CN110208518A - 基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法及其检测试剂盒 - Google Patents
基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法及其检测试剂盒 Download PDFInfo
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Abstract
本发明涉及一种基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法及其检测试剂盒以及应用,所述鸭3型腺病毒抗体间接ELISA检测方法,以制备所得的Fiber1蛋白作为包被抗原包被酶标板,将待测血清样品经稀释后加入酶标板孵育,加入酶标二抗孵育后显色,测定反应后血清样品的OD值;本发明以Fiber1蛋白作为包被抗原建立间接ELISA检测方法,可以快速检测鸭3型腺病毒抗体,具有具有良好的特异性和重复性。对临床样品进行检测,结果表明本方法可以作为鸭3型腺病毒抗体检测的一种方法而运用到生产实际中。
Description
技术领域
本发明涉及一种基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法及其检测试剂盒以及应用。
背景技术
鸭3型腺病毒(Duck adenovirus 3,DAdV-3)是近年来在中国出现的新型鸭腺病毒,具有明显的致病力,感染鸭临床剖检病变为肝脏黄化、质地变脆、肿大、出血及坏死。鸭3型腺病毒具有一般禽腺病毒的基本结构,呈二十面体对称,无囊膜,直径60-80nm,为双链DNA病毒,基因组全长为43842bp,有 2个纤突蛋白(Fiber1和Fiber2)基因。纤突蛋白作为腺病毒的主要结构蛋白之一,具有亚群和型特异性抗原表位,因此它们在腺病毒传染性和致病性方面起着至关重要的作用。Fiber蛋白作为保护性免疫原能够诱导产生中和抗体。Fiber 蛋白还通过介导腺病毒与宿主细胞受体的结合,在病毒感染的初始阶段起重要作用。
近年来鸭3型腺病毒引起鸭群感染发病的形势日益严峻,给养禽业造成了巨大的经济损失,目前急需一种血清学检测方法,用于该病毒感染的临床检测以及今后疫苗免疫后抗体水平的监测,对于该病的有效防控具有十分重要的意义。酶联免疫吸附测定(Enzymelinked immunosorbent assay,ELISA)是将可溶性的抗原或抗体结合到聚苯乙烯等固相载体上,利用抗原抗体结合专一性进行免疫反应的定性和定量检测方法。由于ELISA方法具有很高的敏感性和再现性,并且可以用于大量血清样品的检测,因此该方法是最常用的血清学方法之一。纤突蛋白作为腺病毒的主要结构蛋白之一,具有亚群和型特异性抗原表位,能诱导产生特异性中和抗体。为此,本发明利用原核表达的鸭3型腺病毒Fiber1 蛋白作为包被抗原,建立检测鸭3型腺病毒血清抗体的间接ELISA方法。
发明内容
本发明的目的在于提供一种快速、简便地基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法及其检测试剂盒以及应用。
本发明的目的通过如下技术方案实现:一种Fiber1蛋白的制备方法:以鸭3 型腺病毒的核酸作为模板,利用设计合成的特异性引物进行PCR扩增,回收扩增的PCR产物;将扩增的PCR产物、pET-28a表达载体先用NdeI与XhoI酶切后再连接,构建重组表达质粒;将重组表达质粒转化Rosetta(DE3)感受态细胞,并采用异丙基硫代半乳糖苷诱导表达,将获得的表达产物进行纯化,即得所述 Fiber1蛋白;
其中,所述特异性引物的序列如下:
上游引物F1F:CATATGCTCTGTCCGTTTAGATTCATC;
下游引物F1R:CTCGAGTTATACAATCTTCGCTAGGTACGAGA;
所述Fiber1蛋白的氨基酸序列如SEQ ID NO.3所示。
一种基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,以 Fiber1蛋白作为包被抗原包被酶标板,将待测血清样品经稀释后加入酶标板孵育,加入酶标二抗孵育后显色,测定反应后血清样品的OD值。
一种鸭3型腺病毒抗体间接ELISA检测试剂盒,所述试剂盒以Fiber1蛋白作为ELISA酶标板的包被抗原。
所述的鸭3型腺病毒抗体间接ELISA检测试剂盒,在鸭3型腺病毒血清抗体检测中的应用。
较之现有技术而言,本发明的优点在于:。
1.本发明利用原核表达的鸭3型腺病Fiber1蛋白作为包被抗原,建立了检测鸭3型腺病血清抗体的间接ELISA方法。
2.本发明以Fiber1蛋白作为包被抗原建立间接ELISA检测方法,可以快速检测鸭3型腺病毒抗体,具有良好的特异性和重复性。对临床样品进行检测,结果表明本方法可以作为鸭3型腺病毒抗体检测的一种方法而运用到生产实际中。
3.本发明的检测方法还具有可同时进行大量样品的检测以及检测快速、便捷的优点。
附图说明
图1是Fiber1蛋白纯化的SDS-PAGE分析图。
图2是ELISA方法的特异性分析结果。
具体实施方式
下面结合说明书附图和实施例对本发明内容进行详细说明:
一种Fiber1蛋白的制备方法:以鸭3型腺病毒的核酸作为模板,利用设计合成的特异性引物进行PCR扩增,回收扩增的PCR产物;将扩增的PCR产物、 pET-28a表达载体先用NdeI与XhoI酶切后再连接,构建重组表达质粒;将重组表达质粒转化Rosetta(DE3)感受态细胞,并采用异丙基硫代半乳糖苷诱导表达,将获得的表达产物进行纯化,即得所述Fiber1蛋白;
其中,所述特异性引物的序列如下:
上游引物F1F:CATATGCTCTGTCCGTTTAGATTCATC;
下游引物F1R:CTCGAGTTATACAATCTTCGCTAGGTACGAGA;
其中,上下游引物中下划线部分的序列分别为NdeI和XhoI限制性酶切位点。
所述Fiber1蛋白的氨基酸序列如SEQ ID NO.3所示。
一种基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,以 Fiber1蛋白作为包被抗原包被酶标板,将待测血清样品经稀释后加入酶标板孵育,加入酶标二抗孵育后显色,测定反应后血清样品的OD值。
纤突蛋白作为腺病毒的主要结构蛋白,具有亚群和型特异性抗原表位,能诱导产生特异性中和抗体。本发明利用原核表达的鸭3型腺病Fiber1蛋白作为包被抗原,建立的检测鸭3型腺病血清抗体的间接ELISA方法,能够快速、有效的对鸭3型腺病血清抗体进行检测。
所述的基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,具体包括以下步骤:
(1)包被:以Fiber1蛋白为包被抗原,将包被抗原用包被液稀释后,加入 96孔的ELISA塑胶孔板上,每孔100μL,于4℃包被过夜;之后,弃去包被后孔中多余的抗原,每孔加入200μL的洗涤液洗涤4次,洗完后将板拍干;
(2)封闭:每孔加入200μL封闭剂,37℃,封闭2h;之后,弃去封闭后孔中多余的封闭剂,用洗涤液洗涤4次,洗完后拍干;
(3)加待检血清:每孔加入100μL用样品稀释液稀释过的血清,37℃,孵育1h;之后弃去孵育后孔中多余的血清,用洗涤液洗涤4次,洗完后拍干;
(4)加二抗:每孔加入100μL用样品稀释液稀释过的酶标二抗,37℃,孵育1h;之后,弃去孵育后孔中多余的酶标二抗,用洗涤液洗涤4次,洗完后拍干;
(5)显色:每孔加入100μL显色剂,37℃避光显色10min;
(6)终止:每孔加入100μL终止液;反应终止后置于酶标仪中,在450nm 下,读取每个样品的OD值;
(7)结果判定:待检血清OD值≥0.353时判定为鸭3型腺病毒抗体阳性, OD值<0.353判定为阴性;
其中,步骤(1)中,包被抗原用包被液稀释至4μg/mL。
所述包被液为0.05M、pH9.6的碳酸盐缓冲液;所述洗涤液为PBST缓冲液;所述封闭剂为5%的脱脂乳;所述样品稀释液为PBS缓冲液;所述显色剂为 TMB。所述终止液为0.5M的硫酸溶液;待检血清用样品稀释液按照1:100倍稀释;所述酶标二抗为羊抗鸭IgG酶标二抗;酶标二抗用样品稀释液按1:4000倍稀释。
一种鸭3型腺病毒抗体间接ELISA检测试剂盒,所述试剂盒以Fiber1蛋白作为ELISA酶标板的包被抗原。
所述的鸭3型腺病毒抗体间接ELISA检测试剂盒,它还包括包被液、洗涤液、封闭剂、样品稀释液、酶标二抗、显色剂、终止液、鸭3型腺病毒阳性血清、SPF鸭阴性血清。
所述包被液为0.05M、pH9.6的碳酸盐缓冲液;所述洗涤液为PBST缓冲液;所述封闭剂为5%的脱脂乳;所述样品稀释液为PBS缓冲液;所述显色剂为 TMB。所述终止液为0.5M的硫酸溶液;所述酶标二抗为羊抗鸭IgG酶标二抗;
所述的鸭3型腺病毒抗体间接ELISA检测试剂盒可用于鸭3型腺病毒血清抗体的检测中。
下面结合具体实施例对本发明内容作更细致地阐述:
实施例一:Fiber1蛋白的制备:
1材料:
1.1实验试剂与耗材:
PCR扩增试剂盒Phanta Max Super-Fidelity DNA Polymerase购自南京诺唯赞生物科技有限公司;病毒核酸提取试剂盒EasyPure Viral DNA/RNA Kit、DH5a 感受态菌株、Rosetta(DE3)感受态菌株均购自北京全式金生物技术有限公司; NdeI、XhoI限制性内切酶以及连接酶T4DNAligase购自Thermo Scientific公司; pET-28a表达载体购自Takara公司;羊抗鸭IgG酶标二抗购自KPL公司;TMB 显色剂购自博士德生物技术有限公司;其他常规试剂和耗材购自生工生物工程 (上海)股份有限公司。
1.2毒株与血清:
鸭3型腺病毒(DAdV-3)、鸭瘟病毒(DPV)、禽流感病毒(AIV)、鸭甲肝病毒(DHAV)、番鸭细小病毒(MDPV)、鸭疫里默氏菌(RA)和鸭坦布苏病毒(DTMUV)的鸭源阳性血清以及鸭3型腺病毒株由福建省农业科学院畜牧兽医研究所禽病室保存。
2.鸭3型腺病Fiber1蛋白原核表达
2.1引物设计
利用引物设计软件Oligo7,针对鸭3型腺病毒Fiber1基因设计引物,引物序列如下:
上游引物F1F:CATATGCTCTGTCCGTTTAGATTCATC
下游引物F1R:CTCGAGTTATACAATCTTCGCTAGGTACGAGA
上下游引物有下划线部分的序列分别为NdeI和XhoI限制性酶切位点。
2.2目的基因钓取
2.2.1核酸的制备
利用北京全式金生物技术有限公司的Viral DNA/RNA Kit核酸提取试剂盒,按照说明书的操作方法提取鸭3型腺病毒的核酸。
2.2.2 Fiber1基因的扩增
本发明使用的PCR扩增试剂盒Phanta Max Super-Fidelity DNA Polymerase 购自南京诺唯赞生物科技有限公司。在结合试剂盒推荐的反应体系配置的基础上对PCR反应液进行了优化,优化出的50μL最佳反应体系的具体配置方法见表1,最佳反应条件为:95℃3min,预变性;95℃15s,58℃15s,72℃90s,共30个循环;72℃延伸7min。用胶回收试剂盒纯化PCR产物。
表1 PCR反应液的配置
2.3原核表达载体构建
2.3.1换载酶切
将上述纯化的PCR产物和pET-28a表达载体分别进行酶切,PCR产物和 pET-28a表达载体的50ul酶切体系分别见表2和表3。
表2 PCR产物片段酶切体系
表3表达载体的酶切体系
以上体系放入37℃恒温水浴锅中反应2h,回收酶切的载体pET-28a和目的 DNA片段。
2.3.2目的片段与载体连接
将步骤2.3.1回收纯化好的酶切目的DNA片段和酶切载体pET-28a,进行连接。连接体系为20ul,具体方法见表4。
表4目的片段与载体连接体系
上述连接混合液放在22℃PCR仪1h。
2.3.3转化,筛选克隆
将上述连接液进行转化,转化采用42℃热激法进行,感受态菌株为DH5a。挑取转化后平板上的单菌落于试管中37℃、220rpm/min培养过夜,抽提质粒,采用NdeI,XhoI双酶切鉴定,并将重组质粒送生工生物工程(上海)股份有限公司测序鉴定。
2.3.4阳性重组质粒转化表达菌
取1μL已鉴定为阳性的重组质粒转化Rosetta(DE3)感受态,42℃热击90s 后冰上静置5min后涂布含有终浓度为50μg/mL氨苄霉素和34μg/mL氯霉素的 LB固体平板上,37℃培养过夜。
2.4 Fiber1蛋白诱导表达
挑取表达菌株Rosetta(DE3)的单菌落于装有12mL LB液体培养基的三角瓶中,37℃,220rpm过夜培养。将过夜培养的菌液按1:100比例接种于1L的LB 培养基中,37℃,220rpm培养。当OD值达到0.6时,添加终浓度为0.5mM的 IPTG,220rpm,20℃诱导过夜。4000rpm离心10min收集菌体,弃上清。将收集的细菌菌体用破碎Buffer溶解,冰浴中超声破碎菌体,功率400W,20min,超声2S,暂停6S为一个循环。超声完毕,12000rpm,4℃,离心20min,收集上清进行下一步纯化。
2.5镍琼脂糖凝胶亲和层析法纯化Fiber1蛋白
取5mL Ni-NTA,用5倍柱床体积的Binding Buffer清洗平衡柱子,流速 5mL/min。填料和样品孵育1h后上柱,收集穿透液。5倍柱床体积的Binding Buffer 清洗柱子,流速5mL/min。Wash Buffer洗杂,流速2mL/min,收集洗脱液。Elution Buffer洗脱,流速2mL/min,收集洗脱液。将收集的洗脱液于50mM Tris,300mM NaCl,2mM DTT,pH8.0中透析。透析结束后PEG20000浓缩,0.45μm滤膜过滤后分装1mL/tube,于-80℃冻存。Fiber1蛋白最终纯化SDS-PAGE分析见图1。其中,图1中,M为Protein Marker;1为目的蛋白即Fiber1蛋白。
实施例二:鸭3型腺病毒抗体间接ELISA检测
3.ELISA方法建立
(1)包被:以纯化后的Fiber1蛋白为包被抗原,Fiber1蛋白用0.05M、PH9.6 的碳酸盐缓冲液稀释后加入96孔的ELISA塑胶孔板上,每孔100μL,于4℃包被过夜;弃去包被后孔中多余的抗原,每孔加入200μL的PBST洗涤4次,洗完后将板拍干;
(2)封闭:每孔加入200μL 5%的脱脂乳,37℃,封闭2h。弃去封闭后孔中多余的脱脂乳,用PBST洗涤4次,洗完后拍干;
(3)加待检血清:每孔加入100μL用PBS缓冲液稀释过的鸭血清,37℃,孵育1h;弃去孵育后孔中多余的血清,用PBST洗涤4次,洗完后拍干;
(4)加二抗:每孔加入100μL用PBS缓冲液稀释过的羊抗鸭IgG酶标二抗,37℃,孵育1h。之后,弃去孵育后孔中多余的酶标二抗,用PBST洗涤4 次,洗完后拍干;
(5)显色:每孔加入100μL TMB显色剂,37℃避光显色10min;
(6)终止:鸭3型腺病毒阳性血清样品孔出现蓝绿色,立即加入0.5M的 H2SO4终止反应。反应终止后置于酶标仪中,在450nm读取每个样品的OD值。
用上述方法对包被抗原浓度、血清稀释度以及酶标二抗稀释度进行了优化,优化出的最佳条件如下:包被抗原浓度4μg/mL(即纯化后的Fiber1蛋白用包被液稀释至4μg/mL后,再加入96孔的ELISA塑胶孔板上)、血清1:100倍稀释、酶标二抗1:4000倍稀释。用以上建立的ELISA方法测定35份鸭3型腺病毒阴性血清的OD值,求出平均数为0.230,标准差为0.041,将该平均数加上3倍的标准差作为判定阳性和阴性的临界值,求得临界值为0.353,即若待检血清 OD值≥0.353时判定为鸭3型腺病毒抗体阳性,OD值<0.353判定为阴性。
4.ELISA方法的特异性分析
用上述建立的ELISA方法同时测定鸭3型腺病毒(DAdV-3)、鸭瘟病毒 (DPV)、禽流感病毒(AIV)、鸭甲肝病毒(DHAV)、番鸭细小病毒(MDPV)、鸭疫里默氏菌(RA)和鸭坦布苏病毒(DTMUV)的鸭源阳性血清的OD值,结果如图2所示,只有鸭3型腺病毒的血清检测结果为阳性,其他病原的阳性血清,检测结果均为阴性。
5.临床应用
用以上建立的ELISA检测方法对临床送检的62份鸭血清样品进行检测,检测出鸭3型腺病毒阳性血清16份,阳性率为25.8%。表明鸭3型腺病毒在该次送检的鸭群中有较高的感染率。
序列表
<110> 福建省农业科学院畜牧兽医研究所
<120> 基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法及其检测试剂盒
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
catatgctct gtccgtttag attcatc 27
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<211> 32
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
ctcgagttat acaatcttcg ctaggtacga ga 32
<210> 3
<211> 459
<212> PRT
<213> 人工序列(Artificial sequence)
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Met Leu Cys Pro Phe Arg Phe Ile Gln Tyr Asp Ser Ala Met Met Gln
1 5 10 15
Thr Ala Leu Phe Gly Arg Ser Leu Lys Arg Arg Arg Arg Ala Asn Ser
20 25 30
Asp Thr Glu Leu Glu Ala Glu Lys Gln Ile Lys Pro Glu Thr Thr Thr
35 40 45
Ser Ser Gly Gly Asp Pro Pro Ile Ala Pro Pro Pro Pro Thr Pro Pro
50 55 60
Pro Pro Pro Pro Ala Pro Pro Thr Pro Thr Ile Asp Leu Thr Tyr Pro
65 70 75 80
Tyr Trp Trp Pro Ser Pro Asn Tyr Pro Gly Gly Gly Gly Gly Gly Gly
85 90 95
Gly Ser Cys Thr Cys Gln Gly Asp Pro Leu Gly Pro Ile Val Lys Thr
100 105 110
Asn Thr Gly Phe Asn Ile Arg Leu Gln Ala Pro Val Val Leu Ser Gly
115 120 125
Ser Lys Ala Val Thr Leu Ala Val Asp Asn Thr Leu Gln Thr Asp Gly
130 135 140
Gln Ser Val Gly Val Lys Leu Ser Ser Pro Met Ile Ser Thr Ser Ser
145 150 155 160
Gly Val Thr Leu Asp Thr Gly Asp Gly Leu Thr Phe Asp Ser Gly Arg
165 170 175
Leu Asn Val Ala Cys Thr Pro Arg Arg Ser Leu Lys Val Thr Ser Thr
180 185 190
Gly Leu Asp Ile Val Thr Asp Val Thr Ile Asn Asn Ser Asp Thr Leu
195 200 205
Gly Val Asn Ile Lys Thr Asn Gly Gly Leu Lys Phe Asp Ser Ala Gly
210 215 220
Ile Arg Val Ala Val Asp Gly Thr Thr Val Lys Val Asn Ser Asn Gly
225 230 235 240
Val Leu Gln Ala Leu Pro Gln Val Ser Asn Ala Val Asn Ala Met Glu
245 250 255
Ile Gly Ser Pro Glu Ser Asp Ser Gly Lys Val Tyr Ala Ala Thr Glu
260 265 270
Asp Asn Lys Ile Val Val Thr Arg Pro Tyr Ile Tyr Met Val Ser Ala
275 280 285
Ala Gly Leu Val Asn Gly Val Ile Asn Val Lys Ile Pro Lys Asn Leu
290 295 300
Gln Leu Asp Lys Asn Pro Tyr Thr Pro Ser Gly His Phe Lys Phe Thr
305 310 315 320
Ile Ile Val Pro Phe Asp Lys Gln Phe Asn Val Ala Asn Leu Pro Pro
325 330 335
Tyr Val Thr Ile Pro Asp Thr Val Glu Val Ser Ser Phe Ala Pro Asn
340 345 350
Lys Leu Leu Asp Leu Gly Gly Phe Thr Gly Val Pro Tyr Ile Ser Thr
355 360 365
Asp Leu Glu Asp Glu Thr Thr Asn Trp Tyr Val Asn Gln Asp Ser Glu
370 375 380
Gly Phe Ser Lys Ile Lys Phe Phe Pro Ala Thr Ser Gly Val Thr Val
385 390 395 400
Thr Asp Ala Ser Met Ala Tyr Gly Ile Val Leu Cys Thr Lys Thr Gly
405 410 415
Ala Ser Tyr Ala Ser Asp Ala Leu Ala Phe Ser Phe Asp Ile Thr Met
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Thr Ser Thr Trp Ala Asp Val Pro Thr Gln Ala Ser Met Thr Thr Gly
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Pro Leu Phe Phe Ser Tyr Leu Ala Lys Ile Val
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Claims (10)
1.一种Fiber1蛋白的制备方法:其特征在于:以鸭3型腺病毒的核酸作为模板,利用设计合成的特异性引物进行PCR扩增,回收扩增的PCR产物;将扩增的PCR产物、pET-28a表达载体先用NdeI与XhoI酶切后再连接,构建重组表达质粒;将重组表达质粒转化Rosetta(DE3)感受态细胞,并采用异丙基硫代半乳糖苷诱导表达,将获得的表达产物进行纯化,即得所述Fiber1蛋白;
其中,所述特异性引物的序列如下:
上游引物F1F:CATATGCTCTGTCCGTTTAGATTCATC;
下游引物F1R:CTCGAGTTATACAATCTTCGCTAGGTACGAGA;
所述Fiber1蛋白的氨基酸序列如SEQ ID NO.3所示。
2.一种基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,其特征在于:以Fiber1蛋白作为包被抗原包被酶标板,将待测血清样品经稀释后加入酶标板孵育,加入酶标二抗孵育后显色,测定反应后血清样品的OD值。
3.根据权利要求2所述的基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,其特征在于:具体包括以下步骤:
(1)包被:以Fiber1蛋白为包被抗原,将包被抗原用包被液稀释后,加入96孔的ELISA塑胶孔板上,每孔100μL,于4℃包被过夜;之后,弃去包被后孔中多余的抗原,每孔加入200μL的洗涤液洗涤4次,洗完后将板拍干;
(2)封闭:每孔加入200μL封闭剂,37℃,封闭2h;之后,弃去封闭后孔中多余的封闭剂,用洗涤液洗涤4次,洗完后拍干;
(3)加待检血清:每孔加入100μL用样品稀释液稀释过的血清,37℃,孵育1h;之后弃去孵育后孔中多余的血清,用洗涤液洗涤4次,洗完后拍干;
(4)加二抗:每孔加入100μL用样品稀释液稀释过的酶标二抗,37℃,孵育1h;之后,弃去孵育后孔中多余的酶标二抗,用洗涤液洗涤4次,洗完后拍干;
(5)显色:每孔加入100μL显色剂,37℃避光显色10min;
(6)终止:每孔加入100μL终止液;反应终止后置于酶标仪中,在450nm下,读取每个样品的OD值;
(7)结果判定:待检血清OD值≥0.353时判定为鸭3型腺病毒抗体阳性,OD值<0.353判定为阴性。
4.根据权利要求3所述的基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,其特征在于:步骤(1)中,包被抗原用包被液稀释至4μg/mL。
5.根据权利要求3所述的基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,其特征在于:所述包被液为0.05M、pH9.6的碳酸盐缓冲液。
6.根据权利要求3所述的基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,其特征在于:待检血清用样品稀释液按照1:100倍稀释。
7.根据权利要求3所述的基于Fiber1蛋白检测鸭3型腺病毒抗体的间接ELISA检测方法,其特征在于:所述酶标二抗为羊抗鸭IgG酶标二抗;酶标二抗用样品稀释液按1:4000倍稀释。
8.一种鸭3型腺病毒抗体间接ELISA检测试剂盒,其特征在于:所述试剂盒以Fiber1蛋白作为ELISA酶标板的包被抗原。
9.根据权利要求8所述的鸭3型腺病毒抗体间接ELISA检测试剂盒,其特征在于:它还包括包被液、洗涤液、封闭剂、样品稀释液、酶标二抗、显色剂、终止液、鸭3型腺病毒阳性血清、SPF鸭阴性血清。
10.根据权利要求8所述的鸭3型腺病毒抗体间接ELISA检测试剂盒,在鸭3型腺病毒血清抗体检测中的应用。
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