CN113307866B - 一种抗体组合物及其应用 - Google Patents
一种抗体组合物及其应用 Download PDFInfo
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- CN113307866B CN113307866B CN202110576276.4A CN202110576276A CN113307866B CN 113307866 B CN113307866 B CN 113307866B CN 202110576276 A CN202110576276 A CN 202110576276A CN 113307866 B CN113307866 B CN 113307866B
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Abstract
本发明涉及一种抗体组合物及其应用。所述抗体组合物能够特异性识别SARS‑CoV‑2RBD蛋白、SARS‑CoV‑2N蛋白和流感病毒HA蛋白,本发明利用所述抗体组合物制备试纸,所述试纸包括衬板、样品垫、金标结合垫、硝酸纤维素膜和吸水垫,所述样品垫、金标结合垫、硝酸纤维素膜和吸水垫依次搭接并粘合在衬板上,所述试纸能够同时检测SARS‑CoV‑2病毒与流感病毒,且同时针对SARS‑CoV‑2的核蛋白(N蛋白)和Spike蛋白的RBD区域(RBD蛋白),提高SARS‑CoV‑2病毒检测的灵敏度和特异性。
Description
技术领域
本发明属于生物医学技术领域,涉及一种抗体组合物及其应用。
背景技术
目前,引发季节性流感的主要病毒是甲型H1N1和H3N2亚型流感病毒和乙型流感病毒。由SARS-CoV-2引起的新型冠状病毒肺炎(COVID-19)为一种新型流行病。流感病毒和新型冠状病毒的发病高峰季节有所重叠,且都可以经由空气传播,感染的人体组织相似,如鼻腔、呼吸道和支气管等。在肺组织中,新冠病毒和流感病毒也都对同一种肺细胞(肺泡II型(AT2)细胞)具有偏好性,除此之外,这两种病毒感染后出现的症状也很相似,主要包括发烧、咳嗽或肺部出现炎症特征等。另有研究发现如果同时被两种病毒感染可能会加重机体损伤。因此,同时检测新型冠状病毒和流感病毒两种病毒,一方面可以对相似症状的两种疾病进行鉴别,另一方面,对于共感染的患者来说,可以对下一步的治疗及时提出指导。
目前新型冠状病毒检测产品种类繁多,包括病毒核酸检测和血清学检测。核酸检测可以在病毒感染的早期阶段检测鼻拭子、咽拭子、痰液、粪便或血液等临床样本中的病毒RNA,从而与其他类似病毒区分开来,然而,核酸检测也存在局限性,如工作人员需要经过专业培训,检测过程需要操作生物安全III级实验室内复杂的实验室设施,以及耗费大量时间等。血清学检测主要为新型冠状病毒抗原(S蛋白和N蛋白)ELISA试剂盒和新型冠状病毒中和抗体(IgM、IgG、IgA)胶体金检测试剂盒,抗体反应出现较晚,新冠肺炎抗体检测试剂盒不适用于早期感染的筛查或诊断,ELISA试剂盒诊断时间较长,不适用于大规模快速的诊断筛查。
目前,金纳米颗粒被认为是先进的病毒快速检测技术,且被广泛应用于流感病毒抗原检测,与病毒核酸检测和新冠肺炎抗体检测相比,利用纳米金标记抗体检测抗原具有简单、廉价、快速、灵敏等优点,可实现大规模人群筛查,缓解目前基于聚合酶链反应检测的压力。
CN111426844A公开了一种新型冠状病毒SARS-CoV-2IgG-IgM抗体联检的荧光免疫层析试纸条,该试纸条包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫,样品垫、结合垫、硝酸纤维素膜和吸水垫依次首尾衔接粘贴在底板上,结合垫上包被有SARS-CoV-2结构蛋白-标记物和羊抗兔IgG-标记物,硝酸纤维素膜上设有包被鼠抗人IgG单克隆抗体的检测线T1、包被鼠抗人IgM单克隆抗体的检测线T2和包被兔IgG的质控线C,具有良好的稳定性、操作简单且灵敏度高,但仅能检测新型冠状病毒。
CN212483600U公开了多种呼吸道病原体的IgA抗体检测试纸条,所述试纸条包括依次相连的样品垫、结合垫、反应垫和吸水垫,所述结合垫上包被有胶体金标记的抗人IgA抗体,所述反应垫包括检测区,所述检测区设置有包被SARS-CoV-2抗原的第一检测线,以及包被甲型流感病毒抗原的第二检测线、包被乙型流感病毒抗原的第三检测线、包被呼吸道合胞病毒抗原的第四检测线和包被肺炎病原体抗原的第五检测线中的至少一个。所述试纸条能够在早期对新型冠状病毒肺炎感染者进行筛查,并且对其他呼吸道疾病进行同时检测,但SARS-CoV-2病毒检测的灵敏度和特异性较低。
综上所述,提供一种可以实现同时检测新型冠状病毒和流感病毒的检测试纸,且具备高灵敏度和特异性,对于新型冠状病毒和流感病毒的预防与治疗具有重要意义。
发明内容
针对现有技术的不足和实际需求,本发明提供一种抗体组合物及其应用,所述抗体组合物能够特异性识别SARS-CoV-2RBD蛋白、SARS-CoV-2N蛋白和流感病毒HA蛋白,利用所述抗体组合物制备的联合检测试纸能够实现新型冠状病毒(SARS-CoV-2)与流感病毒的联合检测,且同时针对SARS-CoV-2的核蛋白(N蛋白)和Spike蛋白的RBD区域(RBD蛋白),提高SARS-CoV-2病毒检测的灵敏度和特异性。
为达上述目的,本发明采用以下技术方案:
第一方面,本发明提供一种抗体组合物,所述抗体组合物包括SARS-CoV-2RBD蛋白单克隆抗体、SARS-CoV-2N蛋白单克隆抗体或流感病毒HA蛋白单克隆抗体中的任意一种或至少两种的组合。
所述SARS-CoV-2RBD蛋白单克隆抗体的重链CDR3包括SEQ ID NO:3或SEQ ID NO:4所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的重链CDR3包括SEQ ID NO:7或SEQID NO:8所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的重链CDR3包括SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列。
优选地,所述SARS-CoV-2RBD蛋白单克隆抗体的轻链CDR3包括SEQ ID NO:1或SEQID NO:2所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的轻链CDR3包括SEQ ID NO:5或SEQ ID NO:6所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的轻链CDR3包括SEQID NO:9或SEQ ID NO:10所示的氨基酸序列。
本发明中,所述抗体组合物能够特异性识别SARS-CoV-2RBD蛋白、SARS-CoV-2N蛋白和流感病毒HA蛋白。
SEQ ID NO:1:QQYNHWLTWT。
SEQ ID NO:2:NSRDSSGSHGWV。
SEQ ID NO:3:AKGQQLVQYFDY。
SEQ ID NO:4:ARNDAPPLRFGVPPITTPVWT。
SEQ ID NO:5:VLYMGSGTRV。
SEQ ID NO:6:QQYNTWYT。
SEQ ID NO:7:VRDRGNWFDP。
SEQ ID NO:8:ARLRSGAGRDVFAY。
SEQ ID NO:9:AAWDVSLNGYV。
SEQ ID NO:10:AAWDGSLNGFV。
SEQ ID NO:11:VRAIGAADSY。
SEQ ID NO:12:TRGILQFLEWLLPGDY。
优选地,所述SARS-CoV-2RBD蛋白单克隆抗体的重链CDR1包括SEQ ID NO:13或SEQID NO:14所示的氨基酸序列,所述SARS-CoV-2RBD蛋白单克隆抗体的轻链CDR1包括SEQ IDNO:15或SEQ ID NO:16所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的重链CDR1包括SEQ ID NO:17或SEQ ID NO:18所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的轻链CDR1包括SEQ ID NO:19或SEQ ID NO:20所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的重链CDR1包括SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的轻链CDR1包括SEQ ID NO:23或SEQ ID NO:24所示的氨基酸序列。
SEQ ID NO:13:GFTFSSYA。
SEQ ID NO:14:GFSISTYA。
SEQ ID NO:15:QSVSSN。
SEQ ID NO:16:SLMSHY。
SEQ ID NO:17:GYTFTSYA。
SEQ ID NO:18:GFTFSSYA。
SEQ ID NO:19:SGSVSTSYY。
SEQ ID NO:20:ESVGSN。
SEQ ID NO:21:RFTFSSYW。
SEQ ID NO:22:GFTLGDYG。
SEQ ID NO:23:TSNIGSNP。
SEQ ID NO:24:SSNIGINT。
优选地,所述SARS-CoV-2RBD蛋白单克隆抗体的重链CDR2包括SEQ ID NO:25或SEQID NO:26所示的氨基酸序列,所述SARS-CoV-2RBD蛋白单克隆抗体的轻链CDR2包括SEQ IDNO:27或SEQ ID NO:28所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的重链CDR2包括SEQ ID NO:29或SEQ ID NO:30所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的轻链CDR2包括SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的重链CDR2包括SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的轻链CDR2包括SEQ ID NO:35或SEQ ID NO:36所示的氨基酸序列。
SEQ ID NO:25:ISGSGGST。
SEQ ID NO:26:ITYDGSYK。
SEQ ID NO:27:GAS。
SEQ ID NO:28:GQN。
SEQ ID NO:29:ISAYNGNT。
SEQ ID NO:30:ISYDGSNK。
SEQ ID NO:31:STN。
SEQ ID NO:32:GAS。
SEQ ID NO:33:INQDGSVK。
SEQ ID NO:34:IRSKVYGGTT。
SEQ ID NO:35:SNN。
SEQ ID NO:36:NNN。
优选地,所述SARS-CoV-2RBD蛋白单克隆抗体的重链包括SEQ ID NO:37或SEQ IDNO:38所示的氨基酸序列,所述SARS-CoV-2RBD蛋白单克隆抗体的轻链包括SEQ ID NO:39或SEQ ID NO:40所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的重链包括SEQ IDNO:41或SEQ ID NO:42所示的氨基酸序列,所述SARS-CoV-2N蛋白单克隆抗体的轻链包括SEQ ID NO:43或SEQ ID NO:44所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的重链包括SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列,所述流感病毒HA蛋白单克隆抗体的轻链包括SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列。
SEQ ID NO:37:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGQQLVQYFDYWGQGTLVTVSS。
SEQ ID NO:38:
EVQLVESGGGVVQPGRSLRLSCRASGFSISTYAMNWVRQAPGKGLEWVAAITYDGSYKFYADSVKGRFTISRDNSKNTQYLEMNSLRAEDTAIFYCARNDAPPLRFGVPPITTPVWTSGAEGPRSPSP。
SEQ ID NO:39:
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNHWLTWTFGQGTKVEIK。
SEQ ID NO:40:
SYELTQDPAVSVALGQTVRITCQGDSLMSHYASWYQQKPGQPPVLVIHGQNNRPSGIPDRFSGSRSGDTCSTITGAQAEDEADYYCNSRDSSGSHGWVFGGGTKLTVL。
SEQ ID NO:41:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAISWVRQAPGQGLEWMGWISAYNGNTNSAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCVRDRGNWFDPWGQGTLVTVSS。
SEQ ID NO:42:
EVQLVESGGGVVQPGKSLRISCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVQGRLTISRDNSNNTLYLQLSSLTLEDTAVYYCARLRSGAGRDVFAYWGQGTLVTVSS。
SEQ ID NO:43:
QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCVLYMGSGTRVFGGGTKLTVL。
SEQ ID NO:44:
EIVMTQSPATLSVSPGERATLFCRASESVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAIYHCQQYNTWYTFGQGTKVEIK。
SEQ ID NO:45:
EVQLVESGGGLVQPGGSLRLSCTASRFTFSSYWMTWVRQAPGKGLEWVANINQDGSVKYYLDSVKARFTISRDNAENSLYLLMNSLRAEDTAVYYCVRAIGAADSYWGQGTLVTVSS。
SEQ ID NO:46:
EVQLVESGGGLVQPGRSLRLSCTASGFTLGDYGLSWVRQAPGKGLEWVGFIRSKVYGGTTEYAASVKGRFSISRDDSKSIVYLQMNSLKTEDTAMYYCTRGILQFLEWLLPGDYWGQGTLVTVSS。
SEQ ID NO:47:
QSVLTQPPSASGTPGQRVTISCSGSTSNIGSNPVNWYQQLPGTAPKILIYSNNQCPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDVSLNGYVFGTGTRVTVL。
SEQ ID NO:48:
QSVLTQPPSASGTPGQTVTISCSGSSSNIGINTVNWYQHLPGTAPKLLIYNNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDGSLNGFVFGSGTKVTVL。
第二方面,本发明提供第一方面所述的抗体组合物在制备病毒检测产品中的应用。
优选地,所述检测产品包括检测试纸、检测卡或检测试剂盒中的任意一种。
第三方面,本发明提供一种SARS-CoV-2病毒与流感病毒联合检测试纸,所述试纸包括衬板、样品垫、金标结合垫、硝酸纤维素膜、吸水垫和抗体组合物,其中,所述抗体组合物包括第一SARS-CoV-2RBD蛋白单克隆抗体、第一SARS-CoV-2N蛋白单克隆抗体、第一流感病毒HA蛋白单克隆抗体、第二SARS-CoV-2RBD蛋白单克隆抗体、第二SARS-CoV-2N蛋白单克隆抗体和第二流感病毒HA蛋白单克隆抗体。
优选地,所述衬板的材料包括聚氯乙烯。
优选地,所述样品垫的材料包括玻璃纤维、棉纤维、滤纸纤维或聚酯纤维中的任意一种。
优选地,所述金标结合垫的材料包括玻璃纤维或聚酯纤维。
优选地,所述吸水垫的材料包括吸水滤纸。
优选地,所述金标结合垫包被有胶体金标记的第一SARS-CoV-2RBD蛋白单克隆抗体、胶体金标记的第一SARS-CoV-2N蛋白单克隆抗体和胶体金标记的第一流感病毒HA蛋白单克隆抗体。
优选地,所述胶体金的粒径为35~45nm,包括但不限于36nm、37nm、38nm、39nm、40nm、42nm或44nm。
优选地,所述硝酸纤维素膜设置有包被第二流感病毒HA蛋白单克隆抗体的检测线T1、包被第二SARS-CoV-2RBD蛋白单克隆抗体的检测线T2、包被第二SARS-CoV-2N蛋白单克隆抗体的检测线T3和包被羊抗人单克隆抗体的质控线。
优选地,所述样品垫、金标结合垫、硝酸纤维素膜和吸水垫依次搭接并粘合在衬板上。
优选地,所述抗体组合物为第一方面所述的抗体组合物。
本发明中,所述第一SARS-CoV-2RBD蛋白单克隆抗体和第二SARS-CoV-2RBD蛋白单克隆抗体分别与SARS-Cov-2 RBD蛋白的不同表位结合。所述第一SARS-CoV-2N蛋白单克隆抗体和第二SARS-CoV-2N蛋白单克隆抗体分别与SARS-CoV-2N蛋白的不同表位结合。所述第一流感病毒HA蛋白单克隆抗体和第二流感病毒HA蛋白单克隆抗体分别与流感病毒HA蛋白的不同表位结合。
优选地,所述第一SARS-CoV-2RBD蛋白单克隆抗体可以为第一方面所述的SARS-CoV-2RBD蛋白单克隆抗体中的任意一种,而所述第二SARS-CoV-2RBD蛋白单克隆抗体则为不同的另一种;同理,所述第一SARS-CoV-2N蛋白单克隆抗体可以为第一方面所述的SARS-CoV-2N蛋白单克隆抗体中的任意一种,而所述第二SARS-CoV-2N蛋白单克隆抗体则为不同的另一种;所述第一流感病毒HA蛋白单克隆抗体可以为第一方面所述的流感病毒HA蛋白单克隆抗体中的任意一种,而所述第二流感病毒HA蛋白单克隆抗体则为不同的另一种。
优选地,所述第一SARS-CoV-2RBD蛋白单克隆抗体的重链的氨基酸序列如SEQ IDNO:37,轻链的氨基酸序列如SEQ ID NO:39。
优选地,所述第二SARS-CoV-2RBD蛋白单克隆抗体的重链的氨基酸序列如SEQ IDNO:38,轻链的氨基酸序列如SEQ ID NO:40。
优选地,所述第一SARS-CoV-2N蛋白单克隆抗体的重链的氨基酸序列如SEQ IDNO:42,轻链的氨基酸序列如SEQ ID NO:44。
优选地,所述第二SARS-CoV-2N蛋白单克隆抗重链的氨基酸序列如SEQ ID NO:41,轻链氨基酸序列如SEQ ID NO:43。
优选地,所述第一流感病毒HA蛋白单克隆抗体的重链的氨基酸序列如SEQ ID NO:45,轻链的氨基酸序列如SEQ ID NO:47。
优选地,所述第二流感病毒HA蛋白单克隆抗体的重链的氨基酸序列如SEQ ID NO:46,轻链的氨基酸序列如SEQ ID NO:48。
本发明中,在结合垫上包被胶体金标记的第一SARS-CoV-2RBD蛋白单克隆抗体、胶体金标记的第一SARS-CoV-2N蛋白单克隆抗体和胶体金标记的第一流感病毒HA蛋白单克隆抗体,在检测线T1、T2和T3分别包被第二流感病毒HA蛋白单克隆抗体、第二SARS-CoV-2RBD蛋白单克隆抗体和第二SARS-CoV-2N蛋白单克隆抗体,能够同时特异性针对SARS-CoV-2RBD蛋白、SARS-CoV-2N蛋白和流感病毒HA蛋白,当检测样品中存在上述三种蛋白时,会分别使检测线T2、T3和T1显色,从而实现高灵敏性、特异性地联合检测。
本发明中,当检测样品中存在SARS-CoV-2RBD蛋白时,SARS-CoV-2RBD蛋白首先与结合垫上胶体金标记的第一SARS-CoV-2N蛋白单克隆抗体结合,随后又与检测线T3上的第二SARS-CoV-2N蛋白单克隆抗体结合,使检测线T3显色,反之,当检测样品中不存在SARS-CoV-2RBD蛋白时,检测线T2则不显色;同理,当检测样品中存在SARS-CoV-2N蛋白时,检测线T3显色,当检测样品中存在流感病毒HA蛋白时,检测线T1显色。
第四方面,本发明提供一种如第三方面所述的SARS-CoV-2病毒与流感病毒联合检测试纸的制备方法,所述方法包括以下步骤:
(1)制备第一和第二SARS-CoV-2RBD蛋白单克隆抗体、第一和第二SARS-CoV-2N蛋白单克隆抗体以及第一和第二流感病毒HA蛋白单克隆抗体;
(2)将样品垫和结合垫分别浸泡在样品垫处理液和结合垫处理液中,随后进行烘干处理;
(3)分别取第一SARS-CoV-2RBD蛋白单克隆抗体、第一SARS-CoV-2N蛋白单克隆抗体和第一流感病毒HA蛋白单克隆抗体,加入胶体金溶液中进行标记,随后喷涂到结合垫上,进行烘干处理,得到金标结合垫;
(4)分别取第二SARS-CoV-2RBD蛋白单克隆抗体、第二SARS-CoV-2N蛋白单克隆抗体、第二流感病毒HA蛋白单克隆抗体以及羊抗人单克隆抗体与包被缓冲液混合,并分别包被在硝酸纤维素膜的检测线T2、检测线T3、检测线T1和质控线上,随后进行烘干处理;
(5)将样品垫、金标结合垫、硝酸纤维素膜和吸水垫依次搭接并粘合在衬板上,得到所述SARS-CoV-2病毒与流感病毒联合检测试纸。
优选地,上述各单克隆抗体的制备方法包括以下步骤:
(1’)分离人外周血单核细胞,流式分选其中的浆母B细胞;
(2’)分别对单个浆母B细胞进行逆转录,获得cDNA;
(3’)以cDNA为模板,进行2轮PCR扩增,选择可同时扩增出抗体重链基因和轻链基因的浆母B细胞,并分别对重链基因和轻链基因进行测序;
(4’)以步骤(3’)得到的浆母B细胞的cDNA为模板,扩增抗体重链基因和轻链基因,并构建重组载体,将重组载体转染细胞,进行细胞培养并纯化制备单克隆抗体;
(5’)对所述单克隆抗体进行筛选,得到本发明所需单克隆抗体。
优选地,步骤(2)所述样品垫处理液包括Tris-HCl、PVP-40、BSA或Triton X-100中的任意一种或至少两种的组合。
优选地,步骤(2)所述结合垫处理液包括Tris-HCl、PVP-40、BSA或Tween-20中的任意一种或至少两种的组合。
优选地,步骤(2)所述浸泡的时间为25~35min,包括但不限于26min、27min、28min、29min、30min、31min、32min、33min或34min。
优选地,步骤(2)所述烘干的温度为35~40℃,包括但不限于36℃、37℃、38℃或39℃。
优选地,步骤(2)所述烘干的时间为4~6h。
优选地,步骤(3)所述第一SARS-CoV-2RBD蛋白单克隆抗体与胶体金溶液的配比为(20~30)μg:1mL,包括但不限于21μg:1mL、22μg:1mL、23μg:1mL、24μg:1mL、25μg:1mL、27μg:1mL、28μg:1mL或29μg:1mL。
优选地,步骤(3)所述第一SARS-CoV-2N蛋白单克隆抗体与胶体金溶液的配比为(5~10)μg:1mL,包括但不限于6μg:1mL、7μg:1mL、8μg:1mL或9μg:1mL。
优选地,步骤(3)所述第一流感病毒HA蛋白单克隆抗体与胶体金溶液的配比为(15~25)μg:1mL,包括但不限于16μg:1mL、17μg:1mL、18μg:1mL、19μg:1mL、20μg:1mL、22μg:1mL或24μg:1mL。
优选地,步骤(4)所述包被缓冲液包括Tris-HCl、PVP-40、BSA、Tween-20和NaN3中的任意一种或至少两种的组合。
作为优选的技术方案,所述SARS-CoV-2病毒与流感病毒联合检测试纸的制备方法包括以下步骤:
(1)制备第一和第二SARS-CoV-2RBD蛋白单克隆抗体、第一和第二SARS-CoV-2N蛋白单克隆抗体以及第一和第二流感病毒HA蛋白单克隆抗体;
(2)将样品垫和结合垫分别在样品垫处理液和结合垫处理液中浸泡25~35min,随后于35~40℃进行烘干处理4~6h;
(3)按配比为(20~30)μg:1mL、(5~10)μg:1mL和(15~25)μg:1mL分别将第一SARS-CoV-2RBD蛋白单克隆抗体、第一SARS-CoV-2N蛋白单克隆抗体和第一流感病毒HA蛋白单克隆抗体加入胶体金溶液中进行标记,随后喷涂到结合垫上,于35~40℃、湿度为10%~30%条件下进行烘干处理4~6h,得到金标结合垫;
(4)分别取第二SARS-CoV-2RBD蛋白单克隆抗体、第二SARS-CoV-2N蛋白单克隆抗体、第二流感病毒HA蛋白单克隆抗体以及羊抗人单克隆抗体与包被缓冲液混合,并分别包被在硝酸纤维素膜的检测线T2、检测线T3、检测线T1和质控线上,随后于35~40℃、湿度为10%~30%条件下进行烘干处理4~6h;
(5)将样品垫、金标结合垫、硝酸纤维素膜和吸水垫依次搭接并粘合在衬板上,得到所述SARS-CoV-2病毒与流感病毒联合检测试纸。
针对现有技术,本发明具有以下有益效果:
(1)本发明的抗体组合物能够特异性识别SARS-CoV-2RBD蛋白、SARS-CoV-2N蛋白和流感病毒HA蛋白;
(2)本发明的SARS-CoV-2病毒与流感病毒联合检测试纸,能够同时检测SARS-CoV-2病毒与流感病毒,且同时针对SARS-CoV-2的核蛋白(N蛋白)和Spike蛋白的RBD区域(RBD蛋白),提高SARS-CoV-2病毒检测的灵敏度和特异性;
(3)本发明的SARS-CoV-2病毒与流感病毒联合检测试纸,成本低,使用便捷,具备高特异性和灵敏度,可快速得到结果。
附图说明
图1为本发明SARS-CoV-2病毒与流感病毒联合检测试纸结构示意图,其中,1为样品垫,2为金标结合垫,3为硝酸纤维素膜,4为吸水垫,5为衬板,6为检测线T1,7为检测线T2,8为检测线T3,9为质控线C;
图2为实施例1制备的各单克隆抗体的SDS-PAGE电泳图;
图3为本发明SARS-CoV-2病毒与流感病毒联合检测试纸不同检测结果的判读说明图,(+)为阳性,(-)为阴性,invalid为无效;
图4为分别含有SARS-CoV-2假病毒、SARS病毒、MERS病毒、NL63病毒、OC43病毒、229E病毒或呼吸道合胞病毒(RSV)的标本检测结果图;
图5为分别含有SARS-CoV-2核衣壳、SARS病毒核衣壳(SARS-NP)、MERS病毒核衣壳(MERS-NP)、甲型流感病毒核衣壳(IAV-NP)、B型流感病毒核衣壳(IBV-NP)和呼吸道合胞病毒(RSV)的标本检测结果图;
图6为别含有流感病毒A/California/4/2009(H1N1),A/Puerto Rico/8/1934(H1N1),A/HongKong/4801/2014(H3N2),A/Texas/50/2012(H3N2),B/Colorado/06/2017(Victoria)and B/Phuket/3073/2013(Yamagata)及RSV和PBS的样本检测结果图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例制备本发明所需单克隆抗体,制备方法包括以下步骤:
单抗表达:通过流式分选单个B细胞,再通过单细胞的RT-PCR,将B细胞的mRNA逆转录成cDNA,扩增B细胞的VDJ基因,转染抗体质粒到293真核表达***。用ProteinA琼脂糖珠纯化单克隆抗体,通过SDS-PAGE检查纯化的单抗纯度。
病毒特异单抗功能鉴定:通过ELISA,微量中和实验和噬斑实验,等一系列体外的功能实验鉴定抗体的结合与中和能力。
通过以上方法筛选出分别与流感病毒HA蛋白、SARS-CoV-2RBD蛋白和SARS-CoV-2N蛋白结合力高的单克隆抗体,包括第一SARS-CoV-2RBD蛋白单克隆抗体(CD236,重链和轻链分别为SEQ ID NO:37和SEQ ID NO:39所示)和第二SARS-CoV-2RBD蛋白单克隆抗体(CD217,重链和轻链分别为SEQ ID NO:38和SEQ ID NO:40所示),第一SARS-CoV-2N蛋白单克隆抗体(X-59,重链和轻链分别为SEQ ID NO:42和SEQ ID NO:44所示)和第二SARS-CoV-2N蛋白单克隆抗体(X-23,重链和轻链分别为SEQ ID NO:41和SEQ ID NO:43所示),第一流感病毒HA蛋白单克隆抗体(VA26,重链和轻链分别为SEQ ID NO:45和SEQ ID NO:47所示)和第二流感病毒HA蛋白单克隆抗体(VA20,重链和轻链分别为SEQ ID NO:46和SEQ ID NO:48所示),SDS-PAGE电泳图如图2所示。
实施例2
本实施例提供一种SARS-CoV-2病毒与流感病毒联合检测试纸,如图1所示,所述试纸包括衬板5、样品垫1、金标结合垫2、硝酸纤维素膜3和吸水垫4,所述样品1垫、金标结合垫2、硝酸纤维素膜3和吸水垫4依次搭接并粘合在衬板5上,所述金标结合垫2包被有胶体金标记的第一SARS-CoV-2RBD蛋白单克隆抗体、胶体金标记的第一SARS-CoV-2N蛋白单克隆抗体和胶体金标记的第一流感病毒HA蛋白单克隆抗体,所述硝酸纤维素膜3设置有包被第二流感病毒HA蛋白单克隆抗体的检测线T16、包被第二SARS-CoV-2RBD蛋白单克隆抗体的检测线T27、包被第二SARS-CoV-2N蛋白单克隆抗体的检测线T38和包被羊抗人单克隆抗体的质控线C 9。
所述SARS-CoV-2病毒与流感病毒联合检测试纸的制备方法包括以下步骤:
(1)将样品垫1(玻璃纤维)和结合垫(聚酯纤维)分别在Tris-HCl缓冲液中浸泡30min,随后于37℃进行烘干处理5h;
(2)按配比为25μg/mL、9μg/mL和18μg/mL分别将实施例1制备的第一SARS-CoV-2RBD蛋白单克隆抗体、第一SARS-CoV-2N蛋白单克隆抗体和第一流感病毒HA蛋白单克隆抗体加入胶体金溶液(40nm)中进行标记,随后喷涂到结合垫上,于37℃、湿度为30%条件下进行烘干处理5h,得到金标结合垫2;
(3)分别取实施例1制备的第二SARS-CoV-2RBD蛋白单克隆抗体、第二SARS-CoV-2N蛋白单克隆抗体、第二流感病毒HA蛋白单克隆抗体以及羊抗人单克隆抗体与Tris-HCl缓冲液混合,并分别包被在硝酸纤维素膜3(上海金标NC140)的检测线T27、检测线T38、检测线T16和质控线C 9上,随后于37℃、湿度为10%条件下进行烘干处理5h;
(4)将样品垫1、金标结合垫2、硝酸纤维素膜3和吸水垫4(吸水滤纸)依次搭接并粘合在衬板5上,得到所述SARS-CoV-2病毒与流感病毒联合检测试纸。
试验例1
使用实施例2制备的SARS-CoV-2病毒与流感病毒联合检测试纸对样品进行检测,可出现如图3所示结果,质控线C显色视为有效检测,进行后续判读,当质控线C不显色,则检测无效,不进行判读;当T1或T2中任一条显色,或同时显色,均判定为SARS-CoV-2病毒阳性,而T1和T2均不显色,判定为SARS-CoV-2病毒阴性;T3显色,判定为流感病毒阳性,T3不显色判定为流感病毒阴性;T1或T2中任一条显色,或同时显色,以及T3显色,判定为SARS-CoV-2病毒与流感病毒均为阳性。
试验例2
本试验例使用实施例2制备的检测试纸进行检测试验,结果如图4-图6所示。
图4为分别含有SARS-CoV-2假病毒、SARS假病毒、MERS假病毒、NL63假病毒、OC43假病毒、229E假病毒或呼吸道合胞病毒(RSV)的标本检测结果图(以上所有待检测抗原均来自中山大学陈耀庆课题组),以PBS作为阴性对照,如图4所示,只有检测含有SARS-CoV-2假病毒的样本的试纸的T1检测线显色,而检测其余病毒的试纸的检测线未显色,表明本发明的检测试纸可特异性检测出新型冠状病毒的RBD蛋白,与其他病毒无交叉反应。
图5为分别含有SARS-CoV-2核衣壳、SARS病毒核衣壳(SARS-NP)、MERS病毒核衣壳(MERS-NP)、甲型流感病毒核衣壳(IAV-NP)、B型流感病毒核衣壳(IBV-NP)和呼吸道合胞病毒(RSV)的标本检测结果图,以PBS作为阴性对照,由图5可知,只有检测含有SARS-CoV-2核蛋白样本的试纸的T3检测线显色,而检测其余病毒核蛋白的试纸的检测线未显色,表明本发明的检测试纸可特异性检测出新型冠状病毒的核蛋白,与其他病毒核蛋白无交叉反应。
图6为别含有流感病毒A/California/4/2009(H1N1),A/Puerto Rico/8/1934(H1N1),A/HongKong/4801/2014(H3N2),A/Texas/50/2012(H3N2),B/Colorado/06/2017(Victoria)and B/Phuket/3073/2013(Yamagata)及RSV和PBS样本的检测结果,其中,a为A/California/4/2009(H1N1),b为A/Puerto Rico/8/1934(H1N1),c为A/HongKong/4801/2014(H3N2),d为A/Texas/50/2012(H3N2),e为B/Colorado/06/2017(Victoria),f为B/Phuket/3073/2013(Yamagata),g为RSV,以PBS作为阴性对照,由图6可知,只有检测含有流感病毒试纸的T2检测线显色,而检测其它病毒的试纸的检测线未显色,表明本发明的检测试纸可特异性检测出流感病毒,并且具有良好的广谱性与其他病毒无交叉反应。
终上所述,本发明同时采用针对SARS-CoV-2RBD蛋白、SARS-CoV-2N蛋白单和流感病毒HA蛋白的单克隆抗体,使得SARS-CoV-2病毒与流感病毒联合检测试纸能够同时检测SARS-CoV-2病毒与流感病毒,且具备较高的特异性和灵敏度,此外,所述联合试纸成本低,使用便捷。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 中山大学
<120> 一种抗体组合物及其应用
<130> 20210519
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<212> PRT
<213> 人工序列
<400> 44
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Phe Cys Arg Ala Ser Glu Ser Val Gly Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Ile Tyr His Cys Gln Gln Tyr Asn Thr Trp Tyr Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 45
<211> 117
<212> PRT
<213> 人工序列
<400> 45
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Arg Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asn Ile Asn Gln Asp Gly Ser Val Lys Tyr Tyr Leu Asp Ser Val
50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Tyr
65 70 75 80
Leu Leu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Arg Ala Ile Gly Ala Ala Asp Ser Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 46
<211> 125
<212> PRT
<213> 人工序列
<400> 46
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Gly Asp Tyr
20 25 30
Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Val Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Thr Arg Gly Ile Leu Gln Phe Leu Glu Trp Leu Leu Pro Gly
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 47
<211> 110
<212> PRT
<213> 人工序列
<400> 47
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Ser Asn
20 25 30
Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Ile Leu
35 40 45
Ile Tyr Ser Asn Asn Gln Cys Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Val Ser Leu
85 90 95
Asn Gly Tyr Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu
100 105 110
<210> 48
<211> 110
<212> PRT
<213> 人工序列
<400> 48
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Thr Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ile Asn
20 25 30
Thr Val Asn Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Gly Ser Leu
85 90 95
Asn Gly Phe Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu
100 105 110
Claims (33)
1.一种抗体组合物,其特征在于,所述抗体组合物包括SARS-CoV-2 RBD蛋白单克隆抗体、SARS-CoV-2 N蛋白单克隆抗体或流感病毒HA蛋白单克隆抗体中的任意一种或至少两种的组合;
所述SARS-CoV-2 RBD蛋白单克隆抗体的重链CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:25和SEQ ID NO:3所示,轻链CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:15、SEQ ID NO:27和SEQ ID NO:1所示;
或,所述SARS-CoV-2 RBD蛋白单克隆抗体的重链CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:26和SEQ ID NO:4所示,轻链CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:28和SEQ ID NO:2所示;
所述SARS-CoV-2 N蛋白单克隆抗体的重链CDR1、CDR2和CDR3的氨基酸序列分别如SEQID NO:18、SEQ ID NO:30和SEQ ID NO:8所示,轻链CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:32和SEQ ID NO:6所示;
所述流感病毒HA蛋白单克隆抗体的重链CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO:21、SEQ ID NO:33和SEQ ID NO:11所示,轻链CDR1、CDR2和CDR3的氨基酸序列分别如SEQID NO:23、SEQ ID NO:35和SEQ ID NO:9所示;
或,所述流感病毒HA蛋白单克隆抗体的重链CDR1、CDR2和CDR3的氨基酸序列分别如SEQID NO:22、SEQ ID NO:34和SEQ ID NO:12所示,轻链CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:24、SEQ ID NO:36和SEQ ID NO:10所示。
2.根据权利要求1所述的抗体组合物,其特征在于,所述SARS-CoV-2 RBD蛋白单克隆抗体的重链氨基酸序列如SEQ ID NO:37所示,轻链氨基酸序列如SEQ ID NO:39所示;
或,所述SARS-CoV-2 RBD蛋白单克隆抗体的重链氨基酸序列如SEQ ID NO:38所示,轻链氨基酸序列如SEQ ID NO:40所示;
所述SARS-CoV-2 N蛋白单克隆抗体的重链氨基酸序列如SEQ ID NO:42所示,轻链氨基酸序列如SEQ ID NO:44所示;
所述流感病毒HA蛋白单克隆抗体的重链氨基酸序列如SEQ ID NO:45所示,轻链氨基酸序列如SEQ ID NO:47所示;
或,所述流感病毒HA蛋白单克隆抗体的重链氨基酸序列如SEQ ID NO:46所示,轻链氨基酸序列如SEQ ID NO:48所示。
3.权利要求1或2所述的抗体组合物在制备新型冠状病毒和/或流感病毒检测产品中的应用。
4.根据权利要求3所述的应用,其特征在于,所述检测产品包括检测试纸、检测卡或检测试剂盒中的任意一种。
5.一种SARS-CoV-2病毒与流感病毒联合检测试纸,其特征在于,所述试纸包括衬板、样品垫、金标结合垫、硝酸纤维素膜、吸水垫和抗体组合物;
其中,所述抗体组合物包括第一SARS-CoV-2 RBD蛋白单克隆抗体、第一SARS-CoV-2 N蛋白单克隆抗体、第一流感病毒HA蛋白单克隆抗体、第二SARS-CoV-2 RBD蛋白单克隆抗体、第二SARS-CoV-2 N蛋白单克隆抗体和第二流感病毒HA蛋白单克隆抗体;
所述抗体组合物为权利要求1或2所述的抗体组合物。
6.根据权利要求5所述的检测试纸,其特征在于,所述衬板的材料包括聚氯乙烯。
7.根据权利要求5所述的检测试纸,其特征在于,所述样品垫的材料包括玻璃纤维、棉纤维、滤纸纤维或聚酯纤维中的任意一种。
8.根据权利要求5所述的检测试纸,其特征在于,所述金标结合垫的材料包括玻璃纤维或聚酯纤维。
9.根据权利要求5所述的检测试纸,其特征在于,所述吸水垫的材料包括吸水滤纸。
10.根据权利要求5所述的检测试纸,其特征在于,所述金标结合垫包被有胶体金标记的第一SARS-CoV-2 RBD蛋白单克隆抗体、胶体金标记的第一SARS-CoV-2 N蛋白单克隆抗体和胶体金标记的第一流感病毒HA蛋白单克隆抗体。
11.根据权利要求10所述的检测试纸,其特征在于,所述胶体金的粒径为35~45 nm。
12.根据权利要求5所述的检测试纸,其特征在于,所述硝酸纤维素膜设置有包被第二流感病毒HA蛋白单克隆抗体的检测线T1、包被第二SARS-CoV-2 RBD蛋白单克隆抗体的检测线T2、包被第二SARS-CoV-2 N蛋白单克隆抗体的检测线T3和包被羊抗人单克隆抗体的质控线。
13.根据权利要求5所述的检测试纸,其特征在于,所述样品垫、金标结合垫、硝酸纤维素膜和吸水垫依次搭接并粘合在衬板上。
14.根据权利要求5所述的检测试纸,其特征在于,所述第一SARS-CoV-2 RBD蛋白单克隆抗体和第二SARS-CoV-2 RBD蛋白单克隆抗体分别与SARS-Cov-2 RBD蛋白的不同表位结合。
15.根据权利要求14所述的检测试纸,其特征在于,所述第一SARS-CoV-2 RBD蛋白单克隆抗体的重链的氨基酸序列如SEQ ID NO:37,轻链的氨基酸序列如SEQ ID NO:39。
16.根据权利要求14所述的检测试纸,其特征在于,所述第二SARS-CoV-2 RBD蛋白单克隆抗体的重链的氨基酸序列如SEQ ID NO:38,轻链的氨基酸序列如SEQ ID NO:40。
17.根据权利要求5所述的检测试纸,其特征在于,所述第一SARS-CoV-2 N蛋白单克隆抗体和第二SARS-CoV-2 N蛋白单克隆抗体分别与SARS-CoV-2 N蛋白的不同表位结合。
18.根据权利要求17所述的检测试纸,其特征在于,所述第一SARS-CoV-2 N蛋白单克隆抗体的重链的氨基酸序列如SEQ ID NO:42,轻链的氨基酸序列如SEQ ID NO:44。
19.根据权利要求17所述的检测试纸,其特征在于,所述第二SARS-CoV-2 N蛋白单克隆抗重链的氨基酸序列如SEQ ID NO:41,轻链氨基酸序列如SEQ ID NO:43。
20.根据权利要求5所述的检测试纸,其特征在于,所述第一流感病毒HA蛋白单克隆抗体和第二流感病毒HA蛋白单克隆抗体分别与流感病毒HA蛋白的不同表位结合。
21.根据权利要求20所述的检测试纸,其特征在于,所述第一流感病毒HA蛋白单克隆抗体的重链的氨基酸序列如SEQ ID NO:45,轻链的氨基酸序列如SEQ ID NO:47。
22.根据权利要求20所述的检测试纸,其特征在于,所述第二流感病毒HA蛋白单克隆抗体的重链的氨基酸序列如SEQ ID NO:46,轻链的氨基酸序列如SEQ ID NO:48。
23.一种如权利要求5所述的SARS-CoV-2病毒与流感病毒联合检测试纸的制备方法,其特征在于,所述方法包括以下步骤:
(1)制备第一和第二SARS-CoV-2 RBD蛋白单克隆抗体、第一和第二SARS-CoV-2 N蛋白单克隆抗体以及第一和第二流感病毒HA蛋白单克隆抗体;
(2)将样品垫和结合垫分别浸泡在样品垫处理液和结合垫处理液中,随后进行烘干处理;
(3)分别取第一SARS-CoV-2 RBD蛋白单克隆抗体、第一SARS-CoV-2 N蛋白单克隆抗体和第一流感病毒HA蛋白单克隆抗体,加入胶体金溶液中进行标记,随后喷涂到结合垫上,进行烘干处理,得到金标结合垫;
(4)分别取第二SARS-CoV-2 RBD蛋白单克隆抗体、第二SARS-CoV-2 N蛋白单克隆抗体、第二流感病毒HA蛋白单克隆抗体以及羊抗人单克隆抗体与包被缓冲液混合,并分别包被在硝酸纤维素膜的检测线T2、检测线T3、检测线T1和质控线上,随后进行烘干处理;
(5)将样品垫、金标结合垫、硝酸纤维素膜和吸水垫依次搭接并粘合在衬板上,得到所述SARS-CoV-2病毒与流感病毒联合检测试纸。
24.根据权利要求23所述的方法,其特征在于,步骤(2)所述样品垫处理液包括Tris-HCl、PVP-40、BSA或Triton X-100中的任意一种或至少两种的组合。
25.根据权利要求23所述的方法,其特征在于,步骤(2)所述结合垫处理液包括Tris-HCl、PVP-40、BSA或Tween-20中的任意一种或至少两种的组合。
26.根据权利要求23所述的方法,其特征在于,步骤(2)所述浸泡的时间为25~35 min。
27.根据权利要求23所述的方法,其特征在于,步骤(2)所述烘干的温度为35~40℃。
28.根据权利要求23所述的方法,其特征在于,步骤(2)所述烘干的时间为4~6 h。
29.根据权利要求23所述的方法,其特征在于,步骤(3)所述第一SARS-CoV-2 RBD蛋白单克隆抗体与胶体金溶液的配比为20~30μg:1mL。
30.根据权利要求23所述的方法,其特征在于,步骤(3)所述第一SARS-CoV-2 N蛋白单克隆抗体与胶体金溶液的配比为5~10μg:1mL。
31.根据权利要求23所述的方法,其特征在于,步骤(3)所述第一流感病毒HA蛋白单克隆抗体与胶体金溶液的配比为15~25μg:1mL。
32.根据权利要求23所述的方法,其特征在于,步骤(4)所述包被缓冲液包括Tris-HCl、PVP-40、BSA、Tween-20和NaN3中的任意一种或至少两种的组合。
33.根据权利要求23所述的方法,其特征在于,所述方法包括以下步骤:
(1)制备第一和第二SARS-CoV-2 RBD蛋白单克隆抗体、第一和第二SARS-CoV-2 N蛋白单克隆抗体以及第一和第二流感病毒HA蛋白单克隆抗体;
(2)将样品垫和结合垫分别在样品垫处理液和结合垫处理液中浸泡25~35 min,随后于35~40℃进行烘干处理4~6 h;
(3)按配比为20~30μg:1mL、5~10μg:1mL和15~25μg:1mL分别将第一SARS-CoV-2 RBD蛋白单克隆抗体、第一SARS-CoV-2 N蛋白单克隆抗体和第一流感病毒HA蛋白单克隆抗体加入胶体金溶液中进行标记,随后喷涂到结合垫上,于35~40℃、湿度为10%~30%条件下进行烘干处理4~6 h,得到金标结合垫;
(4)分别取第二SARS-CoV-2 RBD蛋白单克隆抗体、第二SARS-CoV-2 N蛋白单克隆抗体、第二流感病毒HA蛋白单克隆抗体以及羊抗人单克隆抗体与包被缓冲液混合,并分别包被在硝酸纤维素膜的检测线T2、检测线T3、检测线T1和质控线上,随后于35~40℃、湿度为10%~30%条件下进行烘干处理4~6 h;
(5)将样品垫、金标结合垫、硝酸纤维素膜和吸水垫依次搭接并粘合在衬板上,得到所述SARS-CoV-2病毒与流感病毒联合检测试纸。
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