CN112048006B - 一种替代中和效力测定的elisa方法及其应用 - Google Patents

一种替代中和效力测定的elisa方法及其应用 Download PDF

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CN112048006B
CN112048006B CN202010937821.3A CN202010937821A CN112048006B CN 112048006 B CN112048006 B CN 112048006B CN 202010937821 A CN202010937821 A CN 202010937821A CN 112048006 B CN112048006 B CN 112048006B
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秦爱建
张昱晗
武奇
钱琨
邵红霞
叶建强
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Abstract

本发明公开了一种IBV特异性中和表位抗原多肽,所述多肽为环状多肽,所述多肽的氨基酸序列为CSCPYVSYGRFCIQPDGSIKQC。本发明还公开了一种IBV特异性抗体及其制备方法。本发明还公开了一种替代中和效力测定的ELISA方法。本发明还公开了一种ELISA检测试剂盒,本发明应用建立的pELISA方法检测IBV抗体,并发现其与抗IBV中和抗体呈正相关,本发明可以用于IBV疫苗免疫效果的评价,IBV感染鸡的抗体水平测定,从而有益于鸡群健康管理等。

Description

一种替代中和效力测定的ELISA方法及其应用
技术方案
本发明属于生物医学技术领域,具体涉及一种替代中和效力测定的ELISA方法及其应用。
背景技术
传染性支气管炎(IB)是由传染性支气管炎病毒(IBV)引起的传染病。IBV感染鸡引起严重的呼吸道和肾脏疾病,导致重大的经济损失。虽然疫苗现在被广泛使用,但是在鸡群中仍然可以观察到IB的流行。
中和抗体是当病原微生物侵入机体时会产生相应的抗体。病原微生物入侵细胞时需要依赖病原体自身表达的特定分子与细胞上的受体结合,才能感染细胞,并进一步扩增。中和抗体是B淋巴细胞产生的某些抗体,能够与病原微生物表面的抗原结合,从而阻止该病原微生物黏附靶细胞受体,防止侵入细胞。中和抗体是由适应性免疫应答细胞分泌的一种可溶性蛋白。病毒侵入机体之后,免疫细胞将中和抗体分泌到血液里,后者与血液里的病毒颗粒结合,阻止病毒感染细胞,破坏病毒颗粒,这样就把病毒“中和”掉了。
目前一般采用细胞培养方法检测血清中的中和抗体滴度,从而评估疫苗在免疫鸡群中的效用,然而,这个过程耗时费力,难以在基层实现。有报道称免疫荧光和酶联免疫吸附试验可以替代病毒的中和试验,可以更快更简单的方法来测定血清中和抗体的滴度,最近的研究表明,狂犬病毒和牛病毒性腹泻病毒糖蛋白ELISA可以分别测定人和牛接种疫苗后血清中的中和抗体效价。间接ELISA和中和试验之间抗体滴度的相关性也在寨卡病毒和人类乳突病毒中进行了研究。
虽然已经开发了用全病毒粒子或重组S1蛋白、N蛋白和非结构蛋白检测抗体的ELISA方法,迄今为止,还没有血清学方法替代传染性支气管炎病毒的中和试验。
发明内容
发明目的:IBV基因组编码4种主要结构蛋白:S、E、M和N,另外还有十五种非结构蛋白和一些附属蛋白。在这些蛋白中,S糖蛋白被认为是携带中和性表位主要的保护性抗原,可以诱导中和抗体的产生,然而,S1在不同毒株的IBV中差异很大。S2是一个高度保守的蛋白,携带广泛的抗原表位,S2蛋白也存在相关中和性抗原表位。我们的研究也表明S2存在一个广谱的中和表位。
本发明中,我们合成了该中和表位的多肽,在建立检测IBV抗体ELISA方法的基础上,发现其与中和抗体滴度具有相关性,可以用于中和抗体的评价测定。
本发明的所述多肽ELISA检测的血清抗体水平与中和抗体水平呈正相关性。
本发明还要解决的技术问题是提供了一种替代中和效力测定的ELISA方法,所述该技术可以应用于疫苗免疫后的中和抗体水平评估。
本发明还要解决的技术问题是提供了一种IBV特异性抗体及其制备方法。
本发明还要要解决的技术问题是提供了IBV特异性中和表位抗原多肽在制备IBV检测试剂盒方面的应用。
本发明最后要解决的技术问题是提供了一种ELISA检测试剂盒。
技术方案:为了解决上述技术问题,本发明提供了一种IBV特异性中和表位抗原多肽,所述多肽为环状多肽,所述环状多肽的氨基酸序列为CSCPYVSYGRFCIQPDGSIKQC。
本发明内容还包括一种IBV特异性抗体,所述IBV特异性抗体特异性的与所述的IBV特异性中和表位抗原多肽结合。
本发明内容还包括一种IBV特异性抗体的制备方法,将所述的IBV特异性中和表位抗原多肽与佐剂混合,免疫小鼠、鸡或其他实验动物,经2-3次免疫后即可产生抗IBV特异性抗体。
本发明内容还包括一种替代中和效力测定的ELISA方法,包括以下步骤:
1)将所述的IBV特异性中和表位抗原多肽包被酶标板,包被后用兔血清封闭;
2)洗涤多次后,加入鸡血清共同孵育;
3)再次洗涤多次,再与山羊抗鸡IgG酶标抗体共同孵育,经多次洗涤后,用TMB底物显色;
4)最后用0.1%SDS终止反应,用酶联免疫吸附分析仪检测650nm的吸光度,从而检测出个体中IBV的中和抗体水平。
本发明内容还包括所述的IBV特异性中和表位抗原多肽在制备IBV检测试剂盒方面的应用。
本发明内容还包括一种ELISA检测试剂盒,所述ELISA试剂盒包括权利要求1所述的IBV特异性中和表位抗原多肽。
其中,所述ELISA试剂盒包括以下组分:包被所述的IBV特异性中和表位抗原多肽的酶标板、酶标抗体、底物溶液、终止液、洗涤液、阴性对照和阳性对照。
其中,所述酶标抗体为山羊抗鸡IgG酶标抗体G。
其中,所述底物溶液为TMB底物显色溶液。
其中,所述阴性对照为SPF鸡血清。
其中,所述阳性对照为SPF鸡免疫IBV后的血清。
有益效果:本发明应用建立的pELISA方法可以检测出目前已知所有类型IBV的抗体,而常规ELISA仅能检测出经典IBV毒株诱导产生的抗体,并发现本方法与抗IBV中和抗体呈正相关,本发明可以用于IBV疫苗免疫效果的评价,IBV感染鸡的抗体水平测定,从而有益于鸡群健康管理等。
附图说明
图1环状多肽与不同病毒血清样本反应后用酶联免疫吸附分析仪检测650nm的吸光度;
图2不同病毒株免疫血清样品的中和抗体与ELISA抗体的相关性比较;
图3M41和CK/CH/2010/JT1感染鸡后中和抗体和ELISA抗体具有很好的相关性比较。
具体实施方式
下面结合具体的实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于申请所附权利要求书所限定的范围。
除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1
肽的合成:按常规方法合成环状多肽CSCPYVSYGRFCIQPDGSIKQC,色谱鉴定纯度大于95%。
按常规方法合成多肽SCPYVSYGRFCIQPDGSIKQ,色谱鉴定纯度大于95%。
pELISA方法:将合成的环状多肽CSCPYVSYGRFCIQPDGSIKQC和多肽SCPYVSYGRFCIQPDGSIKQ在0.1μg/mL用pH9.60.05M的碳酸盐缓冲液中分别包被ELISA板,在4℃条件下过夜包被。用8%兔血清在37℃下封闭3小时。PBST清洗3次后,将100μL抗IBV鸡血清(PBST进行1∶200稀释)加入,在37℃下孵育1小时,然后清洗5次,再与100μL山羊抗鸡IgG酶标抗体(Jackson公司产品)在37℃下孵育1小时。经5次洗涤后,用100μL TMB底物在37℃显色15min。100μL 1%的SDS终止反应。用酶联免疫吸附分析仪检测650nm的吸光度。
该方法与抗禽流感病毒(AIV)、禽白血病病毒(ALV)、禽网状内皮增生病毒(REV)、鹅瘟病毒(GPV)、鸡传染性法氏囊病病毒(IBDV)、传染性喉气管炎病毒(ILTV)、马立克氏病病毒(MDV)、鸡产蛋下降综合症病毒(EDV)等病毒的阳性血清样本没有反应性。如图1,是环状多肽CSCPYVSYGRFCIQPDGSIKQC与不同病毒血清终止反应后用酶联免疫吸附分析仪检测650nm的吸光度,0.2是阴阳性分界值。从下面表1的650nm吸光度值可以看出,环状多肽OD值更高,敏感性和特异性更好。多肽SCPYVSYGRFCIQPDGSIKQ没有加C的保存期较短,环状多肽CSCPYVSYGRFCIQPDGSIKQC加C稳定且保存期长。
表1.不同形式多肽的检测效果
Figure BDA0002671301750000041
临床样本分析:250份临床(某养殖企业)免疫鸡血清样本中,233份血清样本pELISA为阳性,17份血清样本免疫荧光(IFA)为阴性,232份血清样本经IFA检测为阳性,18份血清样本经IFA检测为阴性。与IFA相比,pELISA对250份血清样本的准确率为98.8%。因此,pELISA有很好的可靠信。
表2比较pELISA与IFA检测结果
Figure BDA0002671301750000051
从表3可以看出pELISA的重复性效果,批间和批内变异系数很小,说明重复效果好。
表3 pELISA的稳定性检测结果
Figure BDA0002671301750000052
实施例2中和抗体与ELISA抗体的相关性
血清中和实验,将待测定的IBV阳性血清进行56℃灭活30min,倍比梯度稀释,与100TCID50 IBV等体积混合作用1h,混合物加入到含80%培养单层鸡胚肾细胞的96孔板中,在37℃5%CO2培养2h。去除上清液,加入200mL DMEM/F12含2%CFS,48h后观察细胞病变(CPE),计算50%中和效价。用间接免疫荧光法对细胞进行固定和检测。比较不同类型IBV的免疫血清ELISA滴度和中和滴度,评价pELISA与中和试验的相关性。对不同毒株如4/91、M41、H52、QXl87和CK/CH/2014/FJ14强毒株免疫血清样品进行检测。如图2所示,血清样本编号8269(QXl87)、4179(M41)、3923(CK/CH/2014/FJ14)和4204(H52)的pELISA滴度均为1∶1333,中和滴度均为1∶102。M41血清编号4179、4180和4187的中和滴度分别为1∶1333、1∶1600和1∶2666,ELISA滴度分别为1∶102、1∶124和1∶213。结果表明,试验血清的ELISA效价与中和效价呈正相关,从图2可以看出ELISA与中和抗体滴度呈正相关。
实施例3用经典IBV毒株和分离毒株感染SPF鸡不同时间的抗体相关性
为了进一步评价鸡传染性支气管炎病毒感染和接种后血清酶联免疫吸附试验(ELISA)和中和抗体水平的变化,对14日龄SPF鸡分别感染了IBV CK/CH/2014/FJ14株、CK/CH/2010/JT1株或M41株。采集感染雏鸡不同时间的血清标本,用实施例1的pELISA法测定血清效价。然后将结果与实施例2中的中和法测定的中和滴度进行比较。如图3所示,鸡接种CK/CH/2010/JT1后第7、14、21和28天血清样本的ELISA滴度分别为1∶100、1∶466.6、1∶666.6和1∶1066,而这些样本的中和滴度分别为1∶14.8、1∶56.25、1∶154.5和1∶206(图3b)。随着感染时间的延长,血清的ELISA滴度和中和滴度均增加,在接种M41或CK/CH/2010/JT1后不同时间点采集的血清中,ELISA滴度和中和滴度呈良好的一致性(图3a和b)。从图3可以看出,M41和CK/CH/2010/JT1感染鸡后中和抗体和ELISA抗体具有很好的相关性。
序列表
<110> 扬州大学
<120> 一种替代中和效力测定的ELISA方法及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213> 特异性中和表位抗原多肽(IBV)
<400> 1
Cys Ser Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Gln Pro Asp
1 5 10 15
Gly Ser Ile Lys Gln Cys
20
<210> 2
<211> 20
<212> PRT
<213> 多肽(IBV)
<400> 2
Ser Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Gln Pro Asp Gly
1 5 10 15
Ser Ile Lys Gln
20

Claims (5)

1.一种非诊断目的的替代中和效力测定的ELISA方法,其特征在于,包括以下步骤:
1)将IBV特异性中和表位抗原多肽包被酶标板,包被后用兔血清封闭;所述IBV特异性中和表位抗原多肽为环状多肽,所述环状多肽的氨基酸序列为CSCPYVSYGRFCIQPDGSIKQC,
2)洗涤多次后,加入鸡血清共同孵育;
3)再次洗涤多次,再与山羊抗鸡IgG酶标抗体共同孵育, 经多次洗涤后,用TMB底物显色;
4)最后用0.1%SDS终止反应,用酶联免疫吸附分析仪检测650nm的吸光度,从而检测出个体中IBV的中和抗体水平。
2.IBV特异性中和表位抗原多肽在制备IBV检测试剂盒方面的应用,所述IBV特异性中和表位抗原多肽为环状多肽,所述环状多肽的氨基酸序列为CSCPYVSYGRFCIQPDGSIKQC。
3.根据权利要求2所述的应用,其特征在于,所述检测试剂盒包括以下组分:包被权利要求1所述的IBV特异性中和表位抗原多肽的酶标板、酶标抗体、底物溶液、终止液、洗涤液、阴性对照和阳性对照。
4.根据权利要求3所述的应用,其特征在于,所述酶标抗体为山羊抗鸡IgG酶标抗体。
5.根据权利要求3所述的应用,其特征在于,所述底物溶液为TMB显色溶液。
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