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测总抗还是测亚型?新冠抗体检测试剂盒ELISA法的深度剖析

2020-05-19 16:29

抗体检测作为核酸检测的重要补充手段,能够用于临床诊断以提升新冠肺炎确诊的准确性,也可用于无症状或轻症感染者筛查,有助于新冠流行病学研究和社会复产复工的统筹规划。目前美国FDA已紧急授权12种新冠抗体检测试剂盒,其中胶体金法共3种,化学发光法共4种,ELISA共 5种(表1)。之前我们已介绍过胶体金法和化学发光法的检测效果(文尾可查看原文),今天我们就来看看ELISA方法的灵敏度和特异性究竟怎么样。


表1 获得美国FDA紧急使用授权的新冠抗体检测试剂盒


文献1解读

2020年3月3日,深圳市第三人民医院的张政教授团队与厦门大学夏宁邵教授团队合作在medRxiv预印平台上发表研究论文“Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019”[1],对由北京万泰生物药业股份有限公司(下称万泰生物)生产的三种ELISA抗体检测试剂盒进行了研究,文章要点总结如下:


1. 万泰生物生产的三种试剂盒原理分别为:

1)双抗原夹心法检测新冠病人血清中的总抗,使用的抗原蛋白是新冠S蛋白的RBD;

2)捕获法检测新冠病人血清中的IgM,使用的抗原蛋白是HRP标记的RBD;

3)间接法检测新冠病人血清中的IgG,使用的抗原蛋白是新冠N蛋白。


2. 对173例新冠确诊病人的血清样本进行检测,总抗、IgM和IgG试剂盒的灵敏度分别为93.1% (161/173) 、82.1% (142/173) 和64.7% (112/173) ;其中总抗试剂盒未检出的12例血清样本来自发病早期的病人。


3. 总抗、IgM和IgG试剂盒的特异性分别为99.1% (211/213),98.6% (210/213) 和99.0% (195/197) 。


4. 总抗、IgM和IgG试剂盒检出的阳性样本的发病中位时间分别为11天、12天和14天。


5. 发病7天内的病例样本中,核酸检测的灵敏度为66.7%,ELISA抗体检测的灵敏度只有38.3%;但在发病8-14天的病例样本中,核酸检测的灵敏度下降到54.0%,而ELISA抗体检测的灵敏度迅速提高,总抗、IgM和IgG试剂盒的灵敏度分别为89.6% 、73.3% 和54.1%;在发病15-39天的病例样本中,ELISA总抗、IgM和IgG试剂盒的灵敏度分别达到100.0% 、94.3% 和79.8%,核酸检测的灵敏度只有45.5%。


6.在新冠肺炎的不同发病时期,将核酸检测与ELISA总抗检测联合使用,能够大幅提升新冠肺炎的确诊准确率。


文献2解读


与上述研究的内容类似,来自丹麦的团队于2020年4月9日在medRxiv预印平台上发表题为“Evaluation of nine commercial SARS-CoV-2 immunoassays”[2]的论文,对商业化的新冠ELISA抗体检测试剂盒进行了评估,论文内容精简如下:


1. 研究人员使用已获得欧盟CE认证的新冠ELISA抗体检测试剂盒进行研究,试剂盒分别为由万泰生物生产的总抗试剂盒和由德国Euroimmun生产的IgA/IgG 试剂盒,Euroimmun的试剂盒原理为利用间接法检测新冠病人血清中的IgA/IgG,使用的抗原蛋白为新冠S蛋白S1亚基(Euroimmun生产的ELISA IgG试剂盒已获得了美国FDA的紧急使用授权,见表1)。


2.  研究人员根据万泰生物和Euroimmun的ELISA试剂盒产品中推荐的cut-off值对新冠阳性和阴性血清的实验检测数值进行分析,发现使用万泰生物的总抗试剂盒能够根据检测值将阳性血清和阴性血清有效区分开,而使用Euroimmun的IgA/IgG试剂盒,阳性血清和阴性血清的检测值区分度较小,因此,对部分血清样本而言,研究人员无法根据检测结果对新冠病毒感染情况进行准确判断(图1)。


图1 万泰生物和Euroimmun的ELISA试剂盒检测血清样本的区分度分析


3. 万泰生物的总抗试剂盒灵敏度为93% (28/30) , 特异性为100% (82/82) ;Euroimmun的IgA和IgG试剂盒灵敏度分别为93% (28/30) 和67% (20/30) ,特异性分别为93%(76/82),96% (79/82)。


4. Euroimmun的IgA 和IgG试剂盒会与含有其它呼吸道病毒的血清样本有交叉反应,如引起普通感冒的冠状病毒HKU1和引起呼吸道感染的腺病毒。


总结


上述由中国和丹麦学者发表的论文都对万泰生物的新冠ELISA总抗试剂盒进行了研究,两项研究的实验结果非常相近,相互支持彼此的结论。此外,两篇论文分别对万泰生物的新冠ELISA总抗试剂盒同万泰生物的IgM/IgG及Euroimmun的IgA/IgG试剂盒进行了比较,发现总抗试剂盒的灵敏度和特异性都好于抗体亚型试剂盒,说明新冠总抗检测可与核酸检测联用提高新冠肺炎确诊的准确率,也可独立使用进行新冠病毒感染病例的筛查。


万泰生物和Euroimmun抗体检测试剂盒中的N蛋白和S蛋白是新冠病毒的重要结构蛋白,目前N蛋白和S蛋白的S1亚基和RBD成为市场上新型冠状病毒抗体检测的主要抗原工具。


ACROBiosystems开发了包括His、Fc、mFc、His&Avi等不同标签的S1、N、RBD蛋白,以及多种突变类型的RBD蛋白,可用于胶体金法、化学发光法、ELISA中以助于新型冠状病毒的检测。目前ACRO已给多家开发新型冠状病毒检测试剂盒的公司提供了高质量的抗原蛋白,获得了一致好评。


货号:NUN-C51H9



产品:SARS-CoV-2 (COVID-19) Nucleocapsid protein, His Tag

(我们也有适用于血清学检测的该产品形式,点此联系。)

N蛋白与Anti-N蛋白抗体的结合活性验证灵敏度为0.02 ng/mL。

Immobilized SARS-CoV-2 Nucleocapsid protein, His Tag (Cat. No. NUN-C51H9) at 2 μg/mL (100 μL/well) can bind SARS-CoV-2 NP Antibody, Rabbit MAb (CLN27) with a linear range of 0.02-0.3 ng/mL.


申请Protocol


货号:S1N-C52H4



产品:SARS-CoV-2 (COVID-19) S1 protein, His Tag (MALS verified)

(我们也有适用于血清学检测的该产品形式,点此联系)

S1蛋白与ACE2蛋白的结合活性验证灵敏度为0.2 ng/mL。

Immobilized SARS-CoV-2 S1 protein, His Tag (Cat. No. S1N-C52H4) at 2 μg/mL (100 μL/well) can bind Human ACE2, Fc Tag (Cat. No.AC2-H5257) with a linear range of 0.2-6 ng/mL.


申请Protocol


货号:SPD-C52H3



产品:SARS-CoV-2 (COVID-19) S protein RBD, His Tag (MALS verified)

(我们也有适用于血清学检测的该产品形式,点此联系)

Immobilized Human ACE2, Fc Tag (Cat. No. AC2-H5257) at 2 μg/mL (100 μL/well) can bind SARS-CoV-2 S protein RBD, His Tag (Cat. No. SPD-C52H3) with a linear range of 0.008-0.125 μg/mL (QC tested).


申请Protocol


上文3个产品我们也有适用于血清学检测的该产品形式,点此可直接联系。


产品列表

(点击下方图片或文尾阅读原文

可查看在线产品更多信息及预购产品预计上线时间!)

kits  For research use only.
Molecule Cat. No. Species Product Description Protocol/Order
S protein RBD EP-105 SARS-CoV-2 SARS-CoV-2 Inhibitor Screening Kit

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S1 protein MBS-K001 SARS-CoV-2 SARS-CoV-2 Spike S1 protein-coupled magnetic beads

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S protein RBD MBS-K002 SARS-CoV-2 SARS-CoV-2 Spike protein RBD-coupled magnetic beads

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TAS-K003 SARS-CoV-2 Anti-SARS-CoV-2 neutralizing antibody titer serologic assay kit

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S1 protein SP-12 SARS-CoV-2 SARS-CoV2 Spike S1 protein-Coated Plates, Clear, 96-Well

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S protein RBD RP-13 SARS-CoV-2 SARS-CoV2 Spike protein RBD-Coated Plates, Clear, 96-Well

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S1 protein TAS-K001 SARS-CoV-2 SARS-CoV-2 antibody titer assay kit (Spike protein S1)

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Antibody
Molecule Cat. No. Species Product Description Protocol/Order
S protein RBD SAD-S35 Human Anti-SARS-CoV-2 neutralizing antibody (Human IgG1)

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Molecule Cat. No. Species Host Product Description Protocol/Order
ACE2 AC2-H82E6 Human HEK293 Biotinylated Human ACE2 / ACEH Protein, His,Avitag™ (MALS verified)

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AC2-H82F9 Human HEK293 Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag™

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AC2-H5257 Human HEK293 Human ACE2 / ACEH Protein, Fc Tag (MALS verified)

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AC2-C52H7 Cynomolgus HEK293 Cynomolgus ACE2 / ACEH Protein, His Tag

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AC2-H52H8 Human HEK293 Human ACE2 / ACEH Protein, His Tag (MALS verified)

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AC2-R5246 Rat HEK293 Rat ACE2 / ACEH Protein, His Tag (MALS verified)

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AC2-M5248 Mouse HEK293 Mouse ACE2 / ACEH Protein, His Tag (MALS verified)

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AC2-P5248 Paguma larvata HEK293 Paguma larvata ACE2 / ACEH Protein, His Tag

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S protein SPN-C52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein (R683A, R685A), His Tag

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SPN-C52H8 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein (R683A, R685A) , His Tag, active trimer (MALS verified)

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SPN-S52H5 SARS HEK293 SARS S protein (R667A), His Tag

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S1 protein S1N-C52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, His Tag

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S1N-C82E8 SARS-CoV-2 HEK293 Biotinylated SARS-CoV-2 (COVID-19) S1 protein, His,Avitag™ (MALS verified)

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S1N-S52H5 SARS HEK293 SARS S1 protein, His Tag (MALS verified)

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S1N-C52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, His Tag (MALS verified)

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S1N-C5255 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, Fc Tag

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S1N-C5257 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, Mouse IgG2a Fc Tag

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S protein RBD SPD-C82E9 SARS-CoV-2 HEK293 Biotinylated SARS-CoV-2 (COVID-19) S protein RBD, His,Avitag™ (MALS verified)

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SPD-C5255 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD, Fc Tag (MALS verified)

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SPD-S52H6 SARS HEK293 SARS S protein RBD, His Tag (MALS verified)

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SPD-C52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD, His Tag (MALS verified)

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SPD-C5259 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD, Mouse IgG2a Fc Tag

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SPD-S52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (V367F), His Tag

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SPD-S52H5 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (N354D), His Tag

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SPD-S52H7 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (W436R), His Tag

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SPD-S52H8 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (R408I), His Tag

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SPD-S52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (N354D, D364Y), His Tag

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Nucleocapsid protein NUN-C51H9 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Nucleocapsid protein, His Tag

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NUN-C5227 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) Nucleocapsid protein, His Tag

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NUN-C81Q6 SARS-CoV-2 E.coli Biotinylated SARS-CoV-2 (COVID-19) Nucleocapsid protein, His,Avitag™

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Envelope protein ENN-C5128 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Envelope protein, His Tag

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Papain-like Protease PAE-C5148 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Papain-like Protease Protein, His Tag

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NSP1 NS1-C51H7 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP1 Protein, His Tag

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NSP7 NS7-C51H6 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP7 Protein, His Tag

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NSP8 NS8-C5149 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP8 Protein, His Tag

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NSP7 & NSP8 NS8-C5125 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP7&NSP8 Protein, His Tag

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S1 protein S1N-C5256 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein (D614G), His Tag

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预计上线时间:6月5日

SIN-V52H3 HCoV-NL63 HEK293 HCoV-NL63 S1 protein, His Tag

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SIN-V52H4 HCoV-229E HEK293 HCoV-229E S1 protein, His Tag

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SIN-V52H5 HCoV-OC43 HEK293 HCoV-OC43 S1 protein, His Tag

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SIN-V52H6 HCoV-HKU1(isolate N5) HEK293 HCoV-HKU1(isolate N5) S1 protein, His Tag

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S1 protein CTD S1D-C52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein CTD, His Tag

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S1 protein NTD S1D-C52H6 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein NTD, His Tag

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S protein RBD SPD-C52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (G476S), His Tag

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SPD-C52H5 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (V483A), His Tag

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S2 protein S2N-C5253 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S2 protein, Fc Tag

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S2N-C5254 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S2 protein, Mouse IgG2a Fc Tag

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Envelope protein ENN-C51P3 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Envelope protein, His,BBP Tag

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NSP15 NS5-C5145 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP15 Protein, His Tag

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NSP16 & NSP10 NS0-C51W3 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP16&NSP10 Heterodimer Protein, His Tag&Twin Strep Tag

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预计上线时间:6月1日



>>>>更多新冠病毒蛋白和检测试剂盒信息请点击文尾阅读原文查看



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参考文献:

1. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019

2. Evaluation of nine commercial SARS-CoV-2 immunoassays

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