CN112063765A - 一种核酸抗体双重检测病毒的试剂盒及其制备方法 - Google Patents
一种核酸抗体双重检测病毒的试剂盒及其制备方法 Download PDFInfo
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- G—PHYSICS
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Abstract
本发明涉及一种核酸抗体双重检测病毒的试剂盒及其制备方法。本发明通过针对H1N1序列进行分析获得了特异性针对H1N1检测的RPA引物和探针，并制备成为相应的检测试纸条；同时针对H1N1保守区筛选并获得效果较好的单克隆抗体，将所述单克隆抗体标记量子点和其他抗体标记的硝酸纤维素膜制备获得H1N1流感病毒荧光量子点快速检测试纸，将两个检测方法进行组合使用，能够进一步提高检测的准确性，并且成本低廉，适于大规模推广使用。
Description
技术领域
本发明涉及检测领域，具体的涉及一种核酸抗体双重检测病毒的试剂盒及其制备方法。
背景技术
流行性感冒病毒(简称流感病毒)是一种造成人类及动物患流行性感冒的ＲNA病毒，属正粘液病毒科，分甲、乙、丙3型，呈球形或丝状，直径80～120nm，3型病毒具有相似的生化和生物学特征。甲型流感病毒的各种亚型是根据病毒粒子血凝素和神经氨酸酶抗原性的不同而分的。流感病毒引起急性上呼吸道感染，主要通过患者的飞沫、患者与患者之间的接触或与被污染物品的接触迅速传播，甲型流感病毒传播更加迅速，感染后潜伏期很短，除感染人外，还可感染哺乳动物和禽类;甲型流感病毒的抗原变异性最强，常引起地区间的暴发与流行，甚至导致世界性大流行。乙型流感病毒变异性较柔弱，一般引起局部流行。丙型流感病毒的抗原性比较稳定，通常不会引起严重的疾病，可以散发出现。其中最严重的大流行发生于1918年，共造成约5000万人死亡。流感病毒感染呈季节性流行，每次疫情可造成２０多万人住院治疗。据世界卫生组织（ＷＨＯ）统计，全球每年大约有300万～500万因流感病毒感染的病例以及25万～50万的死亡病例。我国是流感的高发区，也是流感病毒易发生变异的地区之一，2013年3月就暴发了人感染H7N9禽流感的疫情，不仅影响人们的正常生活和工作，还能引起该病的并发症甚至威胁生命。所以建立快速、准确的检测流感病毒的方法，对于流感患者的临床诊断和及时有效的治疗具有重大的意义，对全球的流感流行趋势起到预见性的作用。
目前，流感病毒检测方法主要有流感病毒分离培养、流感病毒核酸检测、血清免疫学检测、环介导等温扩增法、基因芯片技术等。
病毒分离培养法目前仍是病毒鉴定的经典方法。流感病毒经鸡胚培养或MDCK 细胞培养分离后，可通过血凝试验检测，然后采用已知型别的标准血清进行血凝抑制试验，可确定流感病毒的亚型，微量法血凝抑制试验是WHO 在流感监测工作中推荐使用的标准方法。因为红细胞凝集试验( HA) 和红细胞凝集抑制试验( HI) 的特异性好，所以常用于流感病毒亚型的鉴定，不过易受测定血清中非特异性凝集素及抑制素的干扰，是此方法最大的缺点。酶联免疫反应通过检测流感病毒抗原中的核蛋白，从而检测到流感病毒，因为酶的催化作用，可很好地放大试验反应结果，从而提高该方法的灵敏度。此外该方法还有特异性强、快速等优点，可用于流感疑似病例的快速确诊，是目前广泛使用的一种免疫测定方法。ELISA方法成为流感的诊断常用方法。ELISA 虽然有着很高的灵敏性，但特异性相对较差，交叉反应常出现在不同亚型毒株之间，操作程序也比较复杂，故很少应用。近年来采用分子生物学手段直接检测患者呼吸道样本或分离病毒的实验室越来越多。分子生物学方法敏感、快速，能直接检测临床样本，可用于调查呼吸道疾病暴发流行的病因，比较新变异株与疫苗株基因序列的差别，研究病毒基因进化等。但分子生物学方法不能直接研究病毒抗原性变异情况以及人群对新变异株的免疫能力，因此，分子生物学方法可以用作常规病毒诊断的辅助手段，尤其在流感疫情的应急快速诊断上，发挥着主要作用。近年来，LAMP具有灵敏度高、特异性强、简便快速、易于判断等优点，故广泛使用在流感病毒检测技术上，同时还可以检测流感病毒耐药性基因，对于临床治疗及流感流行趋势监控具有重大的意义。但LAMP 也有其自身不足之处，该方法的结果只能有2种: 扩增和不扩增; 以非特异性的焦磷酸盐沉淀或荧光变化判读结果，因此难以实现高通量的多重检测。基因芯片技术由于同时将大量探针固定于支持物上，所以可以一次性对样品大量序列进行检测和分析，从而解决了传统核酸印迹杂交( southern blotting 和northern blotting 等) 技术操作繁杂、自动化程度低、操作序列数量少、检测效率低等不足。而且，通过设计不同的探针阵列，使用特定的分析方法可使该技术具有多种不同的应用价值，如基因表达图谱鉴定、突变检测、多态性分析、基因组文库作图及杂交测序等。但是也有许多不足之处，例如: 实验条件要求高，成本昂贵，且后期结果分析比较复杂，故不能广泛应用于一般的实验室。
因此，开发一种制备简单、成本低廉、使用方便、不需要高精尖仪器的检测方法是目前研究的一个重要方面。
发明内容
本发明克服现有技术的缺陷，提供了一种制备简单、成本低廉、使用方便、不需要高精尖仪器的检测试剂盒。所述试剂盒通过核酸-抗体双重检测方法对流感病毒进行检测。
本发明一方面，构建了针对流感病毒H1N1的核酸检测试剂。
所述核酸检测试剂包含检测引物对和探针，所述引物对的上下游序列如SEQ IDNo：1-2所示，所述探针的序列如SEQ ID No：3所示。
具体的，上游引物(SEQ ID No：1)：GTAAATTCTGTTATTGAAAAGATGAATACACAGTTC
下游引物(SEQ ID No：2)：Biotin-CTTGGCATTGTTTTTTAACTGGCTTCTTACCTTTTC
探针序列(SEQ ID No：3)：FAM-ACTGTTGGTTCTACTGGAAAATGAAAGAACTTTGGACTACCAC, 在探针中间距 5’端35bp的位置处采用dSpacer修饰，dSpacer分子两侧的距5’端 33bp和38bp位置的胸腺嘧啶(dT)分别被荧光基团FAM和淬灭基团BHQ1 取代，并且在探针的3’末端通过阻塞基团C3Spacer修饰。
所述试纸条带有检测线，检测线上固定有分子A；
序列如SEQ ID NO .2所述的引物，其末端带有特异性结合前述分子A的分子B。所述分子A是生物素配体，分子B是生物素。
本发明提供的试纸条法，对应的探针同样带有碱基替代物dSpcacer(通常为四氢呋喃)，同时探针的5’端还带有荧光基团，但不含淬灭基团。扩增时，核酸内切酶IV剪切dSpcacer，留下可延伸3’-OH，DNA聚 合酶以探针为“正向引物”继续延伸合成DNA，与反向引物(带亲和标记， 例如生物素)一起扩增出一种带有双重标记(荧光基团标记、亲和标记)的扩增产物；该产物在侧向流检测试纸中层析，遇到可识别亲和标记的试纸区域(通常为一条线，即“检测线”，带有链霉亲和素)时，则被富集，表现出线形荧光信号。试纸条法由于不依赖荧光定量PCR仪，成本和适用范围更广。
本发明进一步的，提供一种非疾病诊断目的的H1N1检测方法，它是使用前述引物和探针对样品进行扩增，再用核酸检测试纸条检测扩增产物即可。结果检测：结合试纸条进行显色，上述扩增产物吸取5μL-25μL吸取用缓冲液1×PBST稀释10倍-50倍，用相应标记的试纸条进行检测。结果判读：同时出现T线和C线为阳性(+)，仅出现C线为阴性(-)，只出现T线需考虑试纸条是否有效性。
本发明的RPA检测试剂盒，在检测阳性样本时，检测时间可以缩短到20min以内，可很大程度节约检测时间，尤其适用于即时诊断。
本发明另外提供一种H1N1特异性的表位肽，其氨基酸序列如SEQ ID NO：4所示。
1 VNSVIEKMNT QFTAVGKEFN
21 HLEKRIENLN KKVDDGFLDI
41 WTYNAELLVL LENERTLDYH
61 DSNVKNLYEK VRSQLKNNAK （SEQ ID NO：4）
本发明另外提供一种H1N1特异性的表位肽的编码序列，其核苷酸序列如SEQ ID NO：5所示。
1 GTAAATTCTG TTATTGAAAA GATGAATACA CAGTTCACAG CAGTAGGTAA AGAGTTCAAC
61 CACCTGGAAA AAAGAATAGA GAATTTAAAT AAAAAAGTTG ATGATGGTTT CCTGGACATT
121 TGGACTTACA ATGCCGAACT GTTGGTTCTA CTGGAAAATG AAAGAACTTT GGACTACCAC
181 GATTCAAATG TGAAGAACTT ATATGAAAAG GTAAGAAGCC AGTTAAAAAA CAATGCCAAG（SEQID NO：5）
本发明另外提供一种特异性结合H1N1的单克隆抗体3E6。所述的3E6单克隆抗体具有相应的轻链可变区和重链可变区序列。
3E6轻链可变区
DIVLTQSPALMAASAGEKVTITCAVSQSIQSGYLAWYQQKSGISPKPWIYDTSNQAGGVPARFSGAGSGTSYSLTITSMEAEDAATYYCAQGTTSPLSFGAGTKLELK（SEQ ID NO：6）
3E6重链可变区
EVQLEESGTELARPGASVKLALKASGYIFSSYSGQWIKQRPGAGLELIGYPYPGWIDTRYTQKLTGKATLTADKSSSTLYMQLSSLASEDSAVAYCAGSYFSSDAWGLGTTLAVSS（SEQ ID NO：7）
本发明另外提供一种H1N1 流感病毒荧光量子点快速检测试纸，其中将3E6单克隆抗体标记量子点制备而成。
有益效果
本发明通过针对H1N1序列进行分析获得了特异性针对H1N1检测的RPA引物和探针，并制备成为相应的检测试纸条；同时针对H1N1保守区筛选并获得二个效果较好的单克隆抗体，将所述单克隆抗体标记量子点和其他抗体标记的硝酸纤维素膜制备获得H1N1流感病毒荧光量子点快速检测试纸，将二个检测方法进行组合使用，能够进一步提高检测的准确性，并且成本低廉，适于大规模推广使用。
附图说明
图1 RPA检测方法灵敏度评价结果图
图2 检测试纸条结构图
具体实施方式
为了更进一步的说明本发明的目的、技术方案和优点，我们结合以下具体实施例来阐述本发明，这些实施例仅为了更好的说明本发明专利，而不用于限制本发明范围。基于本发明中的实施例，本领域的技术人员在没有做出创造性的情况下所得到的其他所有实施方式均属于本发明所保护的范围。
实施例1 流感病毒H1N1 RPA特异性检测引物的设计
根据H1N1常见株的基因序列进行比对，选择特异性的保守区，作为引物检测的靶标区。其序列如下：
1 GTAAATTCTG TTATTGAAAA GATGAATACA CAGTTCACAG CAGTAGGTAA AGAGTTCAAC
61 CACCTGGAAA AAAGAATAGA GAATTTAAAT AAAAAAGTTG ATGATGGTTT CCTGGACATT
121 TGGACTTACA ATGCCGAACT GTTGGTTCTA CTGGAAAATG AAAGAACTTT GGACTACCAC
181 GATTCAAATG TGAAGAACTT ATATGAAAAG GTAAGAAGCC AGTTAAAAAA CAATGCCAAG。
根据RPA引物设计的规则，发明人对引物进行优化，通过300多次的优化实验，获得了特异性的引物序列，其序列如下所示。
上游引物(SEQ ID No：1)：GTAAATTCTGTTATTGAAAAGATGAATACACAGTTC
下游引物(SEQ ID No：2)：Biotin-CTTGGCATTGTTTTTTAACTGGCTTCTTACCTTTTC
探针序列(SEQ ID No：3)：FAM-ACTGTTGGTTCTACTGGAAAATGAAAGAACTTTGGACTACCAC, 在探针中间距 5’端35bp的位置处采用dSpacer修饰，dSpacer分子两侧的距5’端 33bp和38bp位置的胸腺嘧啶(dT)分别被荧光基团FAM和淬灭基团BHQ1 取代，并且在探针的3’末端通过阻塞基团C3Spacer修饰。
实施例2 RPA检测方法灵敏度评价
以H1N1病毒裂解液提取RNA作为模板，进行 RPA试验，采用筛选的最优引物进行RPA扩增，同时设置超纯水为阴性对照，反应40℃反应时长(即扩增循环数)分别为25min，RPA反应体系为50μl，其中，2μl正反向引物(10μM)，2μl反向引物(10μM)， 0.6μl探针，25μl含重组酶、DNA聚合酶、单链结合蛋白、核酸内切酶IV、逆转录酶的缓冲液，1μl模板和17.9μlddH2O，充分振荡混匀并且瞬离，最后加入2.5μl的280mM醋酸镁 (MgOAc)，将反应管置于实时荧光PCR仪中40℃恒温反应相应时长；结果如图1所示。
如图1所示，当模板浓度为1ng，100pg，10pg时，均出现明显扩增曲线与目的条带，但模板浓度低于1pg时无明显扩增曲线与目的条带，即说明RPA的检测最低限为1pg，具有较好的检测精度。
实施例3 RPA检测试纸条的制备及检测
按照常规方法制备检测试纸条，其中试纸条带有检测线，检测线上固定有生物素配体；SEQ ID NO：2所述的引物，其末端带有特异性结合生物素配体的生物素。具体的检测试纸条按照图2的形式来制备。
将所述的试纸条分别检测H1N1阳性样本，以及酵母菌、沙眼衣原体、淋球菌、金黄色葡萄球菌、大肠埃希菌和阴道乳酸杆菌标本，将所述标本采用引物以及探针进行RPA扩增，反应40℃反应时长(即扩增循环数)分别为25min，RPA反应体系为50μl，其中，2μl正反向引物(10μM)，2μl反向引物(10μM)， 0.6μl探针，25μl含重组酶、DNA聚合酶、单链结合蛋白、核酸内切酶IV、逆转录酶的缓冲液，1μl模板和17.9μlddH2O，充分振荡混匀并且瞬离，最后加入2.5μl的280mM醋酸镁 (MgOAc)，将反应管置于实时荧光PCR仪中40℃恒温反应相应时长。将扩增产物5μL用缓冲液1×PBST稀释10倍，结果显示，H1N1样本同时出现T线和C线为阳性(+)，其余样本仅出现C线为阴性(-)，说明本发明的试纸条具有较好的有效性。
实施例4 H1N1特异性结合的单克隆抗体的制备
将发明人筛选的具有较好免疫活性的免疫原（SEQ ID No：4）（北京博奥森生物技术有限公司合成）采用肌肉注射的方法免疫6～8周龄雌性BALB/c小鼠5只，免疫剂量为10μg的免疫原与 Quick Antibody－Mouse 3W 佐剂混合后小腿肌肉注射，每次间隔3周，共免疫3次，最后1次免疫2周后，断尾采血分离血清，用间接ELISA检测抗血清抗体效价，选择效价最高的1只小鼠准备做细胞融合，并在末次免疫2～3周后以20μg纯抗原加强免疫，3d后无菌取脾进行融合。提前1 d 制备饲养细胞，复苏NS1 骨髓瘤细胞并培养至对数生长期，无菌取小鼠脾脏，获得淋巴细胞，分别计数后按比例混匀离心，在90 s 内加入1 ml 37℃ 预热的PEG-4000 至细胞沉淀上，轻轻混匀，振荡孵育90s，800 r /min 离心5 min，弃上清，加足量的HAT 培养液重悬细胞后加入到已铺有饲养细胞的96 孔板中，置37℃、5%CO2、饱和湿度培养箱中培养，第7 天用新鲜的HAT 培养液半量换液2次，第10 天改用HT培养液。待细胞集落长至孔底的1 /5 时，取细胞上清液，采用间接ELISA 检测。经间接ELISA方法检测细胞培养上清，能分泌抗体的阳性孔有87孔。将阳性效果最明显的2株阳性孔用有限稀释法进行亚克隆，克隆3 次后采用体内诱生法制备腹水，腹水经Protein A 亲和层析纯化得到纯化的单克隆抗体，将纯化腹水进行滴度检测，结果如表1所示。并于0．01 mol /L 磷酸盐缓冲液中透析后，采用BCA 法测定蛋白浓度．抗体分装后于－20℃冻存．并保存备用。
表1 腹水纯化单抗滴度检测（OD492）
| 抗体 | 1:1000 | 1:10000 | 1:100000 | 1:1000000 | 1:10000000 | 阴性对照 |
| 3E6 | 4.91243 | 1.89456 | 0.45461 | 0.12143 | 0.06321 | 0.05314 |
| 5F2 | 4.23107 | 1.54973 | 0.31549 | 0.09254 | 0.05914 | 0.05219 |
结果显示，3E6和5F2二个单克隆抗体均具有较好的效价。
实施例5 3E6中和活力的测定
运用Lenti-pseudovirus方法测试3E6单抗的中和能力。在过滤后的三种H1N1病毒液（A/swine/Jalisco/12-13/2012(H1N1)、Solomon Islands/3/2006（H1N1）、New Caledonia/20/99（H1N1））中加入稀释好的抗体，并以病毒原液作对照，滴加100 μl /孔到前一天准备好的293A 细胞96 孔板中，37℃、5% CO2培养12～16 h，更换新鲜培养液100μl，48 h 后测定细胞内β-gal 活性，活性越低表明抗体中和能力越强。3E6单抗对不同病毒的中和作用均较强。其中3E6对A/swine/Jalisco/12-13/2012(H1N1) 亚型病毒具有较高的中和能力 (表2) 。
表2 单抗3E6与主要H1N1亚型病毒株的中和活性
| HA亚型 | 抗体浓度（0.01μg/mL） |
| A/swine/Jalisco/12-13/2012(H1N1) | 0.974±0.012 |
| Solomon Islands/3/2006（H1N1） | 0.843±0.009 |
| New Caledonia/20/99（H1N1） | 0.865±0.014 |
实施例6 3E6单克隆抗体亲和力常数测定
采用ForteBio Octet QKe生物大分子相互作用分析仪测定SEQ ID NO：4蛋白与3E6单克隆抗体的亲和力常数，基本过程如下：将SEQ ID NO：4蛋白与生物素以摩尔比1：4混匀，室温孵育2h，G25柱去除未反应的生物素，即得到生物素化的蛋白。测定前将仪器提前开机45min以上，将生物传感器置于1×SD buffer(PBS，pH7.4，0.02％Tween 20，0.1％BSA)中水化(Hydrate)至少10min。在黑色96孔板的第1列和第3列每孔加入200μL SD Buffer。用SDBuffer将生物素化的蛋白稀释至50μg/mL，加至第2列，每孔200μL。将待测抗体用SD Buffer至少稀释5个浓度梯度，加入至第4列，每孔200μL，该列最后一个孔加SD Buffer作为对照孔。将96孔板放入Octet QKe生物大分子相互作用仪中，并设定运行程序，将链霉亲和素生物传感器(Streptavidin biosensors)依次在SD buffer中平衡60s，生物素化蛋白中Loading 300s及在SD buffer中平衡120s后，与抗体结合400s并在SD buffer中解离700s。采用ForteBio Octet QKe数据分析软件计算平衡解离常数KD值。结果如表3所示，3E6单克隆抗体能够较好的能与H1N1抗原肽结合。
表3 单克隆抗体解离常数
| 抗体 | 平衡解离常数（nM） |
| 3E6 | 0.48±0.05 |
实施例7 3E6单克隆抗体的可变区蛋白测序
使用Trizol试剂从培养的3E6小鼠单克隆细胞株中提取总RNA。用Taraka的逆转录cDNA试剂盒把总RNA变为CDNA。cDNA进一步在3’端加Poly G。以加尾的cDNA为模板进行抗体可变区的基因扩增。获得了单克隆抗体的轻链可变区序列和重链可变区序列。
轻链可变区
DIVLTQSPALMAASAGEKVTITCAVSQSIQSGYLAWYQQKSGISPKPWIYDTSNQAGGVPARFSGAGSGTSYSLTITSMEAEDAATYYCAQGTTSPLSFGAGTKLELK
重链可变区
EVQLEESGTELARPGASVKLALKASGYIFSSYSGQWIKQRPGAGLELIGYPYPGWIDTRYTQKLTGKATLTADKSSSTLYMQLSSLASEDSAVAYCAGSYFSSDAWGLGTTLAVSS
实施例8 H1N1 流感病毒荧光量子点快速检测试纸的制备及验证
将3E6单克隆抗体标记量子点,抗体HA (H1N1)单克隆抗体（克隆号IT-096艾美捷Abnova货号MAB10130）包被硝酸纤维素膜搭配检测。将其制备成快速检测试纸后，分别对其检出限、交叉反应、准确性测定进行测试。将国家参考品中的最低检出限样品S1，甲型H1N1( 病毒滴度9． 8 × 105TCID50 /L) ，用0．02 mol /L PBS 缓冲液进行1 ∶ 10、1 ∶ 20、1∶ 40、1 ∶ 80、1 ∶ 160、1 ∶ 320、1 ∶ 640、1 ∶1280、1 ∶ 2560 倍稀释后检测，检测结果见表2，在365 nm 紫外灯照射下，样品在1 ∶ 640倍稀释后，试纸检测线仍然可见荧光条带，因此甲型H1N1 流感病毒荧光量子点快速检测试纸的检出限为1.53×103 TCID50 /L。
表4 H1N1 流感病毒荧光量子点快速检测试纸的最低检出限测定
| 稀释倍数 | 结果 |
| 1 ∶ 10 | + |
| 1 ∶ 20 | + |
| 1 ∶ 40 | + |
| 1 ∶ 80 | + |
| 1 ∶ 160 | + |
| 1 ∶ 320 | + |
| 1 ∶ 640 | + |
| 1 ∶1280 | - |
| 1 ∶ 2560 | - |
同时将所属H1N1 流感病毒荧光量子点快速检测试纸检测大肠杆菌、乙型流感病毒、麻疹病毒、腮腺炎病毒、风疹病毒、水痘-带状疱疹病毒、金黄色葡萄球菌、铜绿假单胞菌，观察是否存在交叉反应，检测结果表明H1N1 流感病毒荧光量子点快速检测试纸与所测样品没有交叉反应，具有良好的特异性。
检测100 份鼻咽拭子样本，采用上述试纸条以及实施例3的试纸条与通用RT-PCR检测结果对照，检测结果见表5。
从表5结果可以看出，100份鼻咽拭子样本经通用RT-PCR 检测结果显示，H1N1阳性样本为38个，H3N2为8个，其余为阴性阳性。经H1N1 流感病毒荧光量子点快速检测试纸测试35份为强阳性，3个为弱阳性。经实施例3的试纸条检测38份均为阳性。
表5 样品检测结果
| 样品 | 样品数 | 实施例8测试纸 | 实施例3试纸条结果 | RT-PCR检测结果 |
| H1N1阳性样品 | 38 | 35/3 | 38 | 38 |
| H3N2阳性样品 | 8 | 0 | 0 | 8 |
| 阴性样品 | 54 | 54 | 54 | 54 |
这也说明实施例8和实施例3的方法可以起到良好的补充，加强检测效果的准确度的效果。
序列表
<110> 北京欣颂生物科技有限公司
<120> 一种核酸抗体双重检测病毒的试剂盒及其制备方法
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Claims (5)
1.一种核酸-抗体双重检测H1N1病毒的试剂盒，所述试剂盒含有特异性检测H1N1核酸的RPA试剂盒以及特异性检测H1N1的试纸条；其中，所述RPA试剂盒含有SEQ ID No：1和2的引物以及SEQ ID No：3的探针；所述试纸条为将3E6单克隆抗体标记量子点制备而成的荧光量子点快速检测试纸条；其中，3E6轻链可变区如SEQ ID No：6所示，重链可变区如SEQ IDNo：7所示。
2.一种核酸检测H1N1病毒的试剂盒，所述试剂盒含有特异性检测H1N1核酸的RPA试剂盒，其特征在于所述RPA试剂盒含有SEQ ID No：1和2的引物以及SEQ ID No：3的探针。
3.一种抗体检测H1N1病毒的试剂盒，其特征在于所述试剂盒含有特异性检测H1N1的试纸条；所述试纸条为将3E6单克隆抗体标记量子点制备而成的荧光量子点快速检测试纸条；其中，3E6轻链可变区如SEQ ID No：6所示，重链可变区如SEQ ID No：7所示。
4.一种特异性结合H1N1病毒的单克隆抗体，其特征在于所述单克隆抗体为3E6，其轻链可变区如SEQ ID No：6所示，重链可变区如SEQ ID No：7所示。
5.如权利要求4所述的单克隆抗体在制备检测H1N1病毒的试剂盒或试剂中的用途。
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112646889A (zh) * | 2020-12-29 | 2021-04-13 | 北京欣颂生物科技有限公司 | 一种利用核酸快速筛选联合抗体检测的肺癌快速检测试剂盒 |
| CN112921124A (zh) * | 2021-04-08 | 2021-06-08 | 北京瀚梅生物科技有限公司 | 一种快速检测病毒的试剂盒 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201413328Y (zh) * | 2009-06-02 | 2010-02-24 | 青岛康地恩药业有限公司 | 一种快速检测猪流感h1n1病毒的胶体金试纸条 |
| CN105296673A (zh) * | 2015-12-03 | 2016-02-03 | 江苏省疾病预防控制中心 | 一种甲型流感病毒分子检测试剂盒及其制备方法 |
| US20170037112A1 (en) * | 2015-08-07 | 2017-02-09 | Panasonic Intellectual Property Management Co., Ltd. | Antibody capable of binding to influenza virus |
| CN107922481A (zh) * | 2015-08-20 | 2018-04-17 | 基亚生物科技股份有限公司 | 抗甲型h1n1流感病毒中和抗体 |
| CN111118219A (zh) * | 2020-02-06 | 2020-05-08 | 广州普世利华科技有限公司 | 一种快速检测甲型流感病毒的rda方法及试剂盒 |
-
2020
- 2020-11-11 CN CN202011250970.9A patent/CN112063765B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201413328Y (zh) * | 2009-06-02 | 2010-02-24 | 青岛康地恩药业有限公司 | 一种快速检测猪流感h1n1病毒的胶体金试纸条 |
| US20170037112A1 (en) * | 2015-08-07 | 2017-02-09 | Panasonic Intellectual Property Management Co., Ltd. | Antibody capable of binding to influenza virus |
| CN107922481A (zh) * | 2015-08-20 | 2018-04-17 | 基亚生物科技股份有限公司 | 抗甲型h1n1流感病毒中和抗体 |
| CN105296673A (zh) * | 2015-12-03 | 2016-02-03 | 江苏省疾病预防控制中心 | 一种甲型流感病毒分子检测试剂盒及其制备方法 |
| CN111118219A (zh) * | 2020-02-06 | 2020-05-08 | 广州普世利华科技有限公司 | 一种快速检测甲型流感病毒的rda方法及试剂盒 |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112646889A (zh) * | 2020-12-29 | 2021-04-13 | 北京欣颂生物科技有限公司 | 一种利用核酸快速筛选联合抗体检测的肺癌快速检测试剂盒 |
| CN112921124A (zh) * | 2021-04-08 | 2021-06-08 | 北京瀚梅生物科技有限公司 | 一种快速检测病毒的试剂盒 |
| CN113234143B (zh) * | 2021-05-10 | 2021-12-14 | 合肥金域医学检验实验室有限公司 | 一种快速检测病毒的核酸抗体试剂盒 |
| CN113234143A (zh) * | 2021-05-10 | 2021-08-10 | 北京保图生物技术有限公司 | 一种快速检测病毒的核酸抗体试剂盒 |
| CN113307867B (zh) * | 2021-05-27 | 2022-01-04 | 杭州康佰裕医学检验实验室有限公司 | 一种核酸抗体联合检测致病菌的试剂盒 |
| CN113024667B (zh) * | 2021-05-27 | 2021-11-30 | 湖南艾科瑞生物工程有限公司 | 核酸抗体联合检测致病菌的试剂盒 |
| CN113307867A (zh) * | 2021-05-27 | 2021-08-27 | 北京保图生物技术有限公司 | 一种核酸抗体联合检测致病菌的试剂盒 |
| CN113024667A (zh) * | 2021-05-27 | 2021-06-25 | 北京保图生物技术有限公司 | 核酸抗体联合检测致病菌的试剂盒 |
| CN113402605A (zh) * | 2021-06-18 | 2021-09-17 | 北京保图生物技术有限公司 | 一种快速炎症检测试剂盒 |
| CN113402604A (zh) * | 2021-07-14 | 2021-09-17 | 北京保图生物技术有限公司 | 快速检测病毒的试剂盒及其制备方法 |
| CN113402604B (zh) * | 2021-07-14 | 2022-01-18 | 河北精硕生物科技有限公司 | 快速检测病毒的试剂盒及其制备方法 |
| CN115925894A (zh) * | 2022-07-28 | 2023-04-07 | 北京热燕生物科技有限公司 | 一种冠状病毒单克隆抗体及其制备的检测试剂 |
| CN115925894B (zh) * | 2022-07-28 | 2024-01-16 | 深圳市华晨阳科技有限公司 | 一种冠状病毒单克隆抗体及其制备的检测试剂 |
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