CN108165533B - 分泌抗水稻条纹花叶病毒单克隆抗体的杂交瘤细胞株及其单抗应用 - Google Patents
分泌抗水稻条纹花叶病毒单克隆抗体的杂交瘤细胞株及其单抗应用 Download PDFInfo
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Abstract
本发明公开了一种分泌抗水稻条纹花叶病毒单克隆抗体的杂交瘤细胞株及其单抗的应用。用提纯的水稻条纹花叶病毒(RSMV)为抗原免疫BALB/c小鼠，经细胞融合、筛选、克隆，获得1株能稳定传代并分泌抗RSMV单抗的杂交瘤细胞株1D4，其保藏号为CGMCC No.14898。该杂交瘤细胞株分泌的单抗腹水间接ELISA效价达到10‑10，抗体类型及亚类为IgG1、kappa链。该单抗与RSMV的N蛋白有特异性反应。利用1D4单抗为核心建立检测RSMV的ACP‑ELISA和dot‑ELISA方法，这两种方法检测病叶的灵敏度分别达到1:2621440和1:40960倍稀释(w/v,g/mL)。该杂交瘤细胞株及其单抗的获得和血清学检测方法的建立为该病毒病的检测诊断及科学防控提供了物质和技术支撑。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种分泌抗水稻条纹花叶病毒单克隆抗体的杂交瘤细胞株及其单抗的应用。
背景技术
水稻是我国乃至世界的重要粮食作物之一，而每年水稻病毒病对我国水稻产量造成了严重损失。2015年，在广东省罗定市太平镇田发现了一种新的水稻病毒病害，它的症状不同于已报道各种水稻病毒病，其为弹状病毒科细胞质弹状病毒属一个新种，命名为水稻条纹花叶病毒(Rice stripe mosaic virus,RSMV)。就目前所知，该病毒是迄今为止唯一自然侵染水稻的细胞质弹状病毒，也是首个经叶蝉传播的细胞质弹状病毒。RSMV感染植株轻度矮缩，叶片呈条纹花叶状，且有部分病叶表现扭曲，病株可抽穗，但抽穗不完全且籽粒空瘪。3叶期稻苗经电光叶蝉传毒接种后10天，新叶出现明显黄色条纹，随后叶片表现为花叶，叶尖向内侧卷曲。除了水稻外，在发病田或邻近田块的马唐草和鸭舌草中也检测到RSMV的存在。
RSMV病毒粒子呈杆状，有包膜，大部分成熟的毒粒子大小长300-360nm，宽45-55nm，其中一些断裂的病毒粒子最小长度为130nm；病毒粒子在细胞质中聚集并形成大量的晶格状，几乎充满整个细胞质空间，而细胞核内未观察到病毒粒子。此外，在晶格状附近有电子半透明颗粒及纤维状的病毒基质，这是病毒复制的场所。有些病毒粒子聚集在囊泡内。这些病毒粒子存在于叶肉细胞及维管束系统。在RSMV基因组的互补链上预测有7个ORFs，推测这7个ORFs的编码蛋白与多数弹状病毒的编码蛋白相似，ORF1预测编码病毒N蛋白；ORF2编码病毒的P蛋白；ORF3编码蛋白是一个碱性的膜蛋白，且该蛋白可能具有运动蛋白的功能；ORF4推测编码M蛋白；ORF5预测编码病毒G蛋白；ORF6推测编码一个辅助蛋白；ORF7预测编码病毒的L蛋白。RSMV的传播介体是电光叶蝉，其能以高效持久的方式传播RSMV。
目前，根据症状观察判断田间水稻条纹花叶病毒的发生情况仍然是最主要的方法，但病毒侵染前期水稻没有任何症状，且多种病毒病造成的症状极为相似，另外，常有病毒复合侵染情况，因此依靠症状观察得到的结果缺乏准确性和科学性。电镜观察病毒粒子需要昂贵的仪器，且有些不同病毒粒子的大小和形态有相似性，导致其仅作为一种辅助检测手段。基于PCR方法的分子检测技术虽然灵敏，但是不适于大规模样品的检测。而血清学方法简单易操作、快速、灵敏、成本较低，且适用于田间样品的大规模检测，是目前理想的植物病毒的检测技术。但血清学方法必须依赖于特异、灵敏的优质抗体。目前，国内外未见任何准对该病毒的抗体报道和专利申请。为此，本发明专利主要针对水稻条纹花叶病毒单克隆抗体的制备及其检测应用开展工作，利用杂交瘤技术制备了1株能分泌抗RSMV单抗杂交瘤细胞1D4，并用其分泌的单抗建立了检测该病毒的血清学方法，从而为我国水稻条纹花叶病毒的检测、诊断及病害的科学防控提供物质和技术支持。
发明内容
本发明的目的是克服现有技术的不足，提供一种分泌抗水稻条纹花叶病毒单克隆抗体杂交瘤细胞株及其单抗的应用。
分泌抗水稻条纹花叶病毒单抗杂交瘤细胞1D4，它能分泌抗水稻条纹花叶病毒的特异性单抗，杂交瘤细胞株1D4于2017年12月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.14898。
抗水稻条纹花叶病毒的单克隆抗体腹水间接ELISA效价达10-10，抗体类型及亚类为IgG1、kappa链，该单抗与水稻条纹花叶病毒60kDa的N蛋白有特异性免疫反应，利用ACP-ELISA和dot-ELISA方法分析发现，该单抗检测感染水稻条纹花叶病毒水稻病叶的灵敏度分别达到1:2 621 440和1:40 960倍稀释(w/v,g/mL)。
抗水稻条纹花叶病毒的单克隆抗体与水稻条纹花叶病毒有特异性免疫反应，而与水稻条纹病毒(RSV)、水稻黑条矮缩病毒(RBSDV)、南方水稻黑条矮缩病毒(SRBSDV)、水稻锯齿矮缩病毒(RRSV)、水稻瘤矮病毒(RGDV)以及健康水稻植物组织和非携毒电光叶蝉组织均不发生任何免疫反应。
抗水稻条纹花叶病毒的单克隆抗体在水稻条纹花叶病毒检测上的应用是以单抗为核心建立的各种免疫学检测方法和免疫学检测试剂盒。
本发明与现有技术相比具有的有益效果：1)提供的杂交瘤细胞株1D4分泌抗水稻条纹花叶病毒特异性单克隆抗体，以该单抗为核心建立的dot-ELISA和ACP-ELISA等免疫学方法及用这些方法建立的试剂盒能高度特异、准确、灵敏地检测水稻条纹花叶病毒；2)利用本发明所制备的单抗检测水稻条纹花叶病毒，不需要昂贵的电子显微镜、PCR仪等设备；3)利用本发明所制备的单抗可有效地用于田间水稻及传毒介体电光叶蝉体内RSMV的检测和诊断，也可用于该病毒病的流行病学调查、病毒基因组功能分析、抗性育种、科学防控等方面。
附图说明
图1是dot-ELISA方法检测水稻中RSMV的灵敏度分析；CK+为感染RSMV的水稻病叶粗提液，CK-为健康水稻叶片粗提液。
图2是dot-ELISA方法检测RSMV的特异性分析：1列上下2个点为1个感染RSMV水稻植物样品的2个生物学重复；2列上下2个点为1个感染RSMV电光叶蝉的2个生物学重复；3列上下2个点为1个感染RSV水稻植物样品的2个生物学重复；4列上下2个点为1个感染RBSDV水稻植物样品的2个生物学重复；5列上下2个点为1个感染RRSV水稻植物样品的2个生物学重复；6列上下2个点为1个感染SRBSDV水稻植物样品的2个生物学重复，7列上下2个点为1个感染RGDV水稻植物样品的2个生物学重复；8列上下2个点为1个感健康水稻植物样品的2个生物学重复。
图3是dot-ELISA方法检测田间水稻样品中RSMV的代表性结果。
生物保藏
分泌抗水稻条纹花叶病毒单抗杂交瘤细胞1D4保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏日为2017年12月15日，保藏号为CGMCC No.14898。
具体实施方式
分泌抗水稻条纹花叶病毒单抗杂交瘤细胞株1D4于2017年12月15日保藏于中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCCNo.14898，它能分泌抗水稻条纹花叶病毒的单克隆抗体。
抗水稻条纹花叶病毒的单克隆抗体腹水间接ELISA效价达10-10，抗体类型及亚类为IgG1、kappa链，能与水稻条纹花叶病毒60kDa的N蛋白有特异性免疫反应，利用ACP-ELISA和dot-ELISA方法分析发现，该单抗检测感染RSMV水稻病叶的灵敏度分别达到1:2 621 440和1:40 960倍稀释(w/v,g/mL)。
抗水稻条纹花叶病毒的单克隆抗体与水稻条纹花叶病毒有特异性免疫反应，而与水稻条纹病毒(RSV)、水稻黑条矮缩病毒(RBSDV)、南方水稻黑条矮缩病毒(SRBSDV)、水稻锯齿矮缩病毒(RRSV)、水稻瘤矮病毒(RGDV)以及健康水稻植物组织和非携毒电光叶蝉组织均不发生任何免疫反应。
抗水稻条纹花叶病毒的单抗在该病毒检测上的应用是以单抗为核心建立的各种免疫学检测方法和免疫学检测试剂盒。
本发明提供的杂交瘤细胞株1D4能大量分泌抗水稻条纹花叶病毒的单抗，且其分泌的单抗效价高、特异性强、灵敏度高、稳定性好。以该单抗为核心建立的检测RSMV的高通量的血清学方法可成功应用于田间RSMV的检测，从而为我国水RSMV的检测和诊断、预警和科学防控提供物质和技术支持。
下面结合实施例和附图对本发明作进一步说明。
一、杂交瘤细胞的获得及其单克隆抗体的制备
1.免疫原及检测抗原的制备
以纯化的病毒离子为抗原免疫BALB/c小鼠。病毒粒子的具体提纯方法如下：收集200mg系统发病的水稻叶片，低温剪碎并加入液氮速冻研磨成粉末，加入500mL提取缓冲液Tris-HCl(100mM，PH 8.0)，果汁搅拌机搅拌5min；2层纱布过滤去渣后分装于50mL离心管中，4℃5000rpm离心10min；收集上清液，进行4℃45 000rpm超速离心40min；沉淀用3mL的100mM提取缓冲液悬浮，随后铺于不连续20-40％蔗糖垫上，4℃30 000rpm离心2h；收集病毒带，4℃45 000rpm超速离心40min，沉淀悬浮于1mL的提取缓冲液中，保存于-80℃冰箱备用。
2.免疫动物
用纯化的RSMV病毒离子免疫8周龄BALB/c雌性小鼠。即RSMV提纯病毒50μL和50μL福氏完全佐剂混合，充分乳化后经腹腔注射100μL/只进行一免；间隔3周，取RSMV提纯病毒30μL和30μL福氏不完全佐剂混合，充分乳化后经腹腔注射60μL/只进行二免；再过3周后30μL提纯病毒和50μL生理盐水混匀后进行腹腔注射80μL/只进行三免，三免后2周腹腔注射100μL提纯病毒和50μL生理盐水混匀液，免疫后3天取脾细胞进行融合。
3.细胞融合
取上述免疫小鼠脾细胞与小鼠骨髓瘤细胞(SP2/0)按8:1的比例，在无血清的RPMI-1640培养基中混匀，1 500rpm离心5min去除培养基，用50％PEG(分子量1 500)作为融合剂，在37℃水浴中融合2min，用无血清的RPMI-1640培养基终止融合后1 500rpm离心5min，沉淀用HAT培养基悬浮，分装到96孔细胞板中，并置于37℃含5％CO2的细胞培养箱中培养。
4.杂交瘤细胞、阳性孔的筛选及其克隆
细胞培养箱中培养5d后，用HAT培养基换液一次，第10天用HT培养基换液，等到融合细胞覆盖孔底10-30％时，以感染RSMV水稻病叶的病毒粗提液为抗原包被ELISA板，用常规间接ELISA方法筛选分泌单抗的阳性孔，共获125个阳性孔。选择5个呈强阳性反应的细胞孔，进行3次有限稀释法克隆，最后获得1株能分泌抗RSMV特异性单抗的杂交瘤细胞株1D4。经5个月以上体外传代和多次冻存复苏后，细胞株均能良好生长，并稳定分泌抗体。经扩大培养后，用于腹水制备和液氮保存。
5.单克隆抗体腹水制备及纯化
(1)取8周龄BALB/c小鼠，腹腔注射0.3mL降植烷(Sigma)，7-10d后腹腔注入6×105个杂交瘤细胞，注射后7-10d可见小鼠腹部明显膨大，针头采取腹水，2 000rpm离心1min，收集上清液即为单克隆抗体腹水。
(2)饱和硫酸铵法纯化单抗腹水(以1mL为例)
1)取1mL腹水5 000rpm 4℃离心5min，取上清，加入2mL生理盐水稀释；
2)逐滴加入pH值在7.0-7.4的饱和硫酸铵溶液3mL，边加边搅拌，全部加入后室温继续搅拌10min；4℃静止2-3h；
3)12 000rpm 4℃离心10min，弃上清，沉淀用2mL生理盐水溶解；
4)将抗体装入透析袋，并于0.01M PBS中4℃透析24h，期间每2h换一次PBS溶液；
5)取出透析袋中抗体，12 000rpm 4℃离心10min，上清即为纯化的抗体；取1μL上清用NanoDrop紫外分光仪测定IgG含量，测定完毕后抗体于-80℃冰箱保存。
6.单克隆抗体的类型及亚类鉴定和腹水效价测定
将纯化的单抗与Sigma公司试剂盒的标准抗BALB/c小鼠IgG1、IgG2a、IgG2b、IgG3、IgM、κ和λ轻链抗体进行DAS-ELISA分析，结果显示，1D4单抗类型和亚类为IgG1、kappa链。以提纯的RSMV为包被抗原，用常规间接ELISA方法检测单抗腹水效价，结果表明1D4单抗腹水效价达到10-10。
7.单克隆抗体的特异性检测
用分别感染水稻条纹病毒(RSV)、水稻黑条矮缩病毒(RBSDV)、南方水稻黑条矮缩病毒(SRBSDV)、水稻锯齿矮缩病毒(RRSV)、水稻瘤矮病毒(RGDV)的病叶粗提液分别包被ELISA板，以水稻的健康叶片粗提液、非携毒电光叶蝉的匀浆液为阴性对照，以感染水RSMV的病叶粗提液、RSMV携毒电光叶蝉的匀浆液为阳性对照，用ACP-ELISA方法测定单抗的特异性。ACP-ELISA方法的具体步骤为：上述病毒感染的病叶用液氮在研钵中研磨成粉末，按1:20(w/v,g/mL)倍加入0.05M碳酸盐包被缓冲液继续研磨匀浆，5 000rpm离心3min后上清即为粗提液；1头叶蝉放入eppendorf离心管中，加入150μL0.05M碳酸盐包被缓冲液后用牙签捣烂匀浆；水稻粗提液或叶蝉匀浆液100μL/孔包被ELISA板；4℃过夜或37℃2h，使其吸附于ELISA聚苯乙烯板孔；PBST洗涤3次后用3％的脱脂奶粉封闭30-60min；加入适当稀释的单抗100μL/孔，37℃1-2h；PBST洗涤3次后加入适当稀释的碱性磷酸脂酶(AP，用于水稻样品)或辣根过氧化物酶(HRP，用于叶蝉样品)标记的羊抗鼠IgG二抗(Sigma公司)100μL/孔，37℃1-2h；PBST洗涤4次后用PNPP(用于水稻样品)或TMB(用于叶蝉样品)底物显色，终止反应后用酶标仪读取OD405或OD450的值，以与阴性样品的OD值比值大于2.1为阳性。结果发现，1D4单抗对感染RSMV水稻病叶组织的粗提液、携毒电光叶蝉组织匀浆液有特异性反应，而与感染RSV、RBSDV、RRSV、SRBSDV、RGDV和健康水稻植物组织及非携毒电光叶蝉的匀浆液均无任何免疫反应。
二、检测RSMV的免疫学方法及其试剂盒的建立
1.检测RSMV的ACP-ELISA方法
1.1 ACP-ELISA方法的步骤：
1)水稻植物组织称重后用液氮在研钵中研磨成粉末，按1:20比例(w/v,g/mL)加入0.05M碳酸盐缓冲液(pH 9.6)继续研磨匀浆3min，5 000rpm离心3min，上清即粗提液100μL/孔加入ELISA板中，以感染RSMV水稻病叶粗提液为阳性对照，健康水稻叶片粗提液为阴性对照，37℃2h或4℃过夜包被；
2)PBST洗涤3次后用5％脱脂奶粉封闭30min；
3)加入适当稀释后的单抗腹水，100μL/孔，37℃孵育1h；
4)PBST洗涤3次后加入适当稀释的碱性磷酸脂酶(AP)标记的羊抗鼠IgG二抗(Sigma)，100μL/孔，37℃孵育1h；
5)用PBST洗涤后加入PNPP硝基磷酸盐底物100μL/孔，室温放置30min；
6)肉眼观察，底物颜色变成黄绿色的孔为阳性；或用2M氢氧化钠终止反应后，用酶联免疫检测仪测OD405，以P/N>2.1作为阳性判断标准。
1.2 ACP-ELISA方法的建立及其检测灵敏度和特异性确定
用常规方阵试验确定ACP-ELISA方法中单抗和酶标二抗的最适工作浓度，即ELISA板横向加用封闭液倍比稀释的单抗，纵向加用封闭液倍比稀释AP标记的羊抗鼠IgG二抗进行ACP-ELISA。试验表明1D4单抗及AP酶标二抗的最适工作浓度分别为1:5 000和1:7 000倍稀释。以抗体的最适工作浓度建立检测RSMV的ACP-ELISA方法。对RSMV病叶粗提液从1:20至655 360倍比稀释，分别以相应稀释度的健叶粗提液作阴性对照，分析ACP-ELISA方法的检测灵敏度。结果表明ACP-ELISA方法对1:2 621 440倍稀释(w/v,g/mL)的病叶粗提液仍呈阳性反应，即对病叶的检测灵敏度可达到1:2 621 440倍稀释(w/v,g/mL)，表明建立的ACP-ELISA方法具有很高的灵敏度。用建立的ACP-ELISA方法检测分别感染RSMV、RSV、RBSDV、RRSV、SRBSDV、RGDV水稻病叶和健康水稻植物组织，检测结果发现，建立的ACP-ELISA方法检测感染RSMV病叶呈强阳性反应，而检测感染RSV、RBSDV、RRSV、SRBSDV、RGDV病叶和健康水稻植物组织均呈阴性反应，且阴阳性结果对比差异极显著，说明该方法的特异性很好。
2.dot-ELISA方法的建立及田间检测应用
2.1 dot-ELISA方法检测植物中RSMV的操作步骤
将水稻植物样品的叶片称重后用液氮在研钵中研磨成粉末，按1:20(w/v,g/mL)的比例加入0.01M PBS(pH7.4)后继续研磨、匀浆；匀浆液5 000rpm离心3min；取2.5μL上清点到硝酸纤维素膜(NC)上，同时设置健康和感染RSMV的水稻叶片分别作为阴性和阳性对照；室温干燥10-20min；NC膜浸入到含5％脱脂奶粉的PBST(含0.05％Tween-20的0.01M PBS)封闭液中室温封闭30min；NC膜放入适度稀释的单抗中室温孵育40-60min；用PBST洗膜3-4次，每次3min；NC膜放入适度稀释的AP酶标记的羊抗鼠IgG二抗中室温孵育40-60min；PBST洗膜4-5次，每次3min；66μL NBT和33μL BCIP底物(Promega)加入到10mL底物缓冲液(0.1mol/LTris Cl、0.1mol/L NaCl、0.025mol/L MgCl2、pH 9.5)中混匀，膜放入底物液中显色反应10-20min，肉眼观察结果，待阳性对照显明显紫色，而阴性没有任何显色时，自来水漂洗膜终止反应，拍照记录结果。
2.2 dot-ELISA方法的建立及其检测灵敏度和特异性确定
用常规方阵试验确定dot-ELISA方法中RSMV单抗和AP酶标二抗的最适工作浓度，试验表明1D4单抗及AP酶标二抗的最适工作浓度分别为1:5 000和1:8 000倍稀释。以上述抗体最适工作浓度建立检测RSMV的dot-ELISA方法。灵敏度分析表明，当RSMV病叶稀释到1:40 960(w/v,g/mL)倍时，以1D4单抗建立的dot-ELISA方法检测仍呈现紫色的阳性斑点，即其检测病叶的灵敏度达到1:40 960倍稀释(w/v,g/mL)(图1)。特异性分析表明，该方法检测感染RSMV的水稻植物呈强阳性反应，而检测分别感染RSV、RBSDV、RRSV、SRBSDV、RGDV病叶和健康水稻植物组织均呈阴性反应(图2)。
2.3 dot-ELISA方法的应用
用建立的dot-ELISA方法对2017年从广东水稻田间采集的53株水稻样品进行检测，结果发现，53个样品中有41个样品感染RSMV，代表性检测结果如图3所示。这些样品同时用RT-PCR方法进行RSMV检测，结果表明所有dot-ELISA检测阳性样品中均能扩增到RSMV特异性的基因片段，而所有dot-ELISA检测阴性样品中均未扩增到任何基因片段。RT-PCR产物核酸测序表明阳性样品确实感染RSMV，说明该dot-ELISA方法能准确、可靠地用于植物样品中水稻条纹花叶病毒的检测。
3.水稻条纹花叶病毒dot-ELISA检测试剂盒(检测水稻样品)
1)试剂盒主要成分：
以上试剂均保存于4℃
2)检测水稻样品的操作步骤：
a.将水稻植物样品的叶片称重后用液氮在研钵中研磨成粉末，按1:20的比例(w/v,g/mL)加入0.01M PBS(pH7.4)后继续研磨、匀浆；
b.匀浆液5 000rpm离心3min；
c.取2.5μL上清点样于NC膜上，同时设置健康和感染RSMV的水稻植物组织分别作为阴性和阳性对照，室温干燥10-20min；
d.NC膜浸入到含5％脱脂奶粉的PBST(含0.05％Tween-20的0.01M PBS)封闭液中室温封闭30min；
e.NC膜放入1:4 000倍稀释的RSMV单抗中室温孵育40-60min；
f.用PBST洗膜3-4次，每次3min；
g.NC膜放入1:5 000稀释的AP酶标记羊抗鼠IgG二抗中室温孵育40-60min；
h.PBST洗膜4-5次，每次3min；
i.66μL NBT和33μL BCIP底物加入到10mL底物缓冲液(0.1M Tris Cl、0.1M NaCl、0.025M MgCl2、pH9.5)中，混匀后膜放入底物液中显色，肉眼观察结果，待阳性对照显明显紫色而阴性对照没有任何显色时自来水漂洗膜终止反应，拍照记录结果。
3)保存及有效期：
于2-8℃避光保存，有效期12个月。
4)缓冲液配方：
a.磷酸盐缓冲液(PBS，0.01mol/L，pH7.4)：NaCl 8g,KCl 0.2g,KH2PO4 0.2g,Na2HPO4·12H2O 3g，加蒸馏水950mL溶解后调pH至7.4，定容至1000mL。
b.ELISA洗涤液(0.01M PBST)：1000mL 0.01M PBS中加0.5mL Tween-20。
c.ELISA封闭液：0.01M PBST中加入脱脂奶粉至终浓度5％(w/v,g/mL)。
4.水稻条纹花叶病毒dot-ELISA检测试剂盒(检测电光叶蝉样品)
1)试剂盒主要成分：
以上试剂均保存于4℃下
2)操作步骤：
a.制样：单头电光叶蝉放入250μL eppendorf离心中，每管加入50μL PBS，用牙签捣烂电光叶蝉；
b.点样：用镊子取一张NC膜放至干净的培养皿内，取3μL虫子匀浆液轻点到NC膜上，每取一个样换一个枪头；
c.干燥：样品点膜完成后膜在室温下干燥，约10-20min；
d.封闭：NC膜浸入到含5％脱脂奶粉的PBST(含0.05％Tween-20的0.01M PBS)封闭液中室温封闭30min；
e.一抗孵育：NC膜放入1:4 000倍稀释的RSMV单抗中室温孵育40-60min；
f.洗涤：用PBST洗膜3-4次，每次3min；
g.二抗孵育：NC膜放入1:5 000稀释的HRP酶标记羊抗鼠IgG二抗中室温孵育40-60min；
h.洗涤：用PBST洗膜4-5次，每次3min；最后用PBS洗膜1次；
i.显色：将洗好的NC膜用滤纸吸干后放入新培养皿中，加入适量TMB显色底物液，淹没NC膜，盖上报纸遮光，肉眼观察结果，待阳性对照显蓝色明显，阴性没有任何显色时记录检测结果，显色时间约5-15min；
3)保存及有效期：
于2-8℃避光保存，有效期12个月。
4)缓冲液配方：
a.磷酸盐缓冲液(PBS，0.01mol/L，pH7.4)：NaCl 8g,KCl 0.2g,KH2PO4 0.2g,Na2HPO4·12H2O 3g，加蒸馏水950mL溶解后调pH至7.4，定容至1000mL。
b.ELISA洗涤液(0.01M PBST)：1000mL 0.01M PBS中加0.5mL Tween-20
c.ELISA封闭液：0.01M PBST中加入脱脂奶粉至终浓度5％(w/v,g/mL)。
Claims (3)
1. 一种分泌抗水稻条纹花叶病毒单克隆抗体的杂交瘤细胞株1D4，其特征在于能分泌抗水稻条纹花叶病毒的特异性单抗，杂交瘤细胞株1D4于2017年12月 15日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.14898。
2.一种如权利要求1所述的杂交瘤细胞株1D4分泌的抗水稻条纹花叶病毒的单克隆抗体。
3.一种如权利要求2所述的抗水稻条纹花叶病毒的单克隆抗体在该病毒检测上的应用，其特征在于以该单抗为核心建立各种免疫学检测方法和免疫学检测试剂盒。
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