CN114487396A - 用于检测猪圆环病毒2型的红色乳胶微球免疫层析试纸条及其制备方法和应用 - Google Patents
用于检测猪圆环病毒2型的红色乳胶微球免疫层析试纸条及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了用于检测猪圆环病毒2型的红色乳胶微球免疫层析试纸条及其制备方法和应用。所述的试纸条包括PVC底板,以及在PVC底板上从左到右依次组装的结合垫、硝酸纤维素膜和吸水垫,所述的结合垫上涂覆有红色乳胶微球偶联的抗猪圆环病毒2型单克隆抗体7C7,所述的硝酸纤维素膜上设有包被抗猪圆环病毒2型单克隆抗体9H4的检测线以及包被山羊抗鼠IgG的质控线。本发明中标记抗体和捕获抗体是挑选的最为广谱性的单抗,故对三种基因型的PCV2都具有较好的检测敏感性,与其他的病毒均无交叉反应。因此,本发明成功筛选出PCV2的广谱性单抗且成功建立了检测PCV2抗原的红色乳胶微球免疫层析试纸条,为后续的PCV2流行病学调查和净化提供了新的技术手段。
Description
技术领域
本发明涉及一种彩色乳胶微球免疫层析试纸条及其制备方法和应用,特别涉及一种用于检测猪圆环病毒2型的红色乳胶微球免疫层析试纸条及其制备方法和应用。本发明属于病毒检测技术领域。
背景技术
猪圆环病毒2型病毒(porcine circovirs type 2,PCV2)属于圆环病毒科圆环病毒属的一员,是一种无囊膜的单股环状DNA病毒,呈二十面体对称。主要包含三个开放阅读框:ORF1、ORF2和ORF3。其中,ORF1编码病毒复制有关的蛋白Rep和Rep’,参与病毒DNA的复制。ORF2编码病毒的唯一结构蛋白,Cap蛋白,60个Cap蛋白构建成一个二十面体病毒外壳。ORF3与诱导细胞凋亡有关并参与病毒致病过程。猪圆环病毒2型病毒直径17nm左右,是目前已知的最小的能感染哺乳动物的DNA病毒之一。虽然圆环病毒体积小,结构简单,但它的致病性却比较复杂:PCV2感染包括亚临床感染和显性感染,前者感染猪没有明显症状,但是平均日增重会有所下降,后者包括断奶后仔猪多系统衰竭综合征(PostweaningMultisystemic Wasting Syndrome,PMWS)、呼吸系统疾病综合征、生殖障碍和肠道疾病等,这些综合征统称为PCV2相关疾病(Porcine Circovirus Diseases,PCVADs),对全世界养猪业造成了严重影响。
目前,基于PCV2全基因或ORF2序列的遗传演化分析,PCV2分为六种基因型,PCV2a~PCV2f,其中PCV2a、PCV2b和PCV2d是主要基因型。2003年以前,PCV2a为主要的流行毒株;从2004年开始,PCV2b毒株占据流行毒株的主体。2006年至今,2d毒株逐渐增多,目前已经变成了许多国家的流行毒株。这说明PCV2存在明显的基因型的转变。随着猪圆环病毒2型各类疫苗的广泛使用,猪群中PCV2抗原检出率已有明显降低,但由于疫病的复杂性和疫苗质量的参差不齐,还是会发生很多PCV2的显性感染,甚至造成猪只的死亡。根据目前流行毒株的改变极其多样性,所以为PCV2血清学抗原检测方法提出了更高的要求,即广谱性和特异性。因此筛选出广谱特异性针对PCV2病毒的单抗就显得尤为重要。
PCV2抗原的常规检测方法一般包括聚合酶链式反应(PCR)、定量PCR、病毒分离和鉴定、酶联免疫吸附试验(ELISA)、化学发光免疫分析法(CLIA),胶体金免疫层析技术(GICA)。PCR相关方法是最敏感的抗原检测方法,抗原检出率较高,对试验操作人员和实验设备要求较高,并不适用于所有猪场的日常检测。病毒分离和鉴定需要专业的技术人员在专业的实验室中进行,检测周期长,不适用于基层与现场检测。ELISA方法虽然比病毒分离容易,但是也需要专业的仪器进行最终的结果判定。CLIA因为其检测效率高、特异性好,近些年来被逐渐地应用到兽医领域,但此种方法同样需要专业的设备,不适用于田间的大规模普查。与PCR、病毒分离鉴定、ELISA、CLIA相比,GICA既不需要专业的人员设备,检测时间又可以大大缩短,只需要简单地取样并进行视觉测定就可以做出相应判断。免疫层析方法检测PCV2抗原方面,刘玉伟[刘玉伟.猪圆环病毒2型抗原和抗体胶体金检测方法的建立[D].山东师范大学,2019.]研制出一种检测PCV2抗原的胶体金试纸条,但由于同一批次试纸条的稳定性及重复性不理想,目前还在优化。柳亚茹[柳亚茹.猪圆环病毒2型抗原抗体荧光微球免疫层析快速检测方法的建立与应用[D].山东农业大学,2019.]研制了一种荧光微球免疫层析快速检测试纸条,可实现快速的定量检测,但由于微球内部加入了荧光物质,读取结果也需要专业的设备,无法用肉眼直接观察,增加了使用的步骤。彩色乳胶微球是近年来发展较快的新技术,在生物领域中得到广泛应用,彩色微球免疫标记的优点有色彩鲜艳而丰富,彩虹系列色彩均可调整,可实现多重检测;粒度均一、单分散性好、检测结果可重复性强;检测结果直观,肉眼可观察,使用方便;特别是与GICA相比,灵敏度更高。基于以上优势,近些年来乳胶微球免疫层系技术已经被广泛地应用到细菌检测、病毒检测、寄生虫检测等诸多方面。
因此,本发明以真核细胞表达系统制备PCV2b和PCV2d流行毒株Cap蛋白的病毒样颗粒(virus-like particles,VLPs)作为免疫原,利用淋巴细胞杂交瘤技术筛选出针对PCV2广谱特异性的单克隆抗体(Monoclonal antibodies,McAbs),并以此制备了猪圆环病毒2型红色乳胶微球免疫层析试纸条,为该病毒的快速检测提供了技术支持,对该病的防控具有重要意义。
发明内容
本发明的目的在于提供一种用于检测猪圆环病毒2型病毒的红色乳胶微球免疫层析试纸条及其制备方法和应用。
为了达到上述目的,本发明采用了以下技术手段:
本发明用真核表达的PCV2b/MDJ和PCV2d/SDRS VLPs分别作为免疫原,共筛选出8株抗PCV2b和4株抗PCV2d的单克隆抗体,其中具有中和活性的单抗有7株,另外还有广谱性的单克隆抗体6株,这6株单抗能够覆盖绝大多数的PCV2毒株,本发明从这6株广谱性单抗中选择了IPMA抗体效价较高的7C7和4B3以及9H4进行红色乳胶微球免疫层析试纸条的研制,经配对实验验证,最终选择了9H4作为包被抗体,7C7作为标记抗体。由于选用的抗体9H4、7C7都是针对PCV2的单克隆抗体,且都具有针对PCV2a、PCV2b和PCV2d广谱的反应特性,故在特异性和敏感性方面具有较为出色的表现,进而能够提升检测的准确性和灵敏度,减少漏检和错检的发生几率。在敏感性检测结果中,针对PCV2a/CL毒株的最小检测限要明显高于PCV2b/MDJ和PCV2d/LNHC毒株的最小检测限,我们分析是由于9H4和7C7单克隆抗体的免疫原分别是PCV2b和PCV2d的毒株,所以其针对PCV2b和PCV2d毒株的敏感性会更高。这也从侧面反映出PCV2基因型的变化导致了其抗原性产生差异。在符合率试验中,本发明将本研究中的红色乳胶微球免疫层析试纸条与敏感性较高的PCR方法进行比较,试纸条对各类组织样品总的符合率为86.67%,检测敏感性为69.70%,特异性为100%。
具体的,本发明的一种用于检测猪圆环病毒2型的红色乳胶微球免疫层析试纸条,所述的试纸条包括PVC底板,以及在PVC底板上从左到右依次组装的结合垫、硝酸纤维素膜和吸水垫,所述的结合垫上涂覆有红色乳胶微球偶联的抗猪圆环病毒2型病毒单克隆抗体7C7,所述的硝酸纤维素膜设有包被抗猪圆环病毒2型病毒单克隆抗体9H4的检测线以及包被山羊抗鼠IgG的质控线;
其中,所述的抗猪圆环病毒2型单克隆抗体7C7,命名为mAb 7C7,分类命名为鼠源杂交瘤细胞,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,其菌种保藏编号为:CGMCC No.23871,保藏日期为2021年11月18日;
其中,所述的抗猪圆环病毒2型单克隆抗体9H4,命名为mAb9H4,分类命名为鼠源杂交瘤细胞,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,其菌种保藏编号为:CGMCC No.23870,保藏日期为2021年11月18日。
其中,优选的,红色乳胶微球偶联的抗猪圆环病毒2型单克隆抗体7C7按照15ul/cm喷涂于玻璃纤维上作为结合垫,其中,单克隆抗体7C7和红色乳胶微球的标记比例为100μg:1mg;检测线上单克隆抗体9H4和质控线上山羊抗鼠IgG的浓度均0.5mg/ml。
其中,优选的,检测线与质控线的距离为1cm。
其中,优选的,红色乳胶微球偶联的抗猪圆环病毒2型单克隆抗体7C7通过以下方法制备得到:
取1mg红色乳胶微球溶液,80HZ超声5min,用超纯水稀释200倍,然后10000rpm离心10min,洗去上清,沉淀用pH=5.6的1000μl 0.1M的柠檬酸缓冲液溶解,80HZ超声5min;获得的溶液在磁力搅拌下缓慢加入100μl浓度为1mg/ml纯化后的抗猪圆环病毒2型单克隆抗体7C7,室温缓慢搅拌2h后缓慢加入50g/L BSA使其终浓度为10g/L,继续搅拌1小时后10000rpm离心1h;沉淀用0.1M PBS溶解,80HZ超声5min,反复洗涤3次,最终沉淀用0.1M PBS溶解至原体积的1/20,得到红色乳胶微球偶联的抗猪圆环病毒2型病毒单克隆抗体7C7溶液。
进一步的,本发明还提出了所述的红色乳胶微球免疫层析试纸条在制备检测猪圆环病毒2型试剂中的应用。
相较于现有技术,本发明的有益效果是:
本发明提供了一种用于检测猪圆环病毒2型病毒的红色乳胶微球免疫层析试纸条及其制备方法。本发明研制的猪圆环病毒2型红色乳胶微球免疫层析试纸条可以较好地克服其他抗原检测方法耗时时间长、对仪器设备以及人员专业化要求高的缺点,只需要10分钟就能肉眼观察判别阴阳性。由于标记抗体和捕获抗体是挑选的最为广谱性的单抗,故对三种基因型的PCV2都具有较好的敏感性,与其他的病毒均无交叉反应。因此,本发明成功筛选出PCV2的广谱性单抗且成功建立了检测PCV2抗原的红色乳胶微球免疫层析试纸条,为后续的PCV2流行病学调查和净化提供了新的技术手段。
附图说明
图1为采用Western Blot方法检测12株单抗与对应基因型PCV2毒株Cap蛋白的反应特性;
图2为采用微量中和试验检测12株杂交瘤细胞上清液中和PCV2/MDJ或PCV2d/SDRS的能力;
图3为纯化后的单抗或多抗;
其中,M:为蛋白marker;泳道1-8分别为:未纯化的单克隆抗体4B3、纯化的单克隆抗体4B3、未纯化的单克隆抗体7C7、纯化的单克隆抗体7C7、未纯化的单克隆抗体9H4、纯化的单克隆抗体9H4、未纯化的PCV2多克隆抗体1121和纯化的PCV2多克隆抗体1121;
图4为PCV2红色乳胶微球免疫层析试纸条的敏感性检测结果;
图5为PCV2红色乳胶微球免疫层系试纸条的特异性检测结果。
具体实施方式
下面通过实验并结合实施例对本发明做进一步说明,应该理解的是,这些实施例仅用于例证的目的,决不限制本发明的保护范围。
本发明实施例所涉及的细胞系、毒株、实验动物、试剂及其来源:
SP2/0和PK15细胞系,所用毒株PCV2a/LG株,PCV2a/CL株,PCV2b/MDJ株,PCV2d/SDRS株,PCV2d/LNHC株,均由中国农业科学院哈尔滨兽医研究所分离、鉴定和保存。实验动物为6周龄雌性BALB/c鼠,由北京维通利华实验动物技术有限公司提供。弗氏完全佐剂、弗氏不完全佐剂,DMEM培养基,HT、HAT混合盐,PEG融合剂,Rabbit anti-Mouse IgG牛血清白蛋白(BSA)蔗糖均购自Sigma公司,SBAClonotyping System-HRP购自Southern Biotech公司,胎牛血清购自Gibco公司。红色乳胶微球购自苏州为度生物技术有限公司、HiTrapProtein G购自GE公司,柠檬酸三钠、磷酸氢二钠、氯化钾、氯化钠等化学试剂均为国产分析纯。PVC衬板、硝酸纤维素膜、玻璃纤维、吸水纸均为Millpore产品。
实施例1单克隆抗体的制备及鉴定
1方法
1.1单抗的制备
1.1.1小鼠免疫
参照文献(边海桥,黄立平,危艳武,夏德利,朱红振,吴洪丽,于冲,刘建行,冯力,刘长明.猪圆环病毒2型病毒样颗粒的制备及其免疫原性研究[J].中国兽医科学,2021,51(08):1007-1014.)表达和纯化PCV2b/MDJ和PCV2d/SDRS毒株Cap蛋白的病毒样颗粒(virus-like particles,VLPs),对小鼠进行三次免疫。首次免疫时,取Cap蛋白VLPs与等体积的弗氏完全佐剂(FCA)充分乳化至均一状态,颈背部皮下多点注射,每种基因型的蛋白免疫三只小鼠,免疫剂量为100μg/只。间隔两周进行二免,免疫原为VLPs与弗氏不完全佐剂等体积混合乳化,免疫途径和剂量同上。第二次免疫一周后,小鼠下颌静脉采血检测抗体产生情况,抗体效价达到1:3200以上时即可进行下一步融合。细胞融合前3天进行加强免疫,腹腔注射1ml含100μg纯化后的VLPs。
1.1.2细胞融合、阳性杂交瘤的筛选和克隆
无菌操作取小鼠脾细胞,将其用50%PEG与提前预备好的SP2/0进行融合。分别用PCV2b/MDJ毒株和PCV2d/SDRS毒株感染PK15细胞制备96孔IPMA反应板,收取融合后十天的细胞培养液上清进行IPMA检测,具体IPMA反应板制备和检测方法参照文献[LIU C M,IHARAT,NUNOYA T,et al.Development of an ELISAbased on the baculovirus-expressedcapsid protein of porcine circovirus type 2 as antigen[J].Journal ofVeterinary Medical Science,2004,66(3):237-242.],其中酶标二抗为Rabbit anti-Mouse IgG。将与IPMA反应板反应为阳性的杂交瘤细胞扩大培养,同时用有限稀释法进行阳性杂交瘤的克隆3次,将获得的阳性杂交瘤细胞及时冻存。
1.1.3单抗腹水的制备
取6-7周龄BALB/c小鼠,腹腔注射弗氏不完全佐剂,0.5ml/只,与此同时复苏杂交瘤细胞,一周后腹腔注射杂交瘤细胞约1×106个/只。观察小鼠,待到腹部增大,行动迟缓时收集腹水。3000g离心10min取上清,分装-20℃保存备用。
1.2单克隆抗体的鉴定
1.2.1亚类鉴定
取各个阳性杂交瘤细胞分泌的上清液,采用SBA Clonotyping System-HRP抗体亚类检测试剂盒鉴定单抗亚类,按试剂盒说明书进行检测。
1.2.2单克隆抗体效价的测定
参照文献(LIU C M,IHARAT,NUNOYAT,et al.Development of an ELISAbased onthe baculovirus-expressed capsid protein of porcine circovirus type 2asantigen[J].Journal of Veterinary Medical Science,2004,66(3):237-242.)分别制备PCV2a、PCV2b和PCV2d三种基因型的IPMA抗体反应板。将单抗腹水按照二倍梯度稀释(1:1000-1:512000)作为一抗分别加入到三种基因型的IPM A抗体反应板中,37℃孵育1h,PBS洗三次后加入Rabbit anti-Mouse IgG作为二抗,100μl/孔,37℃孵育1h后PBS洗板三次,AEC室温显色二十分钟,光学显微镜下观察结果,判定腹水针对三种PCV2基因型的IPMA抗体效价。
1.2.3单抗反应特性鉴定
按文献[HUANG L,LU Y,WEI Y,et al.Development of a blocking ELISA fordetection of serum neutralizing antibodies against porcine circovirus type 2[J].Journal of Virological Methods,2011,171(1):26-33.]所述方法,取PCV2b/MDJ和PCV2d/SDRS细胞培养物分别加入5×Loading Buffer,沸水中煮10min,然后分别进行SDS-PAGE并转印至NC膜上。以制备的12株杂交瘤上清分别作为一抗,抗PCV2-Cap的mAb 3A10(1:3000)作为阳性对照,SP2/0上清作阴性对照,IRDye 800CW Goat anti-Mouse IgG(H+L)为二抗进行Western-bloting分析,用近红外荧光扫描成像系统Infrared Imaging Systems显示实验结果。
1.2.4单抗中和活性测定
按照文献[Huang L,Sun Z,Xia D,Wei Y,Sun E,Liu C,Zhu H,Bian H,Wu H,FengL,Wang J,Liu C.2020.Neutralization mechanism of a monoclonal antibodytargeting a porcine circovirus type 2 Cap protein conformational epitope.JVirol 94:e01836-19]所述方法,将200μl 104.3TCID50 PCV2b/MDJ的病毒液与等体积的PCV2b基因型8株单抗杂交瘤上清液分别在37℃下孵育1小时,本实验室保存的PCV2猪高免血清作为阳性对照,SP2/0上清为阴性对照,孵育后加至长到50%单层的PK15细胞中感作1h,吸出孔内液体后加入含有2%FBS DMEM,100μl/孔,置37℃韩5%CO2培养箱培养36h后用冷甲醇固定。每株单抗和对照做三个重复。参照文献[Huang L,Sun Z,Xia D,Wei Y,Sun E,Liu C,Zhu H,Bian H,Wu H,F eng L,Wang J,Liu C.2020.Neutralization mechanism ofa monoclonal antibody targ eting a porcine circovirus type 2Cap proteinconformational epitope.J Virol 94:e01836-19]进行IPMA检测,通过光学显微镜计数每孔感染细胞数量,进而按照以下公式计算抗体的中和活性:中和活性(%)=(阴性对照阳性细胞数-被检抗体阳性细胞数)/阴性对照阳性细胞数×100。当杂交瘤上清的平均中和活性大于50%时,认为该株单抗具有中和活性。PCV2d基因型4株单抗中和活性测定方法同上。
2.结果
2.1单克隆抗体的亚类
分别经PCV2b-IPMA和PCV2d-IPMA检测筛选,获得8株抗PCV2b阳性杂交瘤细胞,命名为1G5、2C8、4B3、4C9、6H9、7E2、9H4和10G7,获得4株抗PCV2d阳性杂交瘤细胞,命名为2G8、7B9、7C7和7D1。通过细胞克隆、体外传代及冻存与复苏,证明这12株细胞能够稳定分泌特异性单抗。用SBA Clonotyping System-HRP(Southern Biotech公司)对这12株杂交瘤细胞所分泌的单抗进行了亚类鉴定,结果显示,单抗4B3、4C9、6H9、10G7和7B9的亚类为IgG1,单抗1G5、7E2、7C7和7D1的亚类为IgG2a,9H4和2G8单抗亚类为IgG2b,2C8单抗亚类为IgG3,12个单抗的轻链均为κ型。
2.2单克隆抗体的效价及交叉反应检测
采用PCV2a/LG、PCV2b/MDJ和PCV2d/SDRS三种病毒分别制备IPMA反应板,分别检测12株杂交瘤细胞所制备腹水的抗体效价。结果见表1,单抗2C8、4B3、4C9、6H9、7C7、9H4和10G7均可以与上述三株病毒呈阳性反应,效价最高分别为8000、128000、128000、64000、512000、64000和64000;单抗1G5、7B9、7D1和7E2与PCV2b/MDJ和PCV2d/SDRS毒株呈阳性反应,抗体效价最高均可达到128000。单抗2G8仅与PCV2d毒株呈阳性反应,抗体效价为16000。
表1采用IPMA方法检测单克隆抗体的效价和交叉反应性
备注:”-”表示阴性反应。
2.3单克隆抗体反应特性的鉴定
采用Western-blotting方法检测12株单抗与对应基因型PCV2毒株Cap蛋白的反应特性,结果如图1所示,单抗4B3、4C9、6H9、7E2、10G7、7B9、7C7和7D1均可与PCV2-Cap蛋白在约28kD处产生特异性条带(图1),单抗1G5、2C8、9H4和2G8不发生反应(图1),由此推测,这四株单抗可能识别PCV2-Cap的构象表位。同设的阳性对照和阴性对照均成立。
2.4单抗中和活性测定
采用微量中和试验检测12株杂交瘤细胞上清液中和PCV2b/MDJ或PCV2d/SDRS的能力。检测结果见图2:单抗2C8、9H4、10G7、7B9和7C7具有很好的中和效果,中和活性介于80~100%之间(图2);而单抗4B3、4C9、6H9和7E2的中和活性均低于50%(图2)。同时设置的阴性对照(3A10)和阳性对照(PCV2阳性血清)均成立。
实施例2 PCV2红色乳胶微球免疫层析试纸条的建立
1方法
1.1 PCV2阳性血清1121的制备
取猪圆环病毒2型灭活疫苗(LG株)(哈尔滨维科生物技术有限公司)经颈部肌肉免疫5周龄断奶仔猪(经PCR检测PCV2阴性,IPMA抗体效价小于50),1ml/头,相同剂量和途径间隔21天加强免疫。免疫后14天分别滴鼻感染PCV2b/MDJ毒株和PCV2d/LNHC,1ml/毒株/头。三周后经前腔静脉采血并分离血清,用PBS将血清2倍梯度稀释后,采用PCV2a-IPMA、PCV2b-IPMA和PCV2d-IP MA三种反应板按照文献[LIU C M,IHARA T,NUNOYA T,et al.Development of an ELISA based on the baculovirus-expressed capsid protein of porcineci rcovirus type 2 as antigen[J].Journal of Veterinary Medical Science,2004,66(3):237-242.]进行抗体效价的检测。
1.2单克隆抗体以及多克隆抗体的纯化
根据IPMA测得的抗体效价结果,我们选取其中三株广谱且效价较高的单抗(4B3、7C7和9H4)和PCV2的多克隆抗体1121分别按照HiTrap Protein G亲和层析柱的操作步骤进行纯化,纯化后的抗体进行SDS-PAGE分析。
1.3配对抗体的确定
标记:将纯化后单克隆抗体4B3、7C7和9H4分别稀释至终浓度为1mg/ml,分别与红色乳胶微球进行偶联。偶联步骤为:取1mg红色乳胶微球溶液,80HZ超声5min,用超纯水稀释200倍,然后10000rpm离心10min,洗去上清,沉淀用1000μl 0.1M的柠檬酸缓冲液(PH=5.6)溶解,80HZ超声5min。获得的溶液在磁力搅拌下缓慢加入100μl纯化的单克隆抗体,室温缓慢搅拌2h后缓慢加入50g/L BSA使其终浓度为10g/L,继续搅拌1小时后10000rpm离心1h。沉淀用0.1M PBS溶解,80HZ超声5min,反复洗涤3次,最终沉淀用0.1M PBS溶解至原体积的1/20。将3种红色乳胶微球偶联的单克隆抗体涂在结合垫上并做好标记。
包被:分别将纯化后的单克隆抗体4B3、7C7、9H4和多抗1121稀释至终浓度为500μg/ml,固定于NC膜上作为检测线,将商品化的山羊抗鼠IgG稀释至终浓度为500μg/ml,固定于NC膜上作为质控线,然后室温晾干备用。
组合:将涂有不同标记抗体的结合垫与不同抗体包被的NC反应膜两两组合在PVC底板上制成试纸条试验品,NC反应膜的另一端与吸水垫相接。用含0.5%NP-40的0.05M PBS(pH7.4)将阳性质控品分别进行1:20;1:100和1:1000稀释,以能够检测到最高稀释倍数阳性质控品且无非特异性反应为标准作为最佳配对抗体组合。
1.4红色乳胶微球免疫层析试纸条的制备及条件优化
将稀释好的纯化单抗7C7与红色乳胶微球偶联,然后将红色乳胶微球偶联的单抗按照15ul/cm喷涂于玻璃纤维上,制成结合垫,37℃干燥2h备用。将适当浓度的纯化单抗9H4喷涂在NC膜检测线位置,同时将适当浓度的山羊抗鼠IgG喷涂于质控线位置,两条线间隔1cm,37℃干燥2h备用。最终将样品垫、结合垫、NC膜和吸水垫按顺序装配在PVC底板上,然后用切条机切割成宽度为2.5cm的条状,最终用塑料盖板扣上即制成PCV2红色乳胶微球抗原检测试纸条。
为了获得最佳的检测结果,对层析试纸条制备的各个环节进行优化,包括单克隆抗体7C7的最佳标记浓度,喷涂在检测线和质控线上的单抗9H4和山羊抗鼠IgG的最佳浓度。以PCV2组织培养毒(106.5TCID50/ml)和PK15细胞培养物为阳性和阴性样品,用样品稀释液将阴、阳性样本进行2倍梯度稀释,将样品滴加入检测孔,100ul/孔,室温静置放置10min,判读结果。判读标准:C线显色,T线无色,判为阴性;C线显色,T线显色,判为阳性;C线完全没有显色判为无效。以阴性质控品为阴性,阳性质控品最高稀释倍数检测为强阳性的最低抗体浓度为最佳标记抗体浓度、最佳检测线抗体浓度和最佳质控线抗体浓度。优化完成后制备一批层析试纸条用于后续评价和检测。
1.5使用方法及结果判定
将样本(组织匀浆、肛拭子和细胞培养物)用含0.5%NP-40的0.05M PBS(pH7.4)稀释,取100ul上清加入到检测试纸条加样孔中(吸取及加样时不要产生明显气泡),加样后室温反应10min后判读结果。判读标准:C线显色,T线无色,判为阴性;C线显色,T线显色,判为阳性;C线完全没有显色判为无效。
2 PCV2红色乳胶微球免疫层系抗原检测方法的评价
2.1敏感性试验
选取三株不同基因型的PCV2细胞培养毒:PCV2a/CL(104.5TCID50/0.1ml)、PCV2b/MDJ(104.7TCID50/0.1ml)和PCV2d/LNHC(105.5TCID50/0.1ml)用样品稀释液将上述三种病毒分别作10倍系列稀释(1:10、1:100~1:100000)后,用PCV2红色乳胶微球免疫层析试纸条对不同稀释度的样品进行检测。我们利用检测为阳性的最高稀释倍数计算最小病毒检出量,即最小病毒检出量=病毒毒价/检测为阳性的最高稀释倍数。将层析试纸条检测到的不同基因型参考样本的最小病毒量定为层析试纸条的灵敏度。
2.2特异性试验
取非洲猪瘟病毒、猪瘟病毒、猪呼吸与繁殖综合征病毒、猪细小病毒、猪伪狂犬病毒、传染性胃肠炎病毒、猪流行性腹泻病毒、猪轮状病毒(G9)、猪德尔塔冠状病毒、猪圆环病毒1型、PK15细胞和猪圆环病毒2型细胞培养物,用样品稀释液将上述病毒分别进行3倍稀释,然后用PCV2红色乳胶微球免疫层析试纸条进行检测。
2.3重复性试验
按照优化的条件制备3批层析试纸条,每批随机抽取相同数量的层析试纸条进行敏感性和特异性检验。选取PCV2d/SDRS组织培养毒作为敏感性检测重复实验的样品,用样品稀释液做10倍梯度稀释,然后分别用三批层析试纸条进行检测,每批做三个重复。选取特异性检测中的所有组织培养毒,用样品稀释液分别作三倍稀释,然后分别用三批层析试纸条进行检测,每种病毒每批次层析试纸条检测三次。统计结果,评价该层析试纸条的批间和批内重复性。
2.4符合率试验
为了评价PCV2红色乳胶微球免疫层析试纸条的检测效果,我们以检测PCV2基因组的PCR方法为检测标准。样品处理方法如下,将临床送检的75份病料(肺、脾、淋巴结或粪便)制备成0.1g/ml的乳液和10份细胞培养物分别进行3倍稀释,然后分别用层析试纸条进行检测。对于PCR检测,将上述样品的乳液和细胞培养物按照天根血液/细胞/组织基因组DNA提取试剂盒的说明提取DNA,然后采用TaKaRa Premix Taq试剂盒以PCV2-CR(5’-GGAAGCTTCAGTAATTTATTTCATATGGAA-3’)和PCR-CR(5’-GGAAGCTTTTTTATCACTTCGTAATGGTT-3’)为上下游引物进行PCR扩增,扩增条件为:预变性94℃2min;94℃30s,55℃30s,72℃2min,35循环;72℃7min。通过琼脂糖凝胶电泳检测PCR结果。统计上述两种方法的检测结果并计算其符合率。
2.5层析试纸条与毒价测定的比较
为了进一步评价层析试纸条的检测效果,我们分别选取10份层析试纸条为阳性和10份阴性样品进行PCV2毒价测定,具体操作方法如下:将上述样品2000rpm离心10分钟,然后用0.22μm滤器过滤除菌;再用DMEM培养液将上述样品分别作10倍系列稀释,取未稀释、10-1、10-2、10-3、10-4和10-5 6个稀释度,分别接种到长至50%单层的PK-15细胞中,每个稀释度接种4孔,100μl/孔,同时设正常细胞对照4孔,置37℃、含5%CO2的培养箱中感作1小时,吸去孔内液体,加入含2%FBS的DMEM继续培养72小时,参照文献[Huang L,Sun Z,Xia D,Wei Y,Sun E,Liu C,Zhu H,Bian H,Wu H,Feng L,Wang J,Liu C.2020.Neutralizationmechanism of a monoclonal antibody targeting a porcine circovirus type 2 Capprotein conformational epitope.J Virol 94:e01836-19.]进行免疫过氧化物酶单层细胞染色试验(IPMA),其中一抗为PCV2多抗1121,二抗为HRP标记的SPA。按Reed-Muench法计算TCID50。
3结果
3.1 PCV2阳性血清1121的制备
为了获得高效价的PCV2阳性血清,我们采用免疫疫苗结合人工感染的方式,结果显示,该试验猪血清针对于PCV2a、PCV2b和PCV2d毒株的抗体效价分别为12800、12800和12800,因此经前腔静脉大量采血分离血清,命名为PCV2阳性血清1121。
3.2抗体纯化
为了建立红色乳胶微球免疫层析试纸条,我们将单抗4B3、7C7、9H4和多抗1121进行纯化,纯化的样品经SDS-PAGE检测,结果显示纯化后的单抗或多抗均能在55ku和20~25ku附近检测到重链和轻链的条带(图3),其他位置条带很少,说明抗体纯化效果较好,纯度较高。
3.3配对抗体的确定
为了选择最佳的配对抗体建立红色乳胶微球免疫层析试纸条,红色乳胶微球标记抗体4B3、7C7和9H4与包被单抗4B3、单抗7C7、单抗9H4和多抗1121两两配对制备成层析试纸条检测相同阳性和阴性样品。结果发现,红色乳胶微球标记的单抗7C7分别与包被单抗9H4和7C7的组合检测效果要明显优于其他组合(表2),1000倍稀释的阳性对照均能检测出阳性,并且随着稀释倍数的增加检测线逐渐变淡。其中红色乳胶微球标记的单抗7C7与包被单抗7C7的组合C线在阳性样本20倍和100倍稀释时显色较浅,所以优选单抗9H4包被与红色乳胶微球标记单抗7C7的组合。
表2不同配对抗体制备的红色乳胶微球免疫层析试纸条的检测结果
备注:“+”表示阳性,“-”表示阴性,“±”表示疑似。
其中,所述的抗猪圆环病毒2型单克隆抗体7C7,命名为mAb 7C7,分类命名为鼠源杂交瘤细胞,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,其菌种保藏编号为:CGMCC No.23871,保藏日期为2021年11月18日;
其中,所述的抗猪圆环病毒2型单克隆抗体9H4,命名为mAb9H4,分类命名为鼠源杂交瘤细胞,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,其菌种保藏编号为:CGMCC No.23870,保藏日期为2021年11月18日。
3.4红色乳胶微球免疫层析试纸条的建立
为了优化红色乳胶微球免疫层析试纸条,我们对其单抗7C7的红色乳胶微球的标记、单抗9H4和山羊抗鼠IgG的最适浓度等条件进行了摸索,结果显示:单抗7C7和红色乳胶微球的标记比例为100μg/1mg;检测线抗体9H4和质控线山羊抗鼠IgG最适浓度均0.5mg/ml。
3.5敏感性试验
为了检测红色乳胶微球免疫层系试纸条的检测敏感性,用样品稀释液将PCV2a/CL(104.5TCID50/0.1ml)、PCV2b/MDJ(104.7TCID50/0.1ml)和PCV2d/LNHC(105.5TCID50/0.1ml)组织培养物分别进行十倍梯度稀释(1:10~1:100000),然后进行检测。结果显示,PCV2a/CL、PCV2b/MDJ和PCV2d/LNHC的胶体金效价分别为100、10000和10000(图4),根据病毒的毒价计算出该试纸条对三株病毒的最小检测限分别为102.5TCID50/0.1ml、100.7TCID50/0.1ml和101.5TCID50/0.1ml。从该结果我们发现该试纸条对PCV2b和PCV2d毒株的检测敏感性要明显高于PCV2a的。
3.6特异性试验
采用PCV2乳胶微球试纸条检测非洲猪瘟病毒、猪瘟病毒、猪呼吸与繁殖综合征病毒、猪细小病毒、猪伪狂犬病毒、传染性胃肠炎病毒、流行性腹泻病毒、猪轮状病毒(G9)、猪德尔塔装装病毒、猪圆环病毒1型和PK15细胞的组织培养物进行检测,结果显示(图5),PCV2组织培养物C线和T线均出现特异性条带,检测结果为阳性;其他病原的培养物C线均出现条带,T线均未出现条带,检测结果均为阴性。说明该试纸条具有良好的特异性。
3.7重复性试验
用三批试纸条重复检测阳性、阴性质控样品,3次检测结果完全一致,不同批次间、同一批次内检测结果完全一致。
3.8符合率试验
用PCV2红色乳胶微球免疫层系试纸条和PCR两种方法同时检测75份临床样品,结果显示(表3):PCR检出33份阳性和42份阴性,红色乳胶微球免疫层系试纸条检出23份阳性和52份。在PCR检测42份阴性样品中,试纸条检测均为阴性,在PCR检测阳性的33份样品中,有10份样品红色乳胶微球免疫层析试纸条检测为阴性。两种方法检测均为阳性的样品有23份,均为阴性的样品有42份,总的符合率为86.67%(65/75)。以PCR作为参考标准,则该试纸条的检测敏感性为69.70%(23/33),特异性为100%(42/42).说明该红色乳胶微球免疫层系试纸条具有出色的特异性和较好的敏感性。
表3红色乳胶微球免疫层系试纸条和PCR的符合性试验结果
3.9红色乳胶微球免疫层系试纸条与病毒毒价测定的比较
采用毒价测定的方法进一步评价红色乳胶微球免疫层析试纸条的检测结果,选取10份胶体金阳性病料和10份胶体金阴性病料进行毒价测定,结果见表4,试纸条检测为阴性的,毒价测定结果均小于PCV2毒价测定的检测限,试纸条阳性样品的PCV2毒价介于101.7~105.3TCID50/ml之间。
表4红色乳胶微球免疫层析试纸条和毒价测定方法的比较结果
备注:“+”表示试纸条检测结果为阳性,“-”表示试纸条检测结果为阴性。
实施例3PCV2红色乳胶微球免疫层析试纸条的应用
1方法
1.1临床样品的检测
用实施例2制备的红色乳胶微球免疫层析试纸条检测121份具有呼吸道症状或消瘦症状猪的肺脏或腹股沟淋巴结。肺脏和腹股沟淋巴结通过研磨用DMEM培养液稀释制备成0.1g/ml乳液。上述检测样品经过室温静置5分钟后抽取上清用样品稀释液稀释三倍后按照红色乳胶微球免疫层析试纸条操作步骤进行检测。
1.2 PCV2组织培养物的检测
用红色乳胶微球免疫层析试纸条检测PCV2组织培养物。共选择16株已知毒价分离毒株,每个样品进行10倍梯度稀释(1:10~1:100000),然后按照红色乳胶微球免疫层析试纸条操作步骤进行检测。以最高稀释倍数仍为阳性的倒数判为该检测样品的红色乳胶微球层析试纸条效价。
2结果
2.1临床样品检测
采用PCV2红色乳胶微球免疫层析试纸条检测2018-2021年间实验室保存的源于国内10个省的具有呼吸道症状或消瘦症状猪的肺脏或腹股沟淋巴结以及具有腹泻症状猪的粪便共计121份,检测结果如表5所示,每个省均能检测到阳性样品,阳性率介于11.11~40.00%之间,按照年份来看,近4年来,PCV2阳性率均在20%以上,总的阳性率为24.79%。
表5 PCV2红色乳胶微球免疫层析试纸条检测临床样品的结果
备注:“/”表示未检测
2.2 PCV2组织培养物的检测
采用PCV2红色乳胶微球免疫层析试纸条检测已知毒价的PCV2不同毒株的试纸条效价,结果发现(表6),相同基因型,毒价越高,试纸条效价也高。同时也存在毒价不同试纸条效价相同的现象,这是由该试纸条不能精确定量的缺点所造成的。另外,不同毒株之间可能也会存在抗原性的细微差异,从而造成检测结果的不同。
表6 PCV2组织培养物的检测结果
Claims (5)
1.用于检测猪圆环病毒2型的红色乳胶微球免疫层析试纸条,其特征在于,所述的试纸条包括PVC底板,以及在PVC底板上从左到右依次组装的结合垫、硝酸纤维素膜和吸水垫,所述的结合垫上涂覆有红色乳胶微球偶联的抗猪圆环病毒2型病毒单克隆抗体7C7,所述的硝酸纤维素膜设有包被抗猪圆环病毒2型病毒单克隆抗体9H4的检测线以及包被山羊抗鼠IgG的质控线;
其中,所述的抗猪圆环病毒2型单克隆抗体7C7,命名为mAb 7C7,保藏在中国微生物菌种保藏管理委员会普通微生物中心,其菌种保藏编号为:CGMCC No.23871,保藏日期为2021年11月18日;
其中,所述的抗猪圆环病毒2型单克隆抗体9H4,命名为mAb9H4,保藏在中国微生物菌种保藏管理委员会普通微生物中心,其菌种保藏编号为:CGMCC No.23870,保藏日期为2021年11月18日。
2.如权利要求1所述的红色乳胶微球免疫层析试纸条,其特征在于,红色乳胶微球偶联的抗猪圆环病毒2型单克隆抗体7C7按照15ul/cm喷涂于玻璃纤维上作为结合垫,其中,单克隆抗体7C7和红色乳胶微球的标记比例为100μg:1mg;检测线上单克隆抗体9H4和质控线上山羊抗鼠IgG的浓度均0.5mg/ml。
3.如权利要求1所述的红色乳胶微球免疫层析试纸条,其特征在于,检测线与质控线的距离为1cm。
4.如权利要求1所述的红色乳胶微球免疫层析试纸条,其特征在于,红色乳胶微球偶联的抗猪圆环病毒2型单克隆抗体7C7通过以下方法制备得到:
取1mg红色乳胶微球溶液,80HZ超声5min,用超纯水稀释200倍,然后10000rpm离心10min,洗去上清,沉淀用pH=5.6的1000μl 0.1M的柠檬酸缓冲液溶解,80HZ超声5min;获得的溶液在磁力搅拌下缓慢加入100μl浓度为1mg/ml纯化后的抗猪圆环病毒2型单克隆抗体7C7,室温缓慢搅拌2h后缓慢加入50g/LBSA使其终浓度为10g/L,继续搅拌1小时后10000rpm离心1h;沉淀用0.1M PBS溶解,80HZ超声5min,反复洗涤3次,最终沉淀用0.1M PBS溶解至原体积的1/20,得到红色乳胶微球偶联的抗猪圆环病毒2型病毒单克隆抗体7C7溶液。
5.权利要求1-4任一项所述的红色乳胶微球免疫层析试纸条在制备检测猪圆环病毒2型试剂中的应用。
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