CN117430675A - 用于检测鸡传染性支气管炎的试剂盒的抗原及其制备方法、试剂盒 - Google Patents
用于检测鸡传染性支气管炎的试剂盒的抗原及其制备方法、试剂盒 Download PDFInfo
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Abstract
本发明公开了一种用于检测鸡传染性支气管炎的试剂盒的抗原及其制备方法、试剂盒,涉及生物工程技术领域。本发明利用能够稳定表达重组鸡传染性支气管炎病毒核蛋白(N蛋白)的重组菌,在特定条件下诱导表达重组N蛋白并经亲和层析纯化。用纯化出的重组蛋白作为包被抗原,建立了一种简便、特异的间接ELISA方法。本发明还为检测鸡传染性支气管炎病毒抗体提供了特异、敏感、快速、操作简便的试剂盒。
Description
技术领域
本发明涉及生物工程技术领域,尤其涉及一种用于检测鸡传染性支气管炎的试剂盒的抗原及其制备方法、试剂盒。
背景技术
传染性支气管炎病毒(Infectious bronchitis Virus,IBV)属于冠状病毒科、冠状病毒属、γ冠状病毒属,可引起鸡传染性支气管炎(Infectious bronchitis,IB),表现为鸡呼吸道疾病、肾脏和生殖腺病变。该病给世界各地的养鸡业造成了严重的经济损失,目前主要依靠灭活疫苗和弱毒疫苗防控,但病毒易于突变、重组出现新的毒株而导致该病在鸡群时有发生。目前IBV的血清学诊断方法主要有鸡胚中和试验、琼脂扩散试验(AGPT)、血凝抑制试验(HI)和酶联免疫吸附试验(ELISA)。ELISA相对于其他方法具有灵敏度高、简便快速、特异性强等优点,有利于检测大规模群体的免疫状况,成为在IBV检测中的首选方法。
发明内容
本发明所要解决的技术问题在于,提供一种用于鸡传染性支气管炎检测试剂盒的抗原及其制备方法,其具有良好的免疫原性,表达效率高,纯度高,包被量小,反应灵敏,为试剂盒的制备奠定了良好基础。
本发明还要解决的技术问题在于,提供一种用于检测鸡传染性支气管炎的试剂盒。
本发明还要解决的技术问题在于,提供一种鸡传染性支气管炎的检测方法,,其具有良好的特异性、重复性即灵敏性
为了解决上述技术问题,本发明提供了一种用于检测鸡传染性支气管炎的试剂盒的抗原的制备方法,其包括以下步骤:
(1)采用如核苷酸序列如SEQ ID NO:1所示的上游引物和SEQ ID NO:2所示的下游引物扩增IBV N基因,得到IBV N基因片段;
(2)将IBV N基因整合到质粒载体PET-28b中,得到重组质粒;
(3)将所述重组质粒转化大肠杆菌中,得到重组菌液;
(4)将重组菌液在异丙基β-D-1-硫代吡喃半乳糖苷的诱导下表达重组蛋白,并纯化,即得。
作为上述技术方案的改进,步骤(1)中,采用核苷酸序列如SEQ ID NO:1所示的上游引物,核苷酸序列如SEQ ID NO:2所示的下游引物进行PCR扩增,得到IBV N基因片段;
其中,PCR反应体系包括:Premix TaqTM5μL;上游引物1μL;下游引物1μL;模板1μL;ddH2O 2μL;
PCR反应条件为:95℃预变性3min;95℃变性30s;60℃退火30s;72℃延伸2min;72℃保温5min。
作为上述技术方案的改进,步骤(2)中:
将PET-28b质粒通过BamHⅠ和NotⅠ酶进行酶切;将酶切后的质粒、IBV N基因片段与同源重组酶在16℃反应,得到重组质粒;
其中,酶切反应体系包括:BamHⅠ1μL、NotⅠ1μL、buffer 2μL、模板1μL、ddH2O 15μL,酶切反应条件为:37℃2h;
同源重组反应体系为2×CE Mix 5μL,IBV N基因片段2μL,酶切后PET-28b质粒1μL,ddH2O 2μL。
作为上述技术方案的改进,步骤(4)中,采用HIS标签镍柱亲和层析柱纯化;采用洗涤液洗涤15~25次,洗脱液洗脱15~25次,其中,洗脱液为非变性洗脱液,其咪唑浓度为200~300mM;洗涤液为非变性洗涤液,其咪唑浓度为10~30mM。
相应的,本发明还公开了一种用于检测鸡传染性支气管炎的试剂盒的抗原,其由上述的用于检测鸡传染性支气管炎的试剂盒的抗原的制备方法制备而得。
相应的,本发明还公开了一种用于检测鸡传染性支气管炎的试剂盒,其包括上述的抗原。
作为上述技术方案的改进,还包括酶标板、HRP标记的羊抗鸡IgG抗体、阳性血清、阴性血清、封闭液、洗涤液、样品稀释液、抗体稀释液、显色液和终止液。
其中,封闭液磷酸缓冲液(PBST),其含有1wt%~5wt%的牛血清白蛋白(BSA)或1wt%~10wt%脱脂奶粉或含有酪蛋白;示例性的为含有2wt%BSA的PBST、含有3wt%BSA的PBST、含有4wt%BSA的PBST、含有2wt%脱脂奶粉的PBST、含有4wt%脱脂奶粉的PBST、含有6wt%脱脂奶粉的PBST或含有8wt%脱脂奶粉的PBST,但不限于此。优选的为含有2% BSA的PBST,其pH为7.4。
所述样品稀释液为磷酸缓冲液(PBST),其含有0.25wt%~0.5wt%的牛血清白蛋白(BSA)或含有0.8wt%~1.2wt%的牛血清白蛋白(BSA)与浓度为10wt%~20wt%的胎牛血清(FBS);示例性的为含有0.3wt%BSA的PBST、含有0.4wt%BSA的PBST、含有0.45wt%BSA的PBST、含有1wt%BSA和12wt%FBS的PBST或含有1.1wt%和14wt%FBS的PBST,但不限于此。优选的为含有1wt%BSA和15wt%FBS的PBST。
所述抗体稀释液为磷酸缓冲液(PBST),但不限于此。
所述洗涤液为包括0.01wt%~0.1wt%Tween-20的PBS或PBST,示例性的为包括0.03wt%Tween-20的PBS、包括0.05wt%Tween-20的PBS或包括0.07wt%Tween-20的PBS,但不限于此。优选的为含有0.05wt%Tween-20的PBS,其pH为7.4。
所述终止液为浓度为1M~2M的硫酸溶液,优选的为浓度为2M的硫酸溶液。
其中,阳性血清为抗IBV鸡血清,阴性血清为SPF鸡血清(Specific Pathogen Free无特定病原)。
相应的,本发明还公开了一种鸡传染性支气管炎的检测方法,其包括:
(1)采用含有上述的抗原的包被液包被酶标板,其中,包被浓度为0.25μg/mL~0.5μg/mL,包被液为浓度为0.01M~0.1M的碳酸钠溶液;
(2)去除包被液后采用上述的洗涤液洗涤,并加入上述的封闭液封闭1h~3h;
(3)将待测样品采用上述的样品稀释液以1:125~1:2000的比例稀释孵育后加入酶标板,并采用上述的阳性血清、阴性血清设置阳性血清对照和阴性血清对照;其中,孵育温度为35℃~39℃,孵育时间为45min~120min
(4)将HRP标记的羊抗鸡IgG抗体采用上述的抗体稀释液以1:5000~1:12500的比例稀释孵育后加入酶标板,其中,孵育温度为35℃~39℃,孵育时间为30min~90min;
(5)将上述的显色液加入酶标板,显色5min~15min后加入上述的终止液,测定;
若待测样品的OD450nm值≥0.276,则判定为IBV阳性,反之则为阴性。
实施本发明,具有如下有益效果:
本发明利用能够稳定表达重组鸡传染性支气管炎病毒核蛋白(N蛋白)的重组菌,在特定条件下诱导表达重组N蛋白并经亲和层析纯化。用纯化出的重组蛋白作为包被抗原,建立了一种简便、特异的间接ELISA方法。本发明还为检测鸡传染性支气管炎病毒抗体提供了特异、敏感、快速、操作简便的试剂盒。
附图说明
图1是本发明实施例1中诱导表达后产物的检测图,其中,M为蛋白质标准分子量;1:未诱导阳性菌上清;2:未诱导阳性菌沉淀;3:诱导表达过夜后菌体上清;4:诱导表达过夜后菌体沉淀;
图2是本发明实施例1中重组蛋白纯化过程中产物的检测图,其中,M为蛋白质标准分子量,1为超声破碎后菌体上清;2为过柱流穿液;3为第一次Wash Buffer洗涤液;4为第二次Wash Buffer洗涤液;5为Wash Buffer第三次洗涤液;6为第四次Wash Buffer洗涤液;7为第五次Wash Buffer洗涤液;8为第六次Wash Buffer洗涤液;9为第七次Wash Buffer洗涤液;
图3是本发明实施例1中重组蛋白纯化过程中产物的另一检测图,其中,M为蛋白质标准分子量,1为Elution Buffer第一次洗脱液;2为Elution Buffer第二次洗脱液;3为Elution Buffer第三次洗脱液;4为Elution Buffer第四次洗脱液;5为Elution Buffer第五次洗脱液;6为Elution Buffer第六次洗脱液;7为Elution Buffer第七次洗脱液;
图4是本发明实施例1中IBV N蛋白作为抗原与His单抗反应的western-blot检测结果,其中,M为蛋白质标准分子量,1为诱导后菌液上清,2为诱导后菌液沉淀,3为诱导后总蛋白;
图5是实施例2中不同抗原包被浓度和血清稀释度的检测结果图;
图6是实施例2中不同封闭液和封闭时间的检测结果图;
图7是实施例2中不同一抗稀释液和孵育时间的检测结果图;
图8是实施例2中不同二抗稀释倍数和孵育时间的检测结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
实施例1 IBV N重组蛋白的制备
本实施例提供一种高纯度IBV N重组蛋白的制备方法,具体包括:
1、IBV N基因片段的扩增
利用IBV N特异性引物以IBV-P65毒株(Genbank登录号:DQ001339.1)感染的鸡胚尿囊液抽提的核酸为模板,经RT-PCR方法扩增获得IBV N基因片段(IBV N基因编码的核苷酸序列如SEQ ID NO:3所示;IBV N基因编码的氨基酸序列如SEQ ID NO:4所示)。
具体的,所采用的引物序列如下:
SEQ ID NO:1:N-F TAAGGATCCATGGCAAGCGGTAAAGCA
SEQ ID NO:2:N-R GTGCTCGAGTCAAAGTTCATTCTCTCCTAG
扩增体系如下:
Premix TaqTM5μL;上游引物1μL;下游引物1μL;模板1μL;ddH2O 2μL。
反应条件为:95℃预变性3min;95℃变性30s;60℃退火30s;72℃延伸2min;72℃保温5min。
2、重组质粒的构建
将PET-28b质粒通过BamHⅠ和NotⅠ酶进行酶切;将酶切后的质粒、IBV N基因片段与同源重组酶在16℃反应,得到重组质粒;
其中,酶切反应体系包括:BamHⅠ1μL、NotⅠ1μL、buffer 2μL、PET-28b质粒1μL、ddH2O 15μL。酶切反应条件为:37℃2h。
同源重组反应体系为:2×CE Mix 5μL,IBV N基因片段2μL,酶切后PET-28b载体1μL,ddH2O 2μL。
3、重组质粒转化与重组菌制备
将重组质粒转化到大肠杆菌中,具体转化步骤如下:
BL21(DE3)置于冰上解冻,取10μL重组产物加入到宿主菌中,冰上静置30min;42℃水浴热激30s后,立即置于冰上冷却2min,加入800μL SOC培养基,37℃摇菌1h;取100μL菌液涂布在抗性LB固体培养基,过夜培养。
4、重组蛋白的诱导表达
将重组菌液(1/100)摇菌至OD600值≈0.6时,添加0.1mM IPTG,在16℃、220rpm条件下诱导表达过夜,过夜的菌液在4℃、12000rpm离心10min,弃上清,用1/5原菌液体积的裂解液重悬沉淀,超声破碎仪300W、工作5s、休息10s,超声至澄清即可获得重组蛋白液。
5、重组蛋白纯化
按照碧云天的His标签蛋白纯化试剂盒(耐变性剂型)的使用说明书,进行亲和层析技术纯化2)所得菌液,纯化条件:洗涤液浓度20mM,洗涤20次,1mL/次;洗脱液浓度250mM,洗脱20次,1mL/次。
在诱导表达后取样检测,其结果如图1所示,从图中可以看出,能诱导IBV N蛋白在大肠杆菌中表达。
采用洗涤液、洗脱液纯化后取样进行检测,结果如图2~4所示。从图中可以看出,通过纯化镍柱,有效地纯化出工程菌表达的IBV N重组蛋白。
实施例2用于检测鸡传染性支气管炎的试剂盒的制备
本实施例提供一种用于检测鸡传染性支气管炎的试剂盒,其具体包括:
(1)实施例1制备的IBV N重组蛋白;
(2)封闭液:2%BSA溶液,pH为7.4;
(3)洗涤液:含0.05%Tween-20的PBS,pH为7.4;
(4)样品稀释液:含有1%BSA和15%FBS的PBST;
(5)抗体稀释液:PBST;
(6)辣根过氧化物酶(HRP)标记的羊抗鸡IgG抗体;
(7)显色液:TMB单组份显色液;
(8)终止液:2M H2SO4;
(9)阳性血清:抗IBV鸡血清;
(10)阴性血清:阴性SPF鸡血清(Specific Pathogen Free无特定病原)。
具体的,阳性血清、阴性血清的制备方法如下:
12只3周龄SPF鸡分为3组,分别感染rIBV-siMutBeau和rIBV-Beau-KC(S1)【具体建立方法可参Establishment and Cross-Protection Efficacy of aRecombinant AvianGammacoronavirus Infectious Bronchitis Virus Harboring a Chimeric S1 Subunit,https://doi.org/10.3389/fmicb.2022.897560】,及PBS对照组。在免疫后第7d、14d、21d、28d、35d分别采血分离血清。其中,以攻毒组2中rIBV-Beau-KC(S1)攻毒35d后所分离的血清作为阳性血清,以对照组3中PBS缓冲液攻毒35d后所分离的血清作为阴性血清。
表1阳性血清、阴性血清的制备条件表
实施例3鸡传染性支气管炎病毒间接ELISA方法的建立与优化
一、ELISA方法的建立
(1)抗原包被
向酶标板的每孔中分别加入100μL含量为0.5μg/mL纯化的鸡传染性支气管炎病毒重组N蛋白的抗原溶液,4℃包被过夜;包被液为pH为9.6的0.05M碳酸钠溶液。
(2)封闭
去除包被液后,洗涤液洗涤3次,加入100μL封闭液孵育。其中,封闭液封闭液为含1%BSA,封闭时间为1.5h;
(3)一抗孵育
加入用样品稀释液以1:1000稀释的待检血清(一抗),同时设置阳性和阴性血清对照;孵育,PBST洗板3次。其中,一抗在37℃孵育1.5h。其中,稀释液为1wt%BSA和15wt%FBS的混合液。
(4)二抗孵育
加入用抗体稀释液以1:5000稀释的HRP标记的羊抗鸡IgG二抗,在37℃孵育1.5h。
(5)显色
以加入显色剂反应孔,在37℃显色。其中显色剂为TMB,在室温避光显色15min。
(6)终止
加入2M H2SO4终止反应。
(7)结果判定
采用酶标仪测OD450nm处吸光值判定标准为:对样品OD450nm值≥0.276时判定为IBV阳性,反之则为阴性。
二、ELISA方法的优化
(1)最佳抗原包被浓度和血清稀释度的确定
方阵滴定法确定最佳抗原包被浓度和血清稀释度。结果如图5所示。由图中可以看出,当抗原包被浓度为0.5μg/mL,血清稀释度为1:1000时,S/P值最大,说明该组合为最佳抗原包被浓度和血清稀释度。
(2)最佳封闭液和封闭时间的确定
用含有1% BSA、2% BSA、5% BSA和5%脱脂奶粉的PBST放入恒温培养箱37℃分别封闭0.5h、1h、1.5h、2h,根据P/N值确定最佳封闭液和封闭时间,选择P/N最大的组合确定最佳封闭液和封闭时间,结果如图6所示。从图中可以看出1% BSA的PBST封闭1.5h时,P/N值最大。
(3)最佳一抗稀释液及作用时间的确定
分别以PBST、0.25%BSA的PBST、0.5%BSA的PBST、1%BSA的PBST及1%BSA加15%FBS的PBST作一抗稀释液,37℃进行反应,选定P/N值最大对应的一抗稀释液。结果如图7所示。经分析,当1%BSA加15% FBS的PBST为稀释液,37℃反应1.5h时,P/N值最大。
(4)最佳二抗稀释倍数和孵育时间的确定
加入标记羊抗鸡IgG抗体用PBST稀释至稀释度为1:5000、1:7500、1:10000、1:12500,放入37℃恒温培养箱分别孵育0.5h、1h、1.5h、2h,根据P/N值确定最佳二抗稀释倍数和孵育时间,选择P/N值最大的组合作为最佳二抗稀释倍数和孵育时间。结果如图8所示,从图中可以看出,二抗稀释度为1:5000,孵育1.5h时,P/N值最大。
(5)显色
加入显色液后分别在37℃孵育5min、10min、15min、20min,之后加100μL/孔的2MH2SO4终止,选定P/N值最大对应的TMB底物反应时间。经分析,当底物反应时间为8min~15min,P/N值较大,可实现检测功能。最佳地,当底物反应时间为15min时,P/N值最大。终止用的终止液为1M~2M H2SO4;优选地,为2M H2SO4。
表2显色反应时间试验结果表
因此,最终确定的最佳反应条件为:抗原包被浓度为0.5μg/mL,待检血清稀释倍数为1:1000,酶标抗体稀释倍数为1:5000;1%BSA为封闭液;1%BSA+15% FAB为样品稀释液;待检血清和酶标二抗反应时间分别为1.5h和1.5h,底物反应时间15min。
(6)阴阳性判定标准的确定
选取30份阴性血清,用优化的ELISA反应体系检测血清抗体OD450值。对结果进行统计学分析,得出OD450值的平均值(X)和标准差(S),以此作为ELISA方法阴、阳性判定的临界标准。
表3阴阳性判定标准试验结果表
经计算,以上30份阴性血清平均值X=0.153,标准差S=0.041,ELISA阴阳临界值=X+3S=0.276,所以以OD450值0.276阴阳性血清临界值。
三、ELISA方法的优化
(1)特异性实验
使用建立的ELISA方法最优条件分别检测3份已知的IBV、鸡新城疫病毒(NDV)(Genbank登录号:MT447874.1)、禽流感病毒(AIV)(Re-11株)和鸡传染性法氏囊病毒(IBDV)(BC6/85株)阳性血清,并以IDEXX IBV试剂盒作为对照,分析试剂盒的特异性。检测结果显示,除IBV血清检测为阳性以外,其他病原的阳性血清检测均为阴性,说明该试剂盒的特异性良好,与其它阳性血清无交叉反应。
表4特异性实验结果表
(2)重复性实验
取3块同一时间包被的ELISA酶标板进行批内重复试验,在相同条件下检测9份不同样品并对检测结果进行统计学分析。检测结果显示,批内重复试验CV值在0.35~7.99%,证明该方法在批内具有一定的可重复性。
表5批内重复性实验结果表
取3块不同时间包被的ELISA酶标板进行批间重复试验,在相同条件下检测8份不同样品并对检测结果进行统计学分析。检测结果显示,批间重复试验CV值在2.10~9.43%,证明该方法在批间具有一定可重复性。
表6批间重复性实验结果表
(3)符合率检验
用建立的IBV-N作为包被抗原的ELISA板检测181份不同抗体效价的血清样品,同时与IDEXX公司IBV ELISA抗体检测试剂盒的检测结果相对比,分析该方法的敏感性、特异性和符合率。
相对敏感性(%)={阳性数/(阳性数+假阴性数)}×100%
相对特异性(%)={阴性数/(阴性数+假阳性数)}×100%
总符合率(%)={(阳性数+阴性数)/检测总数}×100%
表7符合率实验结果表
实验结果显示,用建立的IBV-N蛋白包被的ELISA板和检测方法检测181份不同抗体效价的血清样品,检测结果为阳性81份,阴性85份,IDEXX公司IBV ELISA检测试剂盒的检测结果为阳性94份,阴性87份。
由此可知建立的ELISA方法的相对敏感性、相对特异性和总符合率分别是86.17%、97.70%和91.16%。因此,本发明中涉及的以IBV-N1蛋白包被的ELISA板为主的抗体检测试剂盒敏感性、特异性较高,与已有的商品化试剂盒具有较高的符合率。
实施例3N-ELISA方法的初步应用
将实施例2中分别感染rIBV-siMutBeau(简称siMut)和rIBV-Beau-KC(S1)(简称KC(S1)),及PBS对照组,在免疫后第7d、14d、21d、28d、35d分别采集到的鸡血清,同时用本发明涉及的试剂盒和商品化IDEXX ELISA试剂盒(以传染性支气管炎病毒作为抗原包被ELISA板)进行检测,以IDEXX检测结果为标准,计算二者符合率。
实验结果表明,本发明涉及的试剂盒检测结果和IDEXX ELISA试剂盒检测血清阴阳性结果一致,结果见表8。可见本发明涉及的试剂盒检测结果与目前国际上大量应用的试剂盒检测结果基本一致,可应用于传染性支气管炎抗体水平的检测。
表8不同检测方法的检测实验结果表
以上所述的仅为本发明一种较佳实施例而已,不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
Claims (10)
1.一种用于检测鸡传染性支气管炎的试剂盒的抗原的制备方法,其特征在于,包括以下步骤:
(1)采用如核苷酸序列如SEQ ID NO:1所示的上游引物和SEQ ID NO:2所示的下游引物扩增IBV N基因,得到IBV N基因片段;
(2)将IBV N基因整合到质粒载体PET-28b中,得到重组质粒;
(3)将所述重组质粒转化大肠杆菌中,得到重组菌液;
(4)将重组菌液在异丙基β-D-1-硫代吡喃半乳糖苷的诱导下表达重组蛋白,并纯化,即得。
2.如权利要求1所述的用于检测鸡传染性支气管炎的试剂盒的抗原的制备方法,其特征在于,步骤(1)中,采用核苷酸序列如SEQ ID NO:1所示的上游引物,核苷酸序列如SEQ IDNO:2所示的下游引物进行PCR扩增,得到IBV N基因片段;
其中,PCR反应体系包括:Premix TaqTM5μL;上游引物1μL;下游引物1μL;模板1μL;ddH2O2μL;
PCR反应条件为:95℃预变性3min;95℃变性30s;60℃退火30s;72℃延伸2min;72℃保温5min。
3.如权利要求1所述的的用于检测鸡传染性支气管炎的试剂盒的抗原的制备方法,其特征在于,步骤(2)中:
将PET-28b质粒通过BamH Ⅰ和Not Ⅰ酶进行酶切;将酶切后的质粒、IBV N基因片段与同源重组酶在16℃反应,得到重组质粒;
其中,酶切反应体系包括:BamH Ⅰ 1μL、Not Ⅰ 1μL、buffer 2μL、模板1μL、ddH2O 15μL,酶切反应条件为:37℃2h;
同源重组反应体系为2×CE Mix 5μL,IBV N基因片段2μL,酶切后PET-28b质粒1μL,ddH2O 2μL。
4.如权利要求1所述的用于检测鸡传染性支气管炎的试剂盒的抗原的制备方法,其特征在于,步骤(4)中,采用HIS标签镍柱亲和层析柱纯化;采用洗涤液洗涤15~25次,洗脱液洗脱15~25次,其中,洗脱液为非变性洗脱液,其咪唑浓度为200~300mM;洗涤液为非变性洗涤液,其咪唑浓度为10~30mM。
5.一种用于检测鸡传染性支气管炎的试剂盒的抗原,其由如权利要求1~4任一项所述的用于检测鸡传染性支气管炎的试剂盒的抗原的制备方法制备而得。
6.一种用于检测鸡传染性支气管炎的试剂盒,其特征在于,包括如权利要求5所述的抗原。
7.如权利要求6所述的用于检测鸡传染性支气管炎的试剂盒,其特征在于,还包括酶标板、HRP标记的羊抗鸡IgG抗体、阳性血清、阴性血清、封闭液、洗涤液、样品稀释液、抗体稀释液、显色液和终止液。
8.如权利要求7所述的用于检测鸡传染性支气管炎的试剂盒,其特征在于,所述封闭液为磷酸缓冲液,其含有1wt%~5wt%的牛血清白蛋白或1wt%~10wt%的脱脂奶粉;
所述洗涤液为含有0.01wt%~0.1wt%Tween-20的PBS;
所述样品稀释液为磷酸缓冲液,其含有0.25wt%~0.5wt%的牛血清白蛋白或含有0.8wt%~1.2wt%的牛血清白蛋白与浓度为10wt%~20wt%的胎牛血清;
所述抗体稀释液为磷酸缓冲液;
所述显色液为TMB溶液;
所述终止液为浓度为1M~2M的硫酸溶液。
9.如权利要求6所述的用于检测鸡传染性支气管炎的试剂盒,其特征在于,所述封闭液为磷酸缓冲液,其含有2wt%的牛血清白蛋白;
所述洗涤液为含有0.05wt%Tween-20的PBS;
所述样品稀释液磷酸缓冲液,其含有1wt%的牛血清白蛋白与15wt%的胎牛血清;
所述终止液为浓度为2M的硫酸溶液。
10.一种鸡传染性支气管炎的检测方法,其特征在于,包括:
(1)采用含有如权利要求5所述的抗原的包被液包被酶标板,其中,包被浓度为0.25μg/mL~0.5μg/mL,包被液为浓度为0.01M~0.1M的碳酸钠溶液;
(2)去除包被液后采用如权利要求8或9所述的洗涤液洗涤,并加入如权利要求8或9所述的封闭液封闭1h~3h;
(3)将待测样品采用如权利要求8或9所述的样品稀释液以1:125~1:2000的比例稀释孵育后加入酶标板,并采用如权利要求8或9所述的阳性血清、阴性血清设置阳性血清对照和阴性血清对照;其中,孵育温度为35℃~39℃,孵育时间为45min~120min
(4)将HRP标记的羊抗鸡IgG抗体采用如权利要求8或9所述的抗体稀释液以1:5000~1:12500的比例稀释孵育后加入酶标板,其中,孵育温度为35℃~39℃,孵育时间为30min~90min;
(5)将如权利要求8或9所述的显色液加入酶标板,显色5min~15min后加入如权利要求8或9所述的终止液,测定;
若待测样品的OD450nm值≥0.276,则判定为IBV阳性,反之则为阴性。
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