuffer溶解后进行第二次超声破碎,离心收集上清用5mL镍柱按镍琼脂糖亲和层析蛋白纯化方法进行蛋白纯化,收集过柱的蛋白用10%分离胶进行SDS-PAGE电泳检测,将纯度最高的组分利用透析袋按梯度低渗透析法进行复性,收集复性后的蛋白,用PEG20000进行包埋浓缩,浓缩液用0.22μm滤器过滤除菌,用BCA蛋白浓度测定试剂盒对浓缩后的蛋白进行蛋白浓度测定,测得浓缩后的蛋白浓度为710ng/mL。分装后低温保存备用。
2.3.4 FCV ORF1重组非结构蛋白Western Blot鉴定
将表达的FCV ORF1重组非结构蛋白用10%的分离胶进行SDS-PAGE电泳,电泳结束后用转膜仪将分离胶上的蛋白转移到PVDF膜上,放入37℃恒温摇床中封闭2h,分别以猫杯状病毒标准阳性血清和FCV商品化灭活疫苗高免血清作为一抗,将一抗用5%BSA作1:500倍稀释,放入37℃恒温摇床中孵育2h,将HRP标记的兔抗猫IgG进行1:10000倍稀释,37℃恒温摇床中作用1h,最后用ECL显色成像进行Western Blot鉴定。检测结果显示,在55KDa处出现与预期相符的条带(图2)。
以FCV商品化灭活疫苗高免血清作为一抗,HRP标记的兔抗猫IgG作为二抗,表达的FCV ORF1重组非结构蛋白作为抗原进行Western Blot鉴定。结果显示,FCV商品化灭活疫苗高免血清与表达的FCV ORF1重组非结构蛋白不发生交叉反应,实验结果与预期相符(图3)。
实施例2基于ORF1蛋白ELISA抗体检测方法的建立
3.3.1标准阳性血清的制备结果
用间接ELISA方法检测3免后第七天的猫血清抗体效价,检测结果见表2。收集3免后第七天的血清用表达的FCV ORF1重组非结构蛋白作为抗原进行Western blot鉴定,结果显示,在55KDa处出现与预期相符的条带,鉴定结果见图4。
表2猫高免血清抗体效价测定结果
3.3.2蛋白包被浓度和一抗稀释度的筛选结果
将FCV ORF1重组非结构蛋白用PBS稀释至终浓度分别为1μg/mL、2μg/mL、4μg/mL、8μg/mL。将猫阳性血清用抗体稀释液进行倍比稀释后包被96孔酶标板,测定各孔的OD450nm值。结果显示,蛋白包被浓度为2μg/mL,一抗以1:800倍稀释时,P/N值最大,抗原与抗体结合效果最佳(表3)。
表3抗原包被浓度和一抗浓度筛选结果(OD450nm)
3.3.3最佳封闭时间和封闭液的确定结果
将抗原封闭时间分别设为37℃0.5h、37℃1h、37℃1.5h、37℃2h、37℃2.5h、37℃3h,每组设3个复孔,批间及批内重复做三次,取平均值,采用单一变量法对封闭时间进行筛选,以P/N值作为筛选依据,将检测数据利用Excel数据处理软件和SPSS软件对数据进行分析整理。结果显示,37℃封闭1.5h时,P/N值最大,对数据进行统计学分析发现,37℃封闭1h与37℃封闭1.5h两组数据之间P=0.693,大于0.05,组间差异不显著,因此,从节约实验时间角度考虑,选择1h作为抗原最佳封闭时间(图5)。
分别选用1%明胶、1%脱脂奶粉、PBST、5%脱脂奶粉、1%BSA、2%BSA、5%BSA、5%PEG8000作为封闭液,于37℃培养箱封闭1h。结果显示,用1%脱脂奶粉作为封闭液时P/N值最大,效果最佳(图5)。
3.3.4一抗作用时间和抗体稀释液选择结果
将一抗作用时间分别设置为37℃0.5h、37℃1h、37℃1.5h、37℃2h、4℃过夜,采用单一变量法优化一抗孵育时间,利用数据处理软件(Excel和SPSS软件)对实验数据进行整理分析,结果显示,将一抗放入37℃培养箱孵育1.5h时P/N值最大,且与其他组数据均存在显著差异,因此,选择1.5h作为一抗最佳孵育时间(图6)。
将抗体分别用2%明胶、5%明胶、2%BSA、5%BSA、PBST、PBS进行稀释,其余条件固定不变,每组设3个复孔,批间及批内各重复3次,测定OD450nm值,对抗体稀释液进行优化。结果显示,2%明胶作为抗体稀释液时效果最佳(图6)。
3.3.5二抗工作浓度和孵育时间的筛选结果
将酶标二抗按1:2000,1:4000,1:8000,1:10000倍倍比稀释,每组3个重复,采用上述优化后的条件,按2.4.2步骤对酶标二抗最适工作浓度进行优化,筛选出二抗的最适工作浓度。结果显示,将二抗稀释为1:4000时P/N值最高,因此,酶标二抗最适工作浓度为1:4000(图7)。
其余条件不变,将酶标二抗孵育时间分别设置为0.5h、1h、1.5h和2h,放入37℃培养箱孵育,每组设3个复孔,采用单一变量法对酶标二抗孵育时间进行筛选。结果显示,将二抗放入37℃培养箱孵育1h时,P/N值最大,且与其他组之间差异显著,因此,选择1h作为二抗最佳孵育时间(图7)。
3.3.6显色时间的优化结果
采用单一变量法,在加入TMB底物显色液后分别避光反应5min,10min,15min,20min,25min,30min,每组设3个复孔,批间和批内各重复3次,以P/N值最大作为筛选依据,优化底物显色时间。结果显示,底物显色时间为15min时,P/N值最大,故选用15min作为最佳显色时间(图8)。
3.4阴、阳性临界值的确定
利用已经建立的ELISA方法对8份猫阴性血清进行检测,结果显示,8份阴性血清OD450nm平均值(X)为0.125,标准方差(S)为0.025,按照cut-off=X+3S=0.2。当临床样本检测OD450nm值≥0.200时,则判定为阳性,若样本检测OD450nm值<0.200时,则判定为阴性。
3.5重复性试验
利用已经建立好的ELISA抗体检测方法检测6份不同的猫血清样本,批间和批内试验各重复3次,并计算其变异系数。结果根据表4、表5所示,6份猫血清样本的批间和批内试验变异系数均小于10%。
表4批内重复性试验结果
表5批间重复性试验结果
3.6特异性试验
运用已经建立好的ELISA抗体检测方法分别对猫疱疹病毒阳性血清、猫细小病毒阳性血清和FCV商品化灭活疫苗阳性血清进行检测,同时设阴阳性对照。检测结果如表6所示,在阴阳性对照成立的条件下,猫疱疹病毒阳性血清、猫细小病毒阳性血清和FCV灭活疫苗阳性血清的OD450nm值均小于0.200,为阴性,说明建立的ELISA抗体检测方法具有较好的特异性,且与FCV商品化灭活疫苗产生的血清抗体不发生交叉反应。
表6特异性试验结果(OD450nm)
3.7敏感性试验
利用本实验建立的间接ELISA抗体检测方法对两份猫血清样本进行检测,同时设阴性对照和阳性对照,检测结果如表7所示,将两份猫临床样本和标准阳性血清进行1:12800倍稀释时,检测结果仍为阳性,说明本研究建立的ELISA方法敏感性高。
表7敏感性试验结果
注:“+”表示阳性判定,“-”表示阴性判定。
3.8临床样本检测结果
首先,利用本发明建立的ELISA抗体检测方法分别对来自成都地区的70份猫血清样本(已知有22份野毒感染样本,20份疫苗感染样本,28份无病毒感染阴性样本)和FCV商品化灭活疫苗阳性血清进行检测,检测结果显示,70份猫血清样本中共检测到22份阳性,阴性样本为48份,其中FCV商品化灭活疫苗阳性血清检测结果为阴性。
其次,将经本发明建立的ELISA抗体检测方法检测为阳性的22份猫血清样本和FCV商品化灭活疫苗的阳性血清用全病毒包被ELISA抗体检测方法进行检测,结果显示,22份猫血清样本与FCV商品化灭活疫苗的阳性血清检测结果均为阳性。
最后,将经本发明建立的方法检测为阴性的48份样本用全病毒包被ELISA抗体检测方法进行检测,结果显示,48份样本中有28份检测为阴性,其余20份样本检测为阳性(表8)。综上所述,本发明建立的间接ELISA方法可用于区分FCV的野毒感染抗体和FCV商品化灭活疫苗免疫抗体。
表8临床样本检测结果
前述的实例仅是说明性的,用于解释本发明所述方法的一些特征。所附的权利要求旨在要求可以设想的尽可能广的范围,且本文所呈现的实施例仅是根据所有可能的实施例的组合的选择的实施方式的说明。因此,申请人的用意是所附的权利要求不被说明本发明的特征的示例的选择限制。在权利要求中所用的一些数值范围也包括了在其之内的子范围,这些范围中的变化也应在可能的情况下解释为被所附的权利要求覆盖。
Claims (10)
1.一种猫杯状病毒ORF1重组非结构蛋白,其特征在于,其核苷酸序列为SEQ ID NO:1所示。
2.一种猫杯状病毒的间接ELISA抗体检测试剂盒,其特征在于,包括权利要求1所述的以猫杯状病毒ORF1重组非结构蛋白制备的包被抗原。
3.根据权利要求2所述的间接ELISA抗体检测试剂盒,其特征在于,所述抗原的包被量为1~8μg/mL。
4.根据权利要求3所述的间接ELISA抗体检测试剂盒,其特征在于,所述抗原的包被量为2μg/mL。
5.根据权利要求2所述的间接ELISA抗体检测试剂盒,其特征在于,还包括酶标一抗、酶标二抗、标准阳性血清、标准阴性血清、洗涤液、显色液、终止液中的至少一种。
6.根据权利要求5所述的间接ELISA抗体检测试剂盒,其特征在于,所述酶标一抗为FCV商品化灭活疫苗高免血清;酶标二抗为HRP标记的兔抗猫IgG。
7.根据权利要求6所述的间接ELISA检测试剂盒,其特征在于,所述酶标一抗的稀释度为1:100~1600;优选地,所述酶标一抗的稀释度为1:800。
8.根据权利要求6所述的间接ELISA抗体检测试剂盒,其特征在于,所述酶标二抗的稀释度为1:2000~10000;优选地,所述酶标二抗的稀释度为1:4000。
9.根据权利要求5所述的间接ELISA抗体检测试剂盒,其特征在于,所述标准阳性血清是由如下方法制备得到的:
(1)将猫杯状病毒ORF1重组非结构蛋白与完全佐剂混合后,对猫进行皮下注射150~300μg,进行首次免疫;
(2)将猫杯状病毒ORF1重组非结构蛋白与不完全佐剂混合后进行第二、三次免疫,5~10d后收集高免血清,即得。
10.根据权利要求5所述的间接ELISA抗体检测试剂盒,其特征在于,所述封闭液选自明胶、脱脂奶粉、PBST、BSA、PEG8000中的至少一种;优选为脱脂奶粉;进一步为1~5%脱脂奶粉。
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