Study of Fluorescence Immunochromatography Based on Quantum Dots for the Detection of SARS-CoV-2 anti-N Protein IgG Antibody

doi:10.12114/j.issn.1007-9572.2022.0166

Chinese General Practice ›› 2022, Vol. 25 ›› Issue (14): 1741-1749.DOI: 10.12114/j.issn.1007-9572.2022.0166

Special Issue: 新型冠状病毒肺炎最新文章合集

• Article·New Diagnostic and Therapeutic Techniques • Previous Articles Next Articles

Study of Fluorescence Immunochromatography Based on Quantum Dots for the Detection of SARS-CoV-2 anti-N Protein IgG Antibody

1. The PLA Military Medical Research Institution, Beijing 100850, China
2. The PLA Center for Disease Control and Prevention, Beijing 100070, China
3. Beijing Hongjinjiu'an Biotechnology Company, Beijing 102299, China

Received:2022-02-23 Revised:2021-03-17 Published:2022-03-31 Online:2022-04-07
Contact: Tao YU, Wei LIU, Jinpeng GUO
About author:
YU R F, ZOU X C, ZOU D Y, et al. Study of fluorescence immunochromatography based on quantum dots for the detection of SARS-CoV-2 anti-N Protein IgG antibody[J]. Chinese General Practice, 2022, 25 (14) : 1741-1748.

基于量子点标记技术的免疫层析法检测新型冠状病毒N蛋白IgG抗体研究

1. 100850　北京市，中国人民解放军军事医学研究院
2. 100070　北京市，中国人民解放军疾病预防控制中心
3. 102299　北京市，北京弘进久安生物科技有限公司

通讯作者: 余涛, 刘威, 郭金鹏
作者简介:
于仁峰，邹小仓，邹大阳，等.基于量子点标记技术的免疫层析法检测新型冠状病毒N蛋白IgG抗体研究[J].中国全科医学，2022，25（14）：1741-1748. [www.chinagp.net] 作者贡献：于仁峰进行文章的构思与设计，统计学处理，撰写论文；邹大阳、余涛、刘威进行研究的实施与可行性分析；徐雅晴、秦日辉进行数据收集；于仁峰、莫冬冬、段佳慧参与试纸条的制作；于仁峰、邹小仓、贺晓明进行数据整理；于仁峰、李琳昊、王可慧进行结果的分析与解释；王可慧进行论文的修订；刘威负责文章的质量控制及审校；刘威、郭金鹏对文章整体负责，监督管理。

Abstract

Abstract:

Background

Based on the current prevalence of Coronavirus Disease 2019 (COVID-19) , early diagnosis, isolation, and treatment are important methods to prevent and control infectious diseases. The establishment of convenient and efficient immunochromatographic detection techniques is essential for the prevention and control of COVID-19 epidemic.

Objective

To establish a method for the detection of SARS-CoV-2 anti-N protein IgG antibody by immun of luorescence chromatography method based on quantum dots labeling technology in August, 2020. In order to determine whether the detected persons had been infected with COVID-19 or been injected with SARS-CoV-2 inactivated vaccine.

Methods

The prepared rat anti-human secondary antibody and anti-N protein antibody were immobilized on a Nitrocellulose (NC) membrane as detection line (T) and quality control line (C) , respectively. Then the SARS-CoV-2 N protein labeled by quantum dots was evenly sprayed on glass fiber, which was assembled, cut and packaged into test strips after drying. The test strips were used to detect the clinical serum of 35 COVID-19 patients and 50 healthy individuals, the results of the initial screening of the ELISA kit were used as a control to calculate the detection specificity and sensitivity of quantum dots fluorescence immunochromatography. The sensitivity of the test strip was detected by using the N protein antibody standard.

Results

The specificity and sensitivity of the strip were 100.00%, 94.29%, and the susceptibility was 8.53-17.06 ng/ml antibody concentration.

Conclusion

The detection of anti-N protein IgG antibody in serum by quantum dots labeling is simple, fast, with strong sensitivity and specificity.

Key words: COVID-19, SARS-CoV-2, Fluorescence immunochromatography, Quantum dots, IgG antibody, Nucleocapsid (N) protein

摘要：

背景

基于目前新型冠状病毒（SARS-CoV-2）引起的新型冠状病毒肺炎（COVID-19），早诊断、早隔离、早治疗是防控传染的重要方法。建立方便快捷的免疫层析检测技术，对于防控SARS-CoV-2的传播十分必要。

目的

建立基于量子点标记技术的免疫荧光层析法检测抗SARS-CoV-2 N蛋白IgG抗体的方法，判断被检测者是否感染过SARS-CoV-2或者是否接种过SARS-CoV-2灭活疫苗。

方法

2020年8月将制备好的鼠抗人二抗和抗N蛋白抗体固定至硝酸纤维素（NC）膜上，分别作为检测线（T）和质控线（C），然后将量子点标记的SARS-CoV-2 N蛋白均匀喷洒在玻璃纤维上，干燥后组装、切割、包装成试纸条。用该试纸条分别检测35例COVID-19患者和50例健康人的血清，以酶联免疫吸附测定法（ELISA）试剂盒初筛结果作为对照，计算量子点荧光免疫层析法的检测特异度和灵敏度，并利用N蛋白抗体标准品检测试纸条灵敏度。

结果

本研究制备的试纸条特异度为100.00%，灵敏度为94.29%，灵敏性为8.53~17.06 ng/ml抗体浓度。

结论

采用量子点标记技术检测血清中的抗N蛋白IgG抗体，操作简单、快速，灵敏度及特异度强。

关键词: 新型冠状病毒肺炎, 新型冠状病毒, 免疫层析法, 量子点, IgG抗体, N蛋白

Renfeng YU,Xiaocang ZOU,Dayang ZOU, et al. Study of Fluorescence Immunochromatography Based on Quantum Dots for the Detection of SARS-CoV-2 anti-N Protein IgG Antibody[J]. Chinese General Practice, 2022, 25(14): 1741-1749. DOI: 10.12114/j.issn.1007-9572.2022.0166.
于仁峰,邹小仓,邹大阳等. 基于量子点标记技术的免疫层析法检测新型冠状病毒N蛋白IgG抗体研究[J]. 中国全科医学, 2022, 25(14): 1741-1749. DOI: 10.12114/j.issn.1007-9572.2022.0166.

Figures/Tables 15

References 17

[1]	LI H J，WANG Y Y，JI M Y，et al. Transmission routes analysis of SARS-CoV-2：a systematic review and case report[J]. Front Cell Dev Biol，2020，8：618. DOI：10.3389/fcell.2020.00618.
[2]	LU R J，ZHAO X，LI J，et al. Genomic characterisation and epidemiology of 2019 novel coronavirus：implications for virus origins and receptor binding[J]. Lancet，2020，395（10224）：565-574. DOI：10.1016/S0140-6736(20)30251-8.
[3]	LIU S J，LENG C H，LIEN S P，et al. Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates[J]. Vaccine，2006，24（16）：3100-3108. DOI：10.1016/j.vaccine.2006.01.058.
[4]	SHANG B，WANG X Y，YUAN J W，et al. Characterization and application of monoclonal antibodies against N protein of SARS-coronavirus[J]. Biochem Biophys Res Commun，2005，336（1）：110-117. DOI：10.1016/j.bbrc.2005.08.032.
[5]	ZENG W H，LIU G F，MA H，et al. Biochemical characterization of SARS-CoV-2 nucleocapsid protein[J]. Biochem Biophys Res Commun，2020，527（3）：618-623. DOI：10.1016/j.bbrc.2020.04.136.
[6]	SUN B Q，FENG Y，MO X N，et al. Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients[J]. Emerg Microbes Infect，2020，9（1）：940-948. DOI：10.1080/22221751.2020.1762515.
[7]	KUHN J H，LI W，CHOE H，et al. Angiotensin-converting enzyme 2：a functional receptor for SARS coronavirus[J]. Cell Mol Life Sci，2004，61（21）：2738-2743. DOI：10.1007/s00018-004-4242-5.
[8]	HE Y X，LU H，SIDDIQUI P，et al. Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies[J]. J Immunol，2005，174（8）：4908-4915. DOI：10.4049/jimmunol.174.8.4908.
[9]	CHE X Y，HAO W，WANG Y D，et al. Nucleocapsid protein as early diagnostic marker for SARS[J]. Emerg Infect Dis，2004，10（11）：1947-1949. DOI：10.3201/eid1011.040516.
[10]	聂聪，刘艳丽，侯宝翠，等. 利用荧光免疫层析技术检测新冠病毒IgG抗体[J]. 中国国境卫生检疫杂志，2021，44（3）：156-161. DOI：10.16408/j.1004-9770.2021.03.002.
[11]	LE T，ZHANG Z H，WU J，et al. A fluorescent immunochromatographic strip test using a quantum dot-antibody probe for rapid and quantitative detection of 1-aminohydantoin in edible animal tissues[J]. Anal Bioanal Chem，2018，410（2）：565-572. DOI：10.1007/s00216-017-0756-1.
[12]	SHEN B，ZHENG Y F，ZHANG X Y，et al. Clinical evaluation of a rapid colloidal gold immunochromatography assay for SARS-Cov-2 IgM/IgG[J]. Am J Transl Res，2020，12（4）：1348-1354.
[13]	CORMAN V M，LANDT O，KAISER M，et al. Detection of 2019 novel coronavirus （2019-nCoV） by real-time RT-PCR[J]. Eur Commun Dis Bull，2020，25（3）：2000045. DOI：10.2807/1560-7917.ES.2020.25.3.2000045.
[14]	冯燕，卢亦愚，严菊英，等. 荧光定量RT-PCR技术快速检测SARS病毒核酸[J]. 中国病毒学，2005，20（3）：228-231.
[15]	中华人民共和国国家卫生健康委员会.新型冠状病毒肺炎诊疗方案（试行第七版）[J]. 心肺血管病杂志，2020，39（2）：103-107.
[16]	MASTERS P S. Coronavirus genomic RNA packaging[J]. Virology，2019，537：198-207. DOI：10.1016/j.virol.2019.08.031.
[17]	WU C H，CHEN P J，YEH S H. Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription[J]. Cell Host Microbe，2014，16（4）：462-472. DOI：10.1016/j.chom.2014.09.009.

样本浓度	批次	FIT/FIC均值	标准偏差	变异系数	结果
1∶500	第一批次	6.197	0.513 1	0.082 79	+
	第二批次	7.634	0.737 7	0.096 63	+
	第三批次	5.466	0.489 3	0.089 51	+
1∶2 000	第一批次	2.124	0.117 1	0.055 12	+
	第二批次	1.578	0.118 9	0.075 30	+
	第三批次	2.343	0.237 1	0.101 2	+
1∶5 000	第一批次	0.635 0	0.044 82	0.070 58	+
	第二批次	0.451 8	0.039 53	0.087 48	+
	第三批次	0.805 1	0.061 11	0.075 91	+

样本浓度	批次	FIT/FIC均值	标准偏差	变异系数	结果
1∶500	第一批次	6.197	0.513 1	0.082 79	+
	第二批次	7.634	0.737 7	0.096 63	+
	第三批次	5.466	0.489 3	0.089 51	+
1∶2 000	第一批次	2.124	0.117 1	0.055 12	+
	第二批次	1.578	0.118 9	0.075 30	+
	第三批次	2.343	0.237 1	0.101 2	+
1∶5 000	第一批次	0.635 0	0.044 82	0.070 58	+
	第二批次	0.451 8	0.039 53	0.087 48	+
	第三批次	0.805 1	0.061 11	0.075 91	+

病毒	C线	T线	FIT/FIC	FIT/FIC均值	结果
229E	59 241	2 694	0.045 47	0.038 92	_
	63 145	2 645	0.041 88
	48 181	1 417	0.029 40
NL63	91 811	663	0.007 221	0.011 2	_
	75 561	1 167	0.015 45
	77 317	845	0.010 92
HKU1	73 395	1 869	0.025 47	0.018 14	_
	70 475	1435	0.020 36
	97 929	841	0.008 592
OC43	53 188	1 569	0.029 50	0.029 85	_
	62 453	1 513	0.024 23
	64 301	2 304	0.035 831 5
MP-P1	22 859	872	0.038 14	0.020 67	_
	20 388	101	0.004 953
	28 102	532	0.018 93
RSV-F0	35 085	662	0.018 86	0.021 03	_
	24 447	333	0.013 62
	22 412	686	0.030 60
MV-NP	25 076	163	0.006 500	0.016 04	_
	22 791	685	0.030 05
	36 817	426	0.011 57
RV-E1	43 036	800	0.018 58	0.009 195	_
	31 194	122.5	0.003 927
	34 702	176	0.005 071

病毒	C线	T线	FIT/FIC	FIT/FIC均值	结果
229E	59 241	2 694	0.045 47	0.038 92	_
	63 145	2 645	0.041 88
	48 181	1 417	0.029 40
NL63	91 811	663	0.007 221	0.011 2	_
	75 561	1 167	0.015 45
	77 317	845	0.010 92
HKU1	73 395	1 869	0.025 47	0.018 14	_
	70 475	1435	0.020 36
	97 929	841	0.008 592
OC43	53 188	1 569	0.029 50	0.029 85	_
	62 453	1 513	0.024 23
	64 301	2 304	0.035 831 5
MP-P1	22 859	872	0.038 14	0.020 67	_
	20 388	101	0.004 953
	28 102	532	0.018 93
RSV-F0	35 085	662	0.018 86	0.021 03	_
	24 447	333	0.013 62
	22 412	686	0.030 60
MV-NP	25 076	163	0.006 500	0.016 04	_
	22 791	685	0.030 05
	36 817	426	0.011 57
RV-E1	43 036	800	0.018 58	0.009 195	_
	31 194	122.5	0.003 927
	34 702	176	0.005 071

检测方法		总体样本数（例）	核酸诊断（例）
免疫层析试纸条	ELISA试剂盒	总体样本数（例）	阳性	阴性
阳性	阳性	32	32	0
阳性	阴性	1	1	0
阴性	阳性	2	2	0
阴性	阴性	50	0	50

Study of Fluorescence Immunochromatography Based on Quantum Dots for the Detection of SARS-CoV-2 anti-N Protein IgG Antibody

基于量子点标记技术的免疫层析法检测新型冠状病毒N蛋白IgG抗体研究

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 15

References 17

Related Articles 15

Recommended Articles 0

Metrics

Comments

[1]	ZUO Xu, HUANG Zhaolan, LU Biao. Differences between the First and Another 3-day Blood Pressure Levels and Associated Factors in a Self-reported Non-hypertensive Population Aged 35-64 Years [J]. Chinese General Practice, 2023, 26(22): 2771-2777.
[2]	WU Jian, YU Chengcheng, YANG Yinmei, XIA Qingyun, LI Quanman, FU Xiaoli. COVID-19 Vaccination Behavior and Its Influencing Factors among the Elderly in Four Regions of China [J]. Chinese General Practice, 2023, 26(22): 2763-2770.
[3]	HE Mei, LI Hui, MU Lifeng, YANG Ming. Effect of Azovudine on Hepatic and Renal Function in Patients with COVID-19: a Case Series Study [J]. Chinese General Practice, 2023, 26(20): 2476-2481.
[4]	WANG Hai, WANG Zhuoli, PEI Honghong, PAN Longfei. Analysis of Risk Factors and Exploration of Predictors of Serious Cases of COVID-19 in Xi'an during the Period of 2021-2022 [J]. Chinese General Practice, 2023, 26(17): 2132-2137.
[5]	YAN Yuge, HUANG Jiaoling. Visualization Analysis of Primary Healthcare Research during the COVID-19 Pandemic [J]. Chinese General Practice, 2023, 26(16): 2027-2035.
[6]	National Center of Gerontology, Electrocardiology and Cardiac Function Branch of Chinese Geriatric Society, Imaging Group of Cardiovascular Department, Beijing Medical Association, Chinese Medical and Health Culture Association Cardiovascular Health and Science Sports Branch of China Health Culture Association. Expert Consensus for Management of Myocardial Injury, Myocarditis, and Post-infection Condition with Coronavirus Disease 2019 (Second Edition) [J]. Chinese General Practice, 2023, 26(14): 1692-1702.
[7]	HUA Qiaoli, LIU Huiling, XU Xiaohua, ZHENG Danwen, WANG Qian, LIU Yuntao, ZHOU Xin, YANG Rongyuan, DING Banghan, GUO Jianwen, ZHANG Zhongde. Prevalence and Associated Factors of Very Early Cognitive Impairment in COVID-19 Convalescents: a Study Using Data from a Questionnaire Survey [J]. Chinese General Practice, 2023, 26(10): 1234-1240.
[8]	YANG Hui. Prevention and Control of the COVID-19 Epidemic and Management of Chronic Disease [J]. Chinese General Practice, 2023, 26(07): 780-782.
[9]	SHI Weijuan, WANG Fengyan, YANG Yuqiong, XIE Qingxiu, LI Yuqi, LI Shiyue, CHEN Rongchang, ZHANG Dongying, ZHENG Jinping, LIANG Zhenyu. Impact of COVID-19 Pandemic on the Frequency of Acute Exacerbations of Chronic Obstructive Pulmonary Disease [J]. Chinese General Practice, 2023, 26(05): 550-556.
[10]	ZHANG Weizong, YUAN Hong, SUN Jindong, YU Huamin, SHI Mingjuan, HU Haiqiang, HE Haiying, YE Li, ZHANG Huihui, BAI Xinghua, SHEN Chaofeng, TU Sijia, WANG Yang, WANG Gang, ZHAO Xiaofeng, YU Tao, LI Cairong, ZHANG Zhi, ZHOU Donglai, CAI Mengyang, NING Le. Risk Factors of In-hospital Death in COVID-19 Patients: a Meta-analysis [J]. Chinese General Practice, 2023, 26(05): 607-620.
[11]	HUANGFU Xiaojuan, LI Xiaojuan, CHEN Min, LIU Jihong, ZHAI Rui, CAI Zhipeng, LI Litao. A Meta-analysis of the Impact of COVID-19 on Stroke Mortality [J]. Chinese General Practice, 2023, 26(03): 348-355.
[12]	CHANG Chun, SUN Yongchang. Global Strategy for Asthma Management and Prevention: Interpretation of the Updates in 2022 [J]. Chinese General Practice, 2022, 25(35): 4355-4362.
[13]	JIA Dongxia, PENG Junchen, LIU Sitai, LI Lan, ZHENG Hang, HOU Liangping, YANG Xianzheng, HUANG Qiangpei, GAO Chao. Meta Analysis of the Efficacy and Safety of Rennin-angiotensin-aldosterone System Iinhibitors in Patients with COVID-19 Associated with Hypertension [J]. Chinese General Practice, 2022, 25(33): 4117-4122.
[14]	Wendi JIANG, Jinjin WANG, Shanshan LI, Yang YANG, Shuai HAO, Zhihui TONG, Wenpeng CAI. Awareness and Skills of Correctly Donning and Doffing Personal Protective Equipment in SARS-CoV-2 Omicron Containment Workers from a Mobile Cabin Hospital [J]. Chinese General Practice, 2022, 25(30): 3726-3733.
[15]	Yu WU, Jue LIU, Min LIU, Wannian LIANG. Research of Epidemiologic Feature and Control of SARS-CoV-2 Omicron Subvariants BA.4 and BA.5 [J]. Chinese General Practice, 2022, 25(30): 3721-3725.