CN110579607A - 一种fadv a病毒间接elisa抗体检测试剂盒及其应用 - Google Patents
一种fadv a病毒间接elisa抗体检测试剂盒及其应用 Download PDFInfo
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Abstract
本发明属于生物工程技术领域,具体涉及一种FADV A病毒间接ELISA抗体检测试剂盒,用于FADV A病毒抗体的快速检测。本发明所述FADV A病毒间接ELISA检测试剂盒,以表达蛋白fiber2为包被抗原,由于表达的特异性蛋白对于FADV A的特异性高,安全性好,且不含无关杂蛋白,能与FADV A阳性血清进行特异结合,且不与其它病毒的阳性血清发生交叉反应,具有良好的抗原性,是理想的ELISA检测抗体的包被抗原载体,使得制得检测试剂盒具有很高的特异性和敏感性,在禽腺病毒A型的检测工作中发挥重要作用。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种FADV A病毒间接ELISA抗体检测试剂盒,用于FADV A病毒抗体的快速检测。
背景技术
禽腺病毒(Fowl adenovirus,FAdV)是禽体内的一种条件性病原体,一般情况下并不表现出临床症状,但在某些条件下,特别是并发感染时,腺病毒会表现严重的致病性,引起家禽较高发病率和死亡率。自2015年在山东、江苏、湖北等地大规模爆发禽Ⅰ群腺病毒以来,此后逐渐向全国各地蔓延,给全国的养禽业造成巨大的经济损失。
禽腺病毒Ⅰ群主要分为5个基因型(A型-E型),以及12个血清型(1-7,8a,8b,9-11型),这些基因型均普遍存在于家禽体内。病禽可以同时感染多个血清型,也可以在拥有某个血清型病毒抗体的同时还可以排出另外一个血清型的病毒。禽腺病毒Ⅰ群病毒呈现世界性分布,常导致各个年龄段的家禽均易感染。其中,FADV A型多会引起病禽的沙囊糜烂,大多情况下病毒在禽体内复制但不致病,或只是引起较轻微的症状,但当有免疫抑制病或发生混合感染时,则会加剧病情的恶化,严重影响禽群健康。目前,随着养禽业逐渐呈现产业化、集约化和规模化的发展趋势,随之对禽腺病毒病的防控提出了更高的要求,也对如何评价禽腺病毒病的主动免疫产生的免疫抗体的性能和效果提出了新的要求。
现有技术中,禽腺病毒A型常用的血清学检测技术主要包括中和试验、琼扩试验等,中和试验因操作繁琐、费时费力、周期较长等缺陷,通常不利于批量样品的检测。ELISA检测是当前生产中应用最广泛的一种抗体检测方法,具有特异、快速、简便、敏感性高等特点。目前,国外已建立了使用不同血清型毒株作为包被抗原以检测禽腺病毒抗体的ELISA方法并研制成标准的试剂盒,但由于价格昂贵,并未得到广泛的应用。纤突蛋白f2是腺病毒的表面蛋白,在介导病毒感染宿主细胞中起重要作用,能有效刺激机体产生体液免疫,诱导中和抗体的产生。因此,研发一种成本较低、特异性及敏感性高、适合基层应用的ELISA检测试剂盒具有重要的意义。
发明内容
为此,本发明所要解决的技术问题在于提供FADV A病毒间接ELISA抗体检测试剂盒,以实现FADV A病毒抗体的快速检测。
为解决上述技术问题,本发明公开了表达蛋白fiber2用于制备FADV A病毒间接ELISA抗体检测试剂盒的用途,所述表达蛋白fiber2具有如SEQ ID No.1所示的核苷酸序列。
本发明还公开了一种所述的FADV A间接ELISA抗体检测试剂盒,包括以FADV A表达蛋白fiber2包被的ELISA板。
进一步的,所述表达蛋白fiber2包被的ELISA板的制备方法为:用pH9.6的0.05M碳酸盐缓冲液作包被液,将所述合成多肽W8稀释至浓度为1μg/mL,按100μL/孔剂量加入至ELISA反应板中,于37℃下作用2小时,并于4℃包被过夜,拍干后用T20blocking buffer于37℃封闭2小时,以pH7.4、含浓度0.05%吐温-20的PBS进行洗涤,拍干并待其干燥后装入含干燥剂的包装袋保存,备用。
更优的,所述试剂盒还包括样品稀释液、10×浓缩洗涤液、酶结合物工作液、显色液、终止液、阳性对照和阴性对照。
所述的FADV A间接ELISA抗体检测试剂盒,所述试剂盒包括:
所述的FADV A间接ELISA抗体检测试剂盒中:
所述样品稀释液为含浓度0.05%吐温-20的0.01M的磷酸盐缓冲液,pH7.4;
所述10×浓缩洗液为含浓度0.5%吐温-20的0.1M的磷酸盐缓冲液,pH7.4;
所述酶结合物工作液为HRP-羊抗鸭IgG;
所述显色液为体积比1:1的浓度0.2mg/mL四甲基联苯胺(TMB)溶液和含浓度0.5‰过氧化氢尿素的柠檬酸-磷酸盐缓冲液的混合液;
所述终止液为浓度0.31%氢氟酸溶液;
所述阳性对照为FADV A阳性血清,其OD650nm≥1.0,并加入1000U/mL的青链霉素;
所述阴性对照为经筛选获得的阴性血清,其OD650nm≤0.25,并加入1000U/mL的青链霉素。
本发明还公开了所述的FADV A间接ELISA抗体检测试剂盒的使用方法,包括将待检血清加入至所述表达蛋白fiber2包被的ELISA板的步骤。
所述的FADV A间接ELISA抗体检测试剂盒的使用方法,具体包括:将待检血清用所述样品稀释液按1:100比例进行稀释,随后按100μL/孔剂量加入至所述ELISA板中,同时设阴性对照及阳性对照,于37℃孵育45min;反应结束后,弃去反应孔中的液体,每孔加洗涤液350μL,洗涤3-5次,每次间隔1min,拍干;随后每孔加100μL所述酶结合物工作液,于37℃孵育45min;随后加洗涤液洗涤3-5次,每次间隔1min,拍干;并依次加入100μL显色液,于37℃避光孵育15min,并加50μL终止液终止反应,用酶标仪在650nm波长下测定各孔吸光度A值,读值计算并判定结果。
本发明还公开了所述的FADV A间接ELISA抗体检测试剂盒在FADV A抗体的检测领域中的应用。
本发明还公开了一种快速检测FADV A抗体的方法,包括所述的FADV A间接ELISA抗体检测试剂盒对待测血清进行检测的步骤,其检测样本的判断标准为:当待检样本OD650nm值与阴性对照OD650nm值的比值(P/N)大于或等于2.1,且待检样本OD650nm值大于0.333判为阳性。
本发明所述FADV A病毒间接ELISA检测试剂盒,以表达蛋白fiber2为包被抗原,表达蛋白fiber2是腺病毒的表面蛋白,在介导病毒感染宿主细胞中起重要作用,能有效刺激机体产生体液免疫,诱导中和抗体的产生,由于表达的特异性蛋白对于FADV A的特异性高,安全性好,且不含无关杂蛋白,能与FADV A阳性血清进行特异结合,且不与其它病毒的阳性血清发生交叉反应,具有良好的抗原性,是理想的ELISA检测抗体的包被抗原载体,使得制得检测试剂盒具有很高的特异性和敏感性,在禽腺病毒A型的检测工作中发挥重要作用。
本发明所述FADV A病毒间接ELISA检测试剂盒,一次检测即可对FADV A抗体水平作出评价,降低了检测成本大大降低;且该试剂盒操作简便、快速,适合批量样品的检测,大大提高了禽腺病毒血清学诊断的速度。
具体实施方式
实施例1表达蛋白fiber2的特异性验证
本实施例所述表达蛋白fiber2的表达按照本领域技术人员所熟知的表达方法进行。
将表达蛋白fiber2(具有如SEQ ID No.1所示的核苷酸序列)用碳酸盐缓冲液分别稀释至蛋白浓度为1μg/mL,每孔100μL包被,放湿盒37℃作用1h后4℃过夜,弃去液体,以PBST洗涤3次,拍干;随后加入封闭液T20blocking buffer,控制剂量300μL/孔,于37℃封闭2h;弃去液体,加PBST洗涤3次;用样品稀释液将鸡阳性血清(FADV A)和SPF鸡阴性血清分别作1:100稀释,控制剂量100μL/孔,37℃作用1h;弃去液体,以PBST洗涤3次;加入羊抗鸡的二抗,控制剂量100μL/孔,37℃作用30min;弃去液体,PBST洗涤3次;加底物(TMB),控制剂量100μL/孔,避光作用15min;加入终止液,控制剂量50μL/孔,放酶标仪读数,结果见表1所示。
表1表达蛋白的特异性验证结果
上表1所示结果表明,所述表达蛋白fiber2与FADV A阳性血清均能发生特异性反应。
实施例2试剂盒成分配制
所述样品稀释液为含0.05%吐温-20的0.01M pH7.4磷酸盐缓冲液:KH2PO40.2g,NaHPO4·12H2O 2.9g,NaCl 8g,定容至1000mL,再加0.5mL吐温-20;
所述10×浓缩洗涤液为含0.5%吐温-20的0.1M pH7.4磷酸盐缓冲液:KH2PO42g,NaHPO4·12H2O 29g,NaCl 80g,定容至1000mL,再加5mL吐温-20;
所述终止液为0.31%氢氟酸溶液:取氢氟酸0.31mL,双蒸水定容至100mL;
阳性对照:将制备的阳性血清用所述样品稀释液作1:100稀释(其OD650nm≥1.0),加入1000U/mL的青链霉素,无菌过滤,作为禽腺病毒1型ELISA抗体检测试剂盒中的阳性对照;
阴性对照:将筛选获得的阴性血清(OD650nm≤0.25),加入1000U/mL的青链霉素,无菌过滤,作为作为禽腺病毒1型ELISA抗体检测试剂盒中的阴性对照。
所述显色液的配制:称取200mg四甲基联苯胺(TMB),用100mL无水乙醇或DMSO溶解后,以双蒸水定容至1000mL;称取21g柠檬酸(C6H8O7·H2O),28.2g无水磷酸氢二钠(Na2HPO4),6.4mL 0.75%过氧化氢尿素,双蒸水定容至1000mL,调pH值4.5-5.0;控制二者的体积比为1:1。
实施例3检测FADV A抗体的间接ELISA反应条件的确定
本实施例采用方阵试验确定多肽抗原和血清最佳工作浓度。用包被缓冲液将筛选的表达蛋白fiber2作1:10,1:20,1:40,1:80等系列稀释,包被ELISA反应板,控制剂量100μL/孔;抗FADV A的阳性血清和阴性血清用样品稀释液分别作1:50,1:100,1:200,1:400,1:800等系列稀释;进行间接ELISA测定;加入显色液显色,及终止液终止反应;并测定光波长650nm的OD值,结果如表2所示。
取阳性血清OD650nm1.0左右,阴性血清OD650nm0.25左右,且阳性血清OD650nm/阴性血清OD650nm即P/N值大于2.1的抗原浓度和血清稀释度为最佳工作浓度,结果表明血清最佳稀释度为1:100,多肽最佳包被浓度1.0μg/mL。
表2多肽抗原和血清最佳工作浓度的确定(OD650nm值)
序列表
<110> 山东省农业科学院家禽研究所(山东省无特定病原鸡研究中心)
<120> 一种FADV A病毒间接ELISA抗体检测试剂盒及其应用
<130> 案卷号
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1031
<212> DNA
<213> fiber2
<400> 1
cggcagacta atagctcttt cgggctttgt tagcagccgg atctcagtgg tggtggtggt 60
ggtgctcgag aacgggggcg cgggcagcgg gacaggtatg caccaccggt ccgatggcca 120
cggtgccgtt cacggtggga ttgaacagtc cggcactggc gcattggaag gtaaaggcga 180
tgagattgta ggtcgtgttg ttggtggcga ggacgggcgt cacttgcatc cgtacgttga 240
tacttcccgt gttgagcgcg ccggtgaaga taggctggta agactccagt accccgaatc 300
gggaagcgta gtagggtgtg ttttggttca tattgaacac gggaggtgag atggggtttc 360
tagcgggttc aaaagaggtc atactgatgt tgcttggctc tagggtccct tgaccgatgt 420
tggaggggtt gcactgcgtg aggaaggagc tcacccagac ggtaaagtat cgcgcgttct 480
cgtttaccgc gcccgtggga cgcgtaccca tggtggttgt gtcgactttg acgtagagcg 540
aggtcgtcag catgcccatg gtattgatct gctgcaggaa gtagctcccc ttaaacgggt 600
tgttgctaga gctcacaaaa ttgcccgtag gggaggacaa gccgatcgtc ccggatgtaa 660
atgcggccac gctgctagtg ggcgcttggt acagttgcaa agtcccattc gtgactttaa 720
attgggtgtt gtcgtactgc aaccctacgc cgggtgctgt gacggcgagc ggacctgcag 780
catcgacctt cacttccaag gtgttatcga cgatttgtag agactcgtcg acggcaacgc 840
ctatgccttg cgcgctgctc tgtaggaatt cggatccgcg acccatttgc tgtccaccag 900
tcatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat 960
ggctgctgcc catggtatat ctccttctta aagttaaaca aaattattct agagggaatg 1020
accgtttacg c 1031
Claims (10)
1.表达蛋白fiber2用于制备FADVA病毒间接ELISA抗体检测试剂盒的用途,所述表达蛋白fiber2具有如SEQ ID No.1所示的核苷酸序列。
2.一种FADVA病毒间接ELISA抗体检测试剂盒,其特征在于,包括以FADVA表达蛋白fiber2包被的ELISA板。
3.根据权利要求2所述的FADVA病毒间接ELISA抗体检测试剂盒,其特征在于,所述表达蛋白fiber2包被的ELISA板的制备方法为:用pH9.6的0.05M碳酸盐缓冲液作包被液,将所述表达蛋白fiber2稀释至浓度为1μg/mL,按100μL/孔剂量加入至ELISA反应板中,于37℃下作用2小时,并于4℃包被过夜,拍干后用T20blocking buffer于37℃封闭2小时,以pH7.4、含浓度0.05%吐温-20的PBS进行洗涤,拍干并待其干燥后装入含干燥剂的包装袋保存,备用。
4.根据权利要求2或3所述的FADVA病毒间接ELISA抗体检测试剂盒,其特征在于,所述试剂盒还包括样品稀释液、10×浓缩洗涤液、酶结合物工作液、显色液、终止液、阳性对照和阴性对照。
5.根据权利要求4所述的FADVA病毒间接ELISA抗体检测试剂盒,其特征在于,所述试剂盒包括:
6.根据权利要求4或5所述的FADVA病毒间接ELISA抗体检测试剂盒,其特征在于:
所述样品稀释液为含浓度0.05%吐温-20的0.01M的磷酸盐缓冲液,pH7.4;
所述10×浓缩洗液为含浓度0.5%吐温-20的0.1M的磷酸盐缓冲液,pH7.4;
所述酶结合物工作液为HRP-羊抗鸡IgG;
所述显色液为体积比1:1的浓度0.2mg/mL四甲基联苯胺(TMB)溶液和含浓度0.5‰过氧化氢尿素的柠檬酸-磷酸盐缓冲液的混合液;
所述终止液为浓度0.31%氢氟酸溶液;
所述阳性对照为经筛选获得的FADVA阳性血清,其OD650nm≥1.0,并加入1000U/mL的青链霉素;
所述阴性对照为经筛选获得的阴性血清,其OD650nm≤0.25,并加入1000U/mL的青链霉素。
7.权利要求2-6任一项所述的FADVA病毒间接ELISA抗体检测试剂盒的使用方法,其特征在于,包括将待检血清加入至所述表达蛋白fiber2包被的ELISA板的步骤。
8.权利要求7所述的FADVA病毒间接ELISA抗体检测试剂盒的使用方法,其特征在于,具体包括:将待检血清用所述样品稀释液按1:100比例进行稀释,随后按100μL/孔剂量加入至所述ELISA板中,同时设阴性对照及阳性对照,于37℃孵育45min;反应结束后,弃去反应孔中的液体,每孔加洗涤液350μL,洗涤3-5次,每次间隔1min,拍干;随后每孔加100μL所述酶结合物工作液,于37℃孵育45min;随后加洗涤液洗涤3-5次,每次间隔1min,拍干;并依次加入100μL显色液,于37℃避光孵育15min,并加50μL终止液终止反应,用酶标仪在650nm波长下测定各孔吸光度A值,读值计算并判定结果。
9.权利要求2-6任一项所述的FADVA病毒间接ELISA抗体检测试剂盒在FADVA抗体的检测领域中的应用。
10.一种快速检测FADVA病毒抗体的方法,其特征在于,包括以权利要求2-6任一项所述的FADVA病毒间接ELISA抗体检测试剂盒对待测血清进行检测的步骤,其检测样本的判断标准为:当待检样本OD650nm值与阴性对照OD650nm值的比值(P/N)大于或等于2.1,且待检样本OD650nm值大于0.333判为阳性。
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