CN116217711A - 一种SARS-CoV-2检测方法和试剂盒 - Google Patents
一种SARS-CoV-2检测方法和试剂盒 Download PDFInfo
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Abstract
本发明涉及一种SARS‑CoV‑2检测方法和试剂盒。本发明提供SARS‑CoV‑2检测试剂盒,其包括用于检测来自受试者样品中SARS‑CoV‑2的抗体1和抗体2。本发明的方法和试剂盒可以提高检测灵敏度和检出率,尤其可以检出新冠变异株。
Description
技术领域
本发明属于蛋白检测领域。更具体地,涉及一种SARS-CoV-2的检测方法和试剂盒。
背景技术
冠状病毒是一种有囊膜的单股正链RNA病毒。根据冠状病毒的遗传学进化、血清学和宿主特异性差异,冠状病毒分为四个属:α-冠状病毒属、β-冠状病毒属、γ冠状病毒属和δ冠状病毒属。β冠状病毒属的病毒又可以分为A、B、C、D四个组群。α和β冠状病毒主要感染包括人类、家畜、宠物在内的哺乳动物,γ和δ冠状病毒主要感染鸟类及哺乳动物。新型冠状病毒属于β-冠状病毒属,从进化树位置上看,2019-nCoV与SARS和类SARS病毒的类群相邻,它们在进化上共同的外类群是一个寄生于果蝠的HKU9-1冠状病毒。2019-nCoV有包膜,颗粒呈圆形或椭圆形,常为多形性,直径50-200nm。
SARS-CoV-2属于冠状病毒科,为不分节段的单股正链RNA病毒。它编码两个大的重叠开放阅读框(ORF1a和ORF1b),4种结构蛋白(S、E、M和N蛋白),以及9种辅助蛋白。其中,N蛋白是病毒粒子的核心成分,它与病毒基因组RNA结合,将RNA包装成核糖核蛋白(RNP)复合体。除了组装外,N蛋白还在病毒mRNA转录和复制中发挥重要作用,并参与免疫调节。有研究报道N蛋白可以与双链RNA结合而具有RNAi抑制活性,可以对抗宿主RNAi介导的抗病毒反应,同时,N蛋白还可诱导感染后的体液和细胞免疫应答,使其成为早期快速诊断和疫苗研发的关键靶标。
SARS-CoV-2N蛋白全长419个氨基酸,大小为43-50kDa。N蛋白有三个相对保守的结构域,其中一域可与病毒基因组RNA相互缠绕形成病毒核衣壳,在病毒RNA的合成过程中发挥着重要的作用,与病毒基因组复制和调节细胞信号通路有关。N蛋白是一个磷酸化蛋白,磷酸化能够调节N蛋白的构象,增强与病毒蛋白的构象,增强与病毒RNA的亲和力。在核衣壳包装过程中,N蛋白可与M蛋白相互作用,促进核衣壳包装成病毒粒子。感染早期机体就能产生抗N蛋白的高水平抗体,可以利用N蛋白建立快速检测2019-nCoV血清抗体方法。因此N蛋白常作为冠状病毒诊断检测工具,是免疫学快速诊断试剂的核心原料。
发明内容
有鉴于此,本发明的目的是提供一种用于检测SARS-CoV-2N蛋白的检测方法和试剂盒。
在一些实施方案中,本发明可以包括下述一项或多项:
一种抗体对,其中,所述抗体对包括结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗体1和结合SARS-CoV-2核衣壳蛋白第96-103位氨基酸片段的抗体2。
本发明的另一目的是提供了一种SARS-CoV-2抗原检测试剂盒,其用于检测来自受试者样品中的SARS-CoV-2N蛋白,其中,其包括上述的抗体对。
本发明还提供了一种SARS-CoV-2免疫层析试纸条,其中,其包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上设置有T线和C线,所述结合垫上设置有抗体1,T线上包被有抗体2;或所述结合垫上设施有抗体2,T线上包被有抗体1。
本发明还提供了上述抗体对在制备检测SARS-CoV-2的试剂盒中的应用。
本发明的方法和试剂盒可以提高检测灵敏度和检出率,尤其可以检出新冠变异株。
具体实施方式
本发明涉及一种抗体对,其中,所述抗体对包括结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗体1和结合SARS-CoV-2核衣壳蛋白第96-103位氨基酸片段的抗体2。
在一些实施方式中,所述抗体1不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段;在一些实施方式中,所述抗体2结合SARS-CoV-2核衣壳蛋白第65-103位氨基酸片段、及第96-136位氨基酸片段。
在本发明中,术语“抗体”在最广义上使用,其可以包括全长单克隆抗体,双特异性或多特异性抗体,以及嵌合抗体,只要它们展示所需的生物学活性。术语“抗原结合片段”是包含抗体CDR的一部分或全部的物质,其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性,因为其结合至抗原,且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。此类片段选自Fab(由完整的轻链和Fd构成),Fv(由VH和VL构成),scFv(单链抗体,VH和VL之间由一连接肽连接而成)或单域抗体(仅由VH组成)。此类片段可通过重组核酸技术产生,或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。
在一些实施方式中,抗体结合SARS-CoV-2核衣壳蛋白对应的氨基酸片段是指抗体能够结合所述氨基酸片段,但该氨基酸片段不一定是最小结合片段。
在一些实施方式中,所述抗体1用于标记,抗体2用于包被;在一些实施方式中,抗体2用于标记,抗体1用于包被。
在一些实施方式中,所述用于标记的抗体用可检测标记物标记,例如金属粒子,如胶体金,荧光标记,发色团标记,电子致密标记,化学发光标记,放射性标记,酶标记,例如放射性同位素,荧光团,罗丹明,荧光素酶,荧光素,辣根过氧化物酶,碱性磷酸酶,β-半乳糖苷酶,葡糖淀粉酶,溶菌酶,糖类氧化酶,葡萄糖氧化酶,半乳糖氧化酶,葡萄糖-6-磷酸脱氢酶,生物素/抗生物素蛋白,自旋标记,噬菌体标记,例如吖啶酯标记,例如通过接头如生物素-亲和素添加荧光标记如吖啶酯标记。
在一些实施方式中,所述用于包被的抗体连接至固相,例如磁性颗粒,胶乳粒子,ELISA板,微量滴定板,硝酸纤维素膜或微流控芯片。
在一些实施方式中,可以使用任何适当的体外测定,基于细胞的测定,体内测定,动物模型等检测本发明抗体的效果如结合活性和/交叉反应性。在一些实施方式中,所述测定可以包括例如ELISA,FACS结合测定,Biacore,竞争性结合测定等。在一些实施方式中,例如在ELISA中表征本发明的抗体(或其抗原结合片段)与抗原(抗原肽)结合的反应性,例如通过过氧化物酶标记的ELISA法读取405nm处的反应值≥0.5确定为有较好的反应性,可用于免疫测定。
在一些实施方式中,本发明还提供了一种SARS-CoV-2抗原检测试剂盒,其用于检测来自受试者样品中的SARS-CoV-2N蛋白,其中,其包括上述的抗体对。
在一些实施方式中,所述试剂盒是免疫层析试纸条,酶联免疫试剂或化学发光试剂。
在一些实施方式中,所述样品包括健康或病理状态的生物组织、细胞或体液,例如唾液、鼻咽拭子。
在一些实施方式中,本发明还提供了一种SARS-CoV-2免疫层析试纸条,其特征在于,所述试纸条包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上设置有T线和C线,所述结合垫上设置有抗体1,T线上包被有抗体2;或所述结合垫上设施有抗体2,T线上包被有抗体1。
本发明还提供了上述抗体对在制备检测SARS-CoV-2的试剂盒中的应用。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1新冠N蛋白单克隆抗体的制备
1、免疫动物
取8~12周龄与骨髓瘤细胞同种系的BALB/c小鼠,以含蛋白质100μg/只的2019-nCoV N蛋白重组抗原与等量福氏完全佐剂充分混匀,注入小鼠腹腔内,每隔2周100μg/只的2019-nCoV N蛋白重组抗原与等量福氏不完全佐剂充分混匀,多次注入小鼠腹腔内加强免疫。经检测小鼠血清(间接ELISA法),滴度在1∶2000以上者可用于融合,融合前3天经小鼠腹腔内再次加强免疫,剂量为50μg/只。
2、饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天,BALB/c鼠拉颈处死,75%酒精全身浸泡,超净台内,无菌操作下用剪刀剪开腹部皮肤,暴露腹膜,用注射器腹腔注入RPMI 1640基础培养液5mL,反复冲洗,回收冲洗液,1000rpm,离心5分钟,留沉淀,用RPMI1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬,调整细胞浓度1×105个/mL,加入96孔板,150μL/孔,37℃,5%CO2培养过夜。
3、免疫脾细胞的制备
小鼠末次免疫后三天,在无菌条件下取出脾脏,置于平皿中,RPMI 1640基础培养液冲洗一次,放于小烧杯的尼龙网上磨碎过滤,制成细胞悬液。离心,弃上清,RPMI 1640基础培养液重悬,如此重复三次,计数。
4、细胞融合
(1)取40mL HAT培养液,15mL DMEM无血清培养液和1mL50%PEG(M12000)分别置于37℃水浴中预温;
(2)分别取小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)(2~5×107)、上述免疫脾细胞(108)悬液加入50mL离心管中混匀,并加DMEM无血清培养液至40mL。离心10分钟,倒尽上清液,混匀;
(3)将离心管置于37℃预温的水中,取0.7mL预温的50%PEG溶液,静置90秒钟。立即滴加37℃15mL预温的无血清培养液;
(4)补加DMEM无血清培养液至40mL,离心10分钟,倒尽上清液。加40mL含15%~20%胎牛血清的HAT培养液。用吸管混匀,滴加到已含有饲养细胞的4块96孔细胞培养板的小孔中,每孔2滴,置37℃、7%CO2的培养箱中培养。
5、杂交瘤细胞的选择培养
免疫小鼠脾细胞与小鼠骨髓瘤细胞,经PEG处理后,形成多种细胞成分的混合体,其中包括未融合的骨髓瘤细胞和免疫脾细胞;骨髓瘤细胞的共核体和免疫脾细胞的共核体,以及骨髓瘤细胞和免疫脾细胞的异核体。仅后者才能形成杂交瘤细胞。为此,在这多种细胞混合体中必须除去未融合的细胞和同种融合的共核体,并选择出真正的杂交细胞。因此,在细胞融合后第1,3,5,7天用前述的HAT培养液换液培养。
6、特异性抗体的检测及杂交瘤细胞克隆化
吸取每个培养孔的上清液,用间接ELISA法检测出培养液中含特异性识别2019-nCoV N蛋白重组抗原的抗体的培养孔。采用间接ELISA法鉴定细胞培养上清的交叉反应性,用2019-nCoV N蛋白重组抗原包被96孔板,封闭,加入杂交瘤细胞培养液上清孵育,加二抗,测405nm反应值,挑选反应值(0.5以上)比较高的细胞株制备抗体进行下一轮筛选实验。筛选得到以下有较好反应性的抗体。
实施例2抗体结合片段的鉴定
采用不同片段的2019-nCoV N蛋白重组抗原分别包被微孔,以PBS+20%NBS为稀释液,稀释单抗为一抗浓度到1ug/ml,以羊抗鼠IgG-HRP为二抗,依据各单抗对不同抗原的反应情况来确定单抗片段。经统计,筛选得到的抗体分别是针对如下片段:
表1
其中结合44-180aa片段的十一株抗体对片段的反应情况摘取如表2:
表2
抗体 | 6I3 | 4C9 | 5C3 | 1A6 | 1E5 | 9D2 | 8E4 | 6K1 | 6A5 | 2S1 | 6B7 |
反应性 | 2.362 | 2.491 | 2.493 | 2.486 | 2.528 | 2.616 | 2.941 | 2.949 | 2.444 | 2.718 | 2.628 |
表2中反应性即OD405反应值。
实施例3优势抗体进一步片段鉴定
将2019-nCoV N蛋白重组抗原的44-180aa片段进行小片段表达,采用不同片段的2019-nCoV N蛋白重组抗原分别包被微孔,以PBS+20%NBS为稀释液,稀释单抗为一抗浓度到1ug/ml,以羊抗鼠IgG-HRP为二抗,依据各单抗对不同抗原的反应情况来确定单抗片段,具体数据如下:
表3-1
抗体编号 | 6I3 | 9D2 | 4C9 | 5C3 | 1A6 | 1E5 |
44-180 | 2.362 | 2.616 | 2.491 | 2.493 | 2.486 | 2.528 |
44-84 | 0.034 | 0.032 | 0.070 | 0.059 | 0.020 | 0.018 |
65-103 | 0.067 | 2.819 | 0.037 | 0.035 | 0.017 | 0.019 |
96-136 | 0.012 | 0.015 | 0.017 | 0.016 | 0.008 | 0.014 |
130-180 | 0.028 | 0.012 | 0.064 | 0.044 | 0.013 | 0.015 |
抗体识别区段 | 44-180 | 65-103 | 44-180 | 44-180 | 44-180 | 44-180 |
表3-2
从以上结果来看,2S1、6B7两株抗体结合65-103aa和96-136aa,可见其识别的表位为96-103aa;9D2结合65-103aa且不结合96-136aa和44-84aa,可见其识别的表位为85-95aa;其余抗体均只识别44-180aa,且不识别44-84aa,65-103aa,96-136aa,130-180aa。
表3-3
抗体 | 2S1 | 6B7 |
反应性 | 2.615 | 2.440 |
表3-3中反应性为抗体与96-103aa的反应值。
实施例4优势抗体配对性能进一步验证
将以上抗体分别用于包被以及标记,实验过程如下:
1、新冠N抗体标记:取5ml浓度为4/万的胶体金,加入30-40ul 0.2M K2CO3,搅拌5min,加入新冠N标记抗体(所加抗体体积=50μg/抗体浓度),搅拌5min,再加入50ul 10%BSA封闭终止标记;离心10000rpm,7min,去上清,沉淀用金子复溶液复溶,最后用金子复溶液定容到0.5ml(即1/10胶体金溶液体积)。
2、配制金子工作液:用金子复溶液将新冠N标记抗体浓缩金以20%的稀释比例配制成金子工作液,铺于玻璃纤维上。
3、制备干燥好的金子:将铺好的金子放入冻干机冻干(1-2h)或者放37℃干燥房干燥过夜。
4、新冠N抗体包被:将硝酸纤维素膜与PVC底板组装好备用;将新冠N包被抗体稀释至1.0-1.5mg/ml,用喷金画膜仪在NC膜上均匀地划线,放37℃恒温箱60min。
5、制备金标条:用切条机将金标条按需要的宽度切条,组装后加样进行检测。
6、检测:根据色卡比对,肉眼判读显色读值。
7、结果
(1)活性:以下示例配对对大肠杆菌表达的重组抗原及病毒上清液的反应性。
表4
(2)特异性:
配对b对采集的180例正常人鼻拭子、180例正常人唾液标本进行胶体金显色测试,特异性均为100%。
(3)检测突变株:
使用配对b检测主流突变株如下表。
表5
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种抗体对,其特征在于,所述抗体对包括结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗体1和结合SARS-CoV-2核衣壳蛋白第96-103位氨基酸片段的抗体2。
2.如权利要求1所述的抗体对,其特征在于,所述抗体1不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段;
可选地,所述抗体2结合SARS-CoV-2核衣壳蛋白第65-103位氨基酸片段、及第96-136位氨基酸片段。
3.如权利要求1所述的抗体对,其特征在于,所述抗体1用于标记,抗体2用于包被;或抗体2用于标记,抗体1用于包被。
4.如权利要求3所述的抗体对,其特征在于,所述用于标记的抗体用可检测标记物标记,例如金属粒子,如胶体金,荧光标记,发色团标记,电子致密标记,化学发光标记,放射性标记,酶标记,例如放射性同位素,荧光团,罗丹明,荧光素酶,荧光素,辣根过氧化物酶,碱性磷酸酶,β-半乳糖苷酶,葡糖淀粉酶,溶菌酶,糖类氧化酶,葡萄糖氧化酶,半乳糖氧化酶,葡萄糖-6-磷酸脱氢酶,生物素/抗生物素蛋白,自旋标记,噬菌体标记,例如吖啶酯标记,例如通过接头如生物素-亲和素添加荧光标记如吖啶酯标记;
可选地,所述用于包被的抗体连接至固相,例如磁性颗粒,胶乳粒子,ELISA板,微量滴定板,硝酸纤维素膜或微流控芯片。
5.如权利要求1所述的抗体对,其特征在于,其中所述抗体的反应性为ELISA评价OD405≥0.5。
6.一种SARS-CoV-2抗原检测试剂盒,其用于检测来自受试者样品中的SARS-CoV-2N蛋白,其特征在于,所述试剂盒包括如权利要求1-5所述的抗体对。
7.如权利要求6所述的试剂盒,其特征在于,所述试剂盒是免疫层析试纸条,酶联免疫试剂或化学发光试剂。
8.如权利要求6或7所述的试剂盒,其特征在于,所述样品包括健康或病理状态的生物组织、细胞或体液,例如唾液、鼻咽拭子。
9.一种SARS-CoV-2免疫层析试纸条,其特征在于,所述试纸条包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上设置有T线和C线,所述结合垫上设置有抗体1,T线上包被有抗体2;或所述结合垫上设施有抗体2,T线上包被有抗体1。
10.权利要求1-5所述的抗体对在制备检测SARS-CoV-2的试剂盒中的应用。
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