CN112362880A - 一种prrsv的ifa中和抗体检测方法 - Google Patents
一种prrsv的ifa中和抗体检测方法 Download PDFInfo
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Abstract
本发明公开了一种PRRSV的IFA中和抗体检测方法,属于分子生物学检测技术领域。本发明公开的一种PRRSV的IFA中和抗体检测方法,包括将等体积的PRRSV病毒液和临床血清接种到Marc145细胞上,固定、加一抗、加二抗、结果观察。本发明方法利用全病毒和抗体相互作用后感染靶细胞,真实的反应了病毒与中和抗体相互作用的情况。
Description
技术领域
本发明涉及生物技术领域,更具体的说是涉及一种PRRSV的IFA中和抗体检测方法。
背景技术
猪繁殖与呼吸综合症病毒(PRRSV)是一种正链包膜RNA病毒。病毒粒子由核衣壳蛋白(由open reading frame 7或ORF7编码)结合病毒RNA构建的核衣壳核心组成。核衣壳被脂质包膜包裹,其中含有六种结构蛋白:糖蛋白GP2(ORF2a),GP3(ORF3),GP4(ORF4)和GP5(ORF5),以及非糖基化蛋白M(ORF6)和E(ORF2b)。GP5和M被认为是包膜中最丰富的蛋白质,而其他包膜蛋白质的含量较低。故PRRSV感染后的抗体反应非常复杂,总体可分为中和抗体(NA)和非中和抗体(NNA),有研究表明中和抗体效价达到1:8可以有效清除PRRSV病毒血症。
现已建立了多种方法来检测PRRSV特异性抗体,例如酶联免疫吸附测定(ELISA)以及基于免疫荧光和免疫层析带的测定。PRRSV特异性中和抗体(NAb)通常在接种后(dpi)28天出现,但在感染后第一周内产生的非保护性抗体可能对PRRSV感染的早期检测更为有用。这些早期抗体包括对PRRSVN蛋白或nsps等结构蛋白具有特异性的非中和抗体,N蛋白和某些非结构蛋白(nsp1,nsp2和nsp7)已被证明具有高度免疫原性。
目前检测PRRSV特异性抗体的大多数商用ELISA试剂盒(例如IDEXX Herd ChekPRRS ELISA)都采用抗N抗体作为PRRSV感染或改良活病毒(MLV)免疫状态的血清学标志物。尽管诸如ELISA之类的商业测试对于确定血清样品中PRRSV特异性抗体的存在非常敏感,但是ELISA不适合用于抗体水平的定量分析:从ELISA获得的OD值通常在小范围(0.1至2)内变化。此外,大多数ELISA试剂盒使用原核表达的单个重组PRRSV结构抗原(通常为PRRSV-N蛋白)作为包被抗原。因此,此类***无法评估针对其他PRRSV包膜蛋白或非结构蛋白(nsps)的PRRSV特异性抗体反应。在此应注意,由于表达和纯化多种ELISA板包被抗原需要付出巨大的努力,因此可能不会开发使用多种PRRSV抗原的***。此外,由于ELISA包膜抗原在大肠杆菌中表达的一般性质,经常报告假阳性或假阴性结果。因此,提供一种PRRSV的IFA中和抗体检测方法,用于准确检测和定量PRRSV特异性抗体,是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种PRRSV的IFA中和抗体检测方法,用于准确检测和定量PRRSV特异性抗体。
为了实现上述目的,本发明采用如下技术方案:
一种PRRSV的IFA中和抗体检测方法,具体步骤如下:
(1)铺板:把Marc145细胞消化离心后制备成细胞悬液,铺到96孔板上,置于37℃5%CO2培养箱中培养,待细胞完全贴壁,将等体积的PRRSV病毒液和临床血清37℃孵育1h,接种到Marc145细胞上,加入胎牛血清含量为2%的DMEM培养基进行培养,置于CO2培养箱中继续培养48h,观察细胞生长情况,待细胞长满后,取出96孔板;同时设立不接毒正常细胞对照孔;
(2)固定:把取出的96孔板用PBS冲洗2-3次,然后用预冷的无水乙醇于-20℃固定30min,细胞反应板制备成功;
(3)加一抗:把细胞反应板用PBS冲洗2-3次,加入100μl用0.01M PBS 1:400稀释的PRRSV标准阳性血清,置于37℃中作用1h;
(4)加二抗:加入100μl用0.01M PBS(pH7.4)稀释200倍的羊抗猪IgG-FITC荧光抗体,37℃作用1h;
(5)结果观察:用PBS冲洗后置于荧光显微镜下观察显色结果。
进一步,所述PRRSV病毒液的制备方法如下:将PRRSV的病料组织用PBS冲洗,剪碎后按照1g组织样品加入1mL PBS,反复冻融三次,制成病料研磨液。
进一步,所述临床血清的制备方法如下:将采集的血液进行离心,收集血清,用于后期临床血清检测。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种PRRSV的IFA中和抗体检测方法,该方法利用全病毒和抗体相互作用后感染靶细胞,真实的反应了病毒与中和抗体相互作用的情况。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明各病料组织的PCR扩增结果;
其中,M:DL 2000Marker;1:***;2:肝脏;3:脾脏;4:肺脏;5:空白对照(模板为ddH2O);
图2附图为本发明IFA检测PRRSV中和抗体的显色结果;
其中,A为中和抗体试验,B为正常接毒细胞对照;
图3附图为本发明IFA检测不同PRRSV接种量的结果;
其中,A、B、C、D、E分别表示1000、100、10、1、0.1个TCID50病毒接种;
图4附图为本发明IFA检测血清中和抗体效价的结果;
其中,A、B、C、D、E、F分别表示血清稀释倍数为1:4、1:8、1:16、1:32、1:64、1:128。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
血液样品和病料组织采集自周边猪场,将采集的血液进行离心,收集血清,用于后期临床血清检测;病料组织进行研磨,检测PRRSV并分离病毒;细胞株为Marc145细胞(非洲绿猴肾细胞),各种病毒抗体由实验室保存。
实施例1PRRSV分离及鉴定
将疑似PRRSV的病料组织(淋巴、肝脏、脾脏、肺脏)用PBS冲洗,剪碎后按照1g组织样品加入1mL PBS,反复冻融三次,制成病料研磨液(作为PRRSV病毒液使用),使用RNA提取试剂盒,从上述病料中提取PRRSV的RNA,然后把RNA反转录为cDNA。
利用DNAMAN软件设计PRRSV目的基因引物,具体引物序列如下:
PRRSV-F:5’-GGCCAGCCAGTCAATCAG-3’;SEQ ID NO.1;
PRRSV-R:5’-GGCAAACTAAACTCCACAGTG-3’;SEQ ID NO.2。
利用设计好的引物,分别以上述提取的PRRSV的淋巴、肝脏、脾脏、肺脏cDNA为模板进行PCR扩增,体系为25μL,反应条件为:98℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸50s,30个循环;72℃延伸10min,结束后4℃保存。PCR产物进行琼脂糖凝胶电泳,结果见图1。结果显示,以病料提取的cDNA为模板进行PCR扩增,出现一条特异性目的条带,长度约为537bp,与预期大小一致;病料中含有PRRSV。
实施例2建立稳定的IFA中和抗体检测方法
把Marc145细胞消化离心后制备成含1.0×105cells/ml的细胞悬液,每孔100μl铺到96孔板上,置于37℃5%CO2培养箱中培养12h,待细胞完全贴壁,将等体积的PRRSV病毒液(50μl)和临床血清37℃孵育1h,然后接种到96孔板内的Marc145细胞上,培养48h待细胞长满后取出96孔板,用预冷的无水乙醇固定。加入PRRSV标准阳性血清,37℃孵育1h,然后加入羊抗猪IgG-FITC,37℃孵育1h,置荧光显微镜下观察,产生特异性绿色荧光为阴性,反之为阳性。将鉴定为阳性的血清进行倍比稀释,重复上述操作,可获得抗体效价。
(1)铺板:把Marc145细胞消化离心后制备成含1.0×105cells/ml的细胞悬液,每孔100μl铺到96孔板上,置于37℃5%CO2培养箱中培养12h,待细胞完全贴壁,将等体积的PRRSV病毒液和临床血清37℃孵育1h,接种到Marc145细胞上,加入胎牛血清含量为2%的DMEM培养基进行培养,置于CO2培养箱中继续培养48h,观察细胞生长情况,待细胞长满后,取出96孔板;同时设立正常接毒细胞对照孔;
(2)固定:把取出的96孔板用PBS冲洗2-3次,然后用预冷的无水乙醇于-20℃固定30min,也可以长时间保存备用,即细胞反应板制备成功;
(3)加一抗:把96孔板用PBS洗过2-3次,然后加入100μl用0.01M PBS1:400稀释的PRRSV标准阳性血清(IDEXX),置于37℃中作用1.0h;
(4)加二抗:加入100μl用0.01M PBS(pH7.4)稀释200倍的羊抗猪IgG-FITC荧光抗体(索莱宝科技有限公司),37℃作用1h;
(5)结果观察:用PBS冲洗后置于荧光显微镜下观察显色结果,结果见图2。
实施例3IFA反应条件
1)PRRSV的TCID50测定
(1)将Marc145细胞悬液铺到96孔板上,每孔100μL,使细胞量达到2-3×105个/mL,培养12h,直至细胞完全贴壁;
(2)在青霉素瓶或离心管中将PRRSV病毒液作连续10倍的稀释,从10-1-10-10;
(3)将稀释好的病毒接种到细胞长成单层的96孔板上,每一稀释度接种一纵排共8孔,每孔接种100μL;
(4)剩下两纵排不接毒,设正常细胞对照(每孔100μL维持液,维持液为胎牛血清含量为2%的DMEM培养基);
(5)培养48h待细胞长满后取出固定,置于-20℃备用;
(6)利用IFA方法进行检测,观察记录发生病变(CPE)的孔数,结果见表1;(7)TCID50按Reed-Muench两氏法计算。
表1 TCID50测定结果统计
按Reed-Muench两氏法计算TCID50,计算方法如下:
距离比=(高于50%病变率的百分数-50%)/(高于50%病变率的百分数-低于50%病变率的百分数)=(55.5-50)/(55.5-8.3)=0.1
lg TCID50=距离比×稀释度对数之间的差+高于50%病变率的稀释度的对数=0.1×(-1)+(-4)=-4.1。
根据上述数据可得TCID50=10-4.1/0.1ml=10-5.1/ml
含义:将该病毒稀释104.1接种100μl可使50%的细胞发生病变。
2)病毒接种量和固定时间
分别用1000个TCID50、100个TCID50、10个TCID50、1个TCID50、0.1个TCID50的病毒接种Marc145细胞,并设置未接毒对照。利用IFA检测方法分别以阳性和阴性血清作为一抗进行检测,观察结果确定最佳病毒接种量,结果如图3所示。
图3结果表明:100个TCID50的病毒接种量最佳且信号较强。
将PRRSV(最佳病毒接种量)接种到长至60%-70%左右的Marc145细胞瓶内,待细胞长满后将其分别铺到4个96孔板上,在37℃5%CO2培养箱培养12、24、36、48h后,逐一取出固定。然后利用IFA检测方法分别以阳性和阴性血清作为一抗进行检测,观察结果确定固定时间。
结果发现,以100个TCID50的病毒接种Marc145细胞,培养48h后固定制备IFA细胞反应板最佳。
3)血清稀释浓度、羊抗猪IgG-FITC工作浓度
将感染PRRSV的猪血清分别按1:50、1:100、1:200、1:400等进行连续倍比稀释,然后分别作为一抗加入制备好的细胞反应板内,利用IFA抗体检测方法分别进行检测,观察结果确定最佳血清稀释浓度。
将制备好的细胞反应板取出,加入1:400倍稀释的阳性和阴性血清,然后分别加入1:100、1:200、1:300、1:400倍稀释的羊抗猪IgG-FITC,观察实验结果确定最佳二抗稀释浓度。
结果发现,待检血清以1:50开始,可根据抗体强弱情况在此基础上进行连续2倍稀释检测抗体效价,羊抗猪IgG-FITC工作浓度1:200最佳。
实施例4IFA的特异性及敏感性
分别用已知的猪PRRSV阳性血清,阴性健康猪血清、猪伪狂犬病毒(PRV)阳性血清、猪圆环2型病毒(PCV2)阳性血清、猪瘟病毒(CSFV)阳性血清与病毒孵育后进行检测。通过结果确定IFA检测方法的特异性和敏感性。
结果发现,PRRSV只与PRRSV阳性血清发生特异性中和反应,和其它病毒阳性血清无交叉反应,表明IFA检测方法具备特异性。表明IFA中和抗体检测方法敏感性好。
实施例6IFA的批内批间重复性以及保质期
取相同批次和不同批次制备的若干中和抗体检测,检测结果基本一致,表明IFA检测方法的重复性较好。
实施例7IFA临床中和抗体检测
利用建立的PRRSV的IFA中和抗体检测方法对两个猪场的血清进行中和抗体检测,结果见表3。对1号猪场的51份血清进行中和抗体检测,有20份为阳性,27份为阴性,4份疑似阳性;对2号猪场的67份血清进行中和抗体检测,有22份为阳性,39份为阴性,6份疑似阳性。
表3临床中和抗体检测结果
实施例9PRRSV临床血清中和抗体及效价检测
利用上述建立的IFA中和抗体检测方法检测抗体为阳性的血清,并对中和抗体进行效价检测,在96孔培养板中把血清连续倍比稀释为1:2、1:4、1:8、1:16直至1:4096,在上述各孔内加入50μl稀释好的PRRSV病毒液,混匀后放入37℃5%CO2培养箱中作用1h。同时设阴、阳性血清对照,病毒对照和正常细胞对照。
然后重复IFA中和抗体检测方法的步骤,检测血清中和抗体效价。
以其中一份阳性血清为例,检测该血清的中和抗体效价,图4是血清稀释倍数为1:4、1:8、1:16、1:32、1:64、1:128的结果图,可以看出该血清的中和抗体效价为1:32。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
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Claims (3)
1.一种PRRSV的IFA中和抗体检测方法,其特征在于,具体步骤如下:
(1)铺板:把Marc145细胞消化离心后制备成细胞悬液,铺到96孔板上,置于37℃5%CO2培养箱中培养,待细胞完全贴壁,将等体积的PRRSV病毒液和临床血清37℃孵育1h,接种到Marc145细胞上,加入胎牛血清含量为2%的DMEM培养基进行培养,置于CO2培养箱中继续培养48h,观察细胞生长情况,待细胞长满后,取出96孔板;同时设立不接毒正常细胞对照孔;
(2)固定:把取出的96孔板用PBS冲洗2-3次,然后用预冷的无水乙醇于-20℃固定30min,细胞反应板制备成功;
(3)加一抗:把细胞反应板用PBS冲洗2-3次,加入100μl用0.01M PBS 1:400稀释的PRRSV标准阳性血清,置于37℃中作用1h;
(4)加二抗:加入100μl用0.01M PBS(pH7.4)稀释200倍的羊抗猪IgG-FITC荧光抗体,37℃作用1h;
(5)结果观察:用PBS冲洗后置于荧光显微镜下观察显色结果。
2.根据权利要求1所述的一种PRRSV的IFA中和抗体检测方法,其特征在于,所述PRRSV病毒液的制备方法如下:将PRRSV的病料组织用PBS冲洗,剪碎后按照1g组织样品加入1mLPBS,反复冻融三次,制成病料研磨液。
3.根据权利要求1所述的一种PRRSV的IFA中和抗体检测方法,其特征在于,所述临床血清的制备方法如下:将采集的血液进行离心,收集血清,用于后期临床血清检测。
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