CN114544952A - 一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒 - Google Patents
一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒,包括新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板、稀释液、洗涤液、酶标二抗、底物液、终止液、阴性标准品、阳性标准品;该试剂盒中以新型冠状病毒N蛋白、S1蛋白和M蛋白按照特定的比例复配的抗原来包被酶标反应板,可用于快速检测血清中新型冠状病毒IgG抗体并对IgG抗体含量进行定量计算,其灵敏度高,特异性强。
Description
技术领域
本发明属于生物技术领域,具体涉及一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒。
背景技术
新型冠状病毒的传染源主要是新型冠状病毒感染患者,包括无症状感染者。新型冠状病毒受感染患者通常表现为肺炎样症状如发热、干咳和呼吸困难等,和腹泻等胃肠道症状,其次为严重急性期呼吸道感染,部分病例会出现急性呼吸窘迫症合并严重呼吸道并发症,甚至导致死亡。目前,新型冠状病毒感染患者没有特定的治疗方法,早期诊断并及时管控是阻止疫情进一步播散和控制新感染线索的关键。因此,加强疫情监控,及时筛查并确诊新型冠状病毒感染者是首要任务。
病毒的实验诊断主要包括分离培养、核酸检测技术和免疫学抗原抗体检测技术。病毒分离培养是病毒学检测的“金标准”,该法特异性较强,但其费时长,一般至少需1-2周才能观察到病变,且具有实验室生物安全要求高,操作复杂的特点。免疫学抗体检测技术的原理是在病毒感染过程中,人体免疫系统中会产生相应抗体,病毒特异性IgM抗体是人体免疫系统中最先出现的抗体,一般在感染后一周内出现,病毒特异性IgG抗体在感染后2~4周内出现,IgM抗体是近期感染的标志,IgG抗体是既往感染的标志。免疫学抗原抗体检测技术可以准确快速的检测出新型冠状病毒感染患者及无症状感染者。
S蛋白是新型冠状病毒非常重要的表面蛋白,参与病毒感染敏感细胞以及膜融合过程感染宿主细胞,S蛋白包含S1、S2和受体结合域(RBD)。N蛋白在冠状病毒中含量丰富,是一种高度免疫原性蛋白,参与基因组复制和细胞信号通路调节。M蛋白是病毒包膜的组成部分,参与病毒颗粒的组装和释放。
目前,现有技术虽然公开了一些新型冠状病毒的IgG型抗体免疫检测试剂盒,但是,市售的ELISA试剂盒一般用N蛋白、S蛋白、M蛋等单一抗原包被,检测单一抗原相应的IgG抗体,灵敏性和特异性较低。因此,由于2019-nCov-SARS的特殊性质,亟需研发一种特异性好、灵敏度高的新型冠状病毒ELISA检测试剂盒。
发明内容
本发明的目的在于提供一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒,该试剂盒中以新型冠状病毒N蛋白、S1蛋白和M蛋白按照特定的比例复配的抗原来包被酶标反应板,可用于快速检测血清中新型冠状病毒IgG抗体并对IgG抗体含量进行定量计算,其灵敏度高,特异性强。
为实现上述目的,本发明采取的技术方案如下:
一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒,包括新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板、稀释液、洗涤液、酶标二抗、底物液、终止液、阴性标准品、阳性标准品。
所述新型冠状病毒IgG型抗体ELISA定量检测试剂盒中还包括封孔膜。
所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原中,新型冠状病毒N蛋白、S1蛋白和M蛋白的质量比为1.5~2:1.5~2:1,优选为2:2:1;所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原的包被量为0.5~1μg/孔,优选为1μg/孔。
所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板的制备方法包括以下步骤:
(1)将新型冠状病毒N蛋白、S1蛋白和M蛋白混合,得到复配抗原;
(2)复配抗原经包被液稀释后包被酶标板中的微孔,孵育,洗涤;
(3)向微孔中加入稳定缓冲液,孵育,干燥;
(4)向微孔中加入封闭液封闭,即可得到所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板。
所述稀释液为含10%牛血清蛋白和0.1%ProClin300的0.01M pH7.4的PBS缓冲液;
所述洗涤液为含有0.05%Tween 20的0.01M pH7.4的PBST缓冲液。
所述酶标二抗为辣根过氧化物酶标记的兔抗人IgG单抗;所述酶标二抗的滴度为1:5000。
所述底物液为TMB-H2O2尿素溶液;
所述终止液为2mol/L H2SO4溶液。
所述阳性标准品为含人血清抗新型冠状病毒IgG,使用前加入样本稀释液溶解。
所述阴性标准品为含未感染新型冠状病毒人血清IgG,使用前加入样本稀释液溶解。
所述阳性标准品为人血清抗新型冠状病毒IgG标准品,所述人血清抗新型冠状病毒IgG标准品的制备方法为:将10份感染了新型冠状病毒的阳性血清混合,经Protein A管柱亲和层析纯化后浓缩至终浓度为1μg/ml,再经冷冻干燥。
所述阴性标准品的制备方法为:将10份未感染新型冠状病毒血清混合,经ProteinA管柱亲和层析纯化浓缩至终浓度为1μg/ml,再经冷冻干燥。
本发明还提供了一种新型冠状病毒IgG型抗体的检测方法,使用权利要求本发明所述的试剂盒进行检测。
所述试剂盒的使用方法或新型冠状病毒IgG型抗体的检测方法包括以下步骤:
A、用稀释液将血浆或血清样本稀释200倍充分混匀后得到样本液;
B、分别向酶标板的孔中加入100μl稀释液、标准液、阴性对照液、阳性对照液、样本液分别作为空白组、标准组、阴性对照组、阳性对照组、实验组,并做复孔实验,然后用封孔膜封板后,在37℃下孵育30分钟;
C、用蒸馏水将稀释液稀释十倍,然后每孔用稀释的洗涤液300μl冲洗3遍;
D、用稀释液将酶标二抗稀释100倍,各孔分别加入100μl稀释后的酶标二抗,并重复步骤C进行洗涤;
E、各孔加入100μl底物液,20~25℃闭光孵育20~30min;
F、各孔加入100μl终止液,30分钟内,在450nm波长处用酶标仪测定各孔的吸光度值。
本发明试剂盒的检测结果与广州万孚公司新型冠状病毒IgG抗体ELISA检测试剂盒的一致性高,灵敏度为95%,特异度为99.5%
具体实施方式
本发明所涉及的各试剂如下:
包被液(0.05mol/L,pH9.6的碳酸钠-碳酸氢钠缓冲液):Na2CO3 1.5g,NaHCO32.9g,Na2N3 0.2g,加双蒸水至1000ml,调至pH9.45-9.75。
包被稳定液(含1%酪蛋白,5%蔗糖的水溶液):蔗糖5g、酪蛋白1g,加双蒸水至1000ml溶解,调节PH值至7.15-7.25。
封闭液:牛血清白蛋白1g,1×PBS(pH7.4)100ml。
稀释液(含1%牛血清白蛋白的0.01M PBS磷酸盐缓冲液pH7.4),PBS:NaCl 8.0g、KH2PO4 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g,硫柳汞0.1g,防腐剂ProClin300 1g,加双蒸水至1000ml,调至pH7.4,即得。
洗涤液(PBST,pH7.4):NaCl 8.0g、KH2PO4 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween 20 0.5ml、硫柳汞0.1g、加双蒸水至1000ml,调至pH7.4。
酶标二抗:HRP标记鼠抗人IgG单抗,购自abcam公司。
底物液(TMB-H2O2尿素溶液):
①底物液A(3、3‘、5、5‘-四甲基联苯胺,TMB):TMB200mg,无水乙醇100ml,加双蒸水至1000ml。
②底物液B缓冲液(0.1mol/L柠檬酸-0.2mol/L磷酸氢二钠缓冲液,pH5.0~5.4):Na2HPO4 14.60g,柠檬酸9.33g,0.75%过氧化氢尿素6.4ml,加双蒸水至1000ml,调至pH5.0~5.4。
③将底物液A和B按1:1混合,即成TMB-H2O2尿素溶液。
终止液:(2mol/L H2SO4溶液):双蒸水600ml,浓硫酸100ml,加双蒸水至900ml。
人血清抗新型冠状病毒IgG标准品制备:将10份感染了新型冠状病毒的阳性血清混合,经Protein A管柱亲和层析纯化后浓缩至终浓度为1μg/ml,定为1AU单位,纯化时所使用洗脱液为0.1M pH值7.4的PBS缓冲液;按照每瓶1.5ml体积加入到3ml西林瓶中,冷冻干燥48小时,得到标准品;其在使用时加入1.5mL稀释液溶解作为阳性标准液;
阴性标准品制备:将10份未感染新型冠状病毒血清混合,经Protein A管柱亲和层析纯化后浓缩至终浓度为1μg/ml,定为1AU单位,纯化时所使用洗脱液为0.1M pH值7.4的PBS缓冲液;按照每瓶1.5ml体积加入到3ml西林瓶中,冷冻干燥48小时,得到标准品;其在使用时加入1.5mL稀释液溶解作为阴性标准液;
新型冠状病毒N蛋白:购自北京义翘神州科技股份有限公司(货号40588-V07E)。
新型冠状病毒S1蛋白:购自北京义翘神州科技股份有限公司(货号40591-V08H)。
新型冠状病毒M蛋白:购自abcam公司(货号ab286080)。
下面结合实施例对本发明进行详细说明。
实施例1
新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板的制备方法,包括以下步骤:
(1)将新型冠状病毒N蛋白、S1蛋白和M蛋白按照2:2:1的质量比混合,得到复配抗原;
(2)复配抗原经包被液稀释至浓度为100μg/ml后包被酶标板中的微孔,每孔100μl,然后在2-8℃孵育过夜,之后每孔加入300μl洗涤液进行清洗,反复清洗2次;
(3)向微孔中加入100μl包被稳定液,在18-25℃条件下孵育2-4小时,然后抽干;
(4)向微孔中加入200μl封闭液,37℃封闭2小时,即可得到所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板。其用铝箔袋包装,并在袋中放入干燥剂。包装后储存在2-8℃的冷库中。
实施例2
包被抗原的包被量及复配组合优化
(1)取96孔酶标板一块;将新型冠状病毒N蛋白、S1蛋白和M蛋白按照不同的质量比复配抗原,用包被液将复配抗原分别稀释至100μg/ml和10μg/ml,每孔100μl;37℃温箱过夜烘干;取过夜烘干的抗原包被板加200μl封闭液37℃封闭1.5小时,得到梯度稀释抗原包被酶标反应板;
(2)将阴性标准品、阳性标准品用稀释液按质量体积1g:100mL稀释,每孔加入100μl,在37℃下孵育30分钟,用蒸馏水将稀释液稀释十倍即100ml洗涤液中加入900ml蒸馏水,然后每孔用稀释的洗涤液300μl冲洗3遍;
(3)用稀释液将酶标二抗稀释100倍即40μl酶标二抗+3960μl稀释液,各孔分别加入100μl稀释后的酶标二抗,在37℃下孵育30分钟,重复步骤(2)中的洗涤步骤;
(4)各孔加入100μl底物液,室温闭光孵育30min;各孔加入100μl终止液后在酶标仪上读取450nm波长处吸光度,即A值,
(5)选择标准品的A值为1.8~2.0间、阴性对照品的A值小于0.2的包被抗原的稀释度作为最适滴度,选择A值最大的特定比例的复配抗原作为包被抗原,可见最适包被酶标反应板的复配抗原组合为质量比为2:2:1的新型冠状病毒N蛋白、S1蛋白和M蛋白组合,最适抗原包被量为100μg/ml,每孔100μl。
表1复配抗原比例示例
从表1中的抗原包被筛选结果发现,新型冠状病毒S1、N和M包被与单一或两个抗原包被比较,其敏感性更高。
实施例3
酶标二抗最适滴度的选择
a)取96孔酶标板一块,用100ng/ml人IgG 100μl/孔4℃包被过夜,洗涤液洗板3次,350μl/孔,甩干;
b)将HRP标记鼠抗人IgG单抗用稀释液作一系列稀释后分别加入已包被的孔中,100μl/孔,37℃孵育40min,后用洗板3次,350μl/孔,甩干;
c)加底物显色,每孔加入底物液100μl,37℃或室温避光显色15分钟;后加入终止液50μl终止反应,在酶标仪上读取450nm波长处吸光度,即A值,取A值在1.0时的酶标抗体稀释度,作为酶标二抗的最适滴度,其最适滴度为1:5000。
实施例4
一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,包括按照实施例2中的方法制备的新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板、稀释液、洗涤液、酶标二抗、底物液、终止液、阴性标准品、阳性标准品。
并利用该试剂盒定量检测新型冠状病毒IgG型抗体,具体步骤如下:
A、用稀释液将血浆或血清样本稀释200倍即5μl样本+995μl稀释液,充分混匀后得到样本液;
B、分别向酶标板的孔中加入100μl稀释液、标准液、阴性标准液、阳性标准液、样本液分别作为空白组、阳性标准品、阴性标准品、实验组,并做复孔实验,然后用封孔膜封板后,在37℃下孵育30分钟;
C、用蒸馏水将稀释液稀释十倍即100ml洗涤液中加入900ml蒸馏水,然后每孔用稀释的洗涤液300μl冲洗3遍,倒置微孔板于干净的吸水纸上叩击数次;
D、用稀释液将酶标二抗稀释100倍即40μl酶标二抗+3960μl稀释液,各孔分别加入100μl稀释后的酶标二抗,在37℃下孵育30分钟;并重复步骤C进行洗涤;
E、各孔加入100μl底物液,当底物液加入第一板条后开始计时,室温闭光孵育30min;
F、各孔加入100μl终止液;
G、30分钟内,在450nm波长处用酶标仪测定各孔的吸光度值,结果如表1所示:
表2各组吸光值
计算空白组、标准组、阴性对照组和实验组各复孔的平均吸光度值(A),各组的平均吸光度值=测试的各复孔平均吸光值-空白组的平均吸光值。即实验组S1、S2和S3的平均吸光值分别为1.914、1.922和1.894
实施例5
新型冠状病毒IgG型抗体ELISA定量检测试剂盒的特异性实验
将新型冠状病毒标准IgG阳性血清、乙型脑炎病毒IgG阳性血清、麻疹病毒IgG阳性血清、风疹病毒IgG阳性血清、人巨细胞病毒IgG阳性血清和新型冠状病毒标准IgG阴性血清按照实施例2中实验组的测试方法采用本发明实施例2中的新型冠状病毒IgG型抗体ELISA定量检测试剂盒进行检测,新型冠状病毒标准IgG阳性血清、乙型脑炎病毒IgG阳性血清、麻疹病毒IgG阳性血清、风疹病毒IgG阳性血清、人巨细胞病毒IgG阳性血清的P/N值分别为6.23、1.22、1.15、1.27和1.06,说明该试剂盒特异性良好。
上述参照实施例对一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒进行的详细描述,是说明性的而不是限定性的,可按照所限定范围列举出若干个实施例,因此在不脱离本发明总体构思下的变化和修改,应属本发明的保护范围之内。
Claims (10)
1.一种新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,包括新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板、稀释液、洗涤液、酶标二抗、底物液、终止液、阴性标准品、阳性标准品。
2.根据权利要求1所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原中,新型冠状病毒N蛋白、S1蛋白和M蛋白的质量比为1.5~2:1.5~2:1;所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原的包被量为0.5~1μg/孔。
3.根据权利要求1或2所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板的制备方法包括以下步骤:
(1)将新型冠状病毒N蛋白、S1蛋白和M蛋白混合,得到复配抗原;
(2)复配抗原经包被液稀释后包被酶标板中的微孔,孵育,洗涤;
(3)向微孔中加入稳定缓冲液,孵育,干燥;
(4)向微孔中加入封闭液封闭,即可得到所述新型冠状病毒N蛋白、S1蛋白和M蛋白复配抗原包被的酶标反应板。
4.根据权利要求1或2所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述稀释液为含10%牛血清蛋白和0.1%ProClin300的0.01M pH7.4的PBS缓冲液;所述洗涤液为含有0.05%Tween 20的0.01M pH7.4的PBST缓冲液。
5.根据权利要求1或2所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述酶标二抗为辣根过氧化物酶标记的兔抗人IgG单抗;所述酶标二抗的滴度为1:5000。
6.根据权利要求1或2所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述底物液为TMB-H2O2尿素溶液。
7.根据权利要求1或2所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述终止液为2mol/L H2SO4溶液。
8.根据权利要求1或2所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述阴性标准品为含未感染新型冠状病毒IgG血清,使用前加入样本稀释液溶解;所述阳性标准品为含人血清抗新型冠状病毒IgG,使用前加入样本稀释液溶解。
9.根据权利要求1或2所述的新型冠状病毒IgG型抗体ELISA定量检测试剂盒,其特征在于,所述阳性标准品的制备方法为:将10份感染了新型冠状病毒的阳性血清混合,经Protein A管柱亲和层析纯化后浓缩至终浓度为1μg/ml,再经冷冻干燥;所述阴性标准品的制备方法为:将10份未感染新型冠状病毒血清混合,经Protein A管柱亲和层析纯化浓缩至终浓度为1μg/ml,再经冷冻干燥。
10.一种新型冠状病毒IgG型抗体的检测方法,其特征在于,使用权利要求1-9任意一项所述的试剂盒进行检测。
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