非洲猪瘟病毒p72蛋白抗体全自动化学发光酶免疫检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.03.034

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (3): 1355-1365.doi: 10.11843/j.issn.0366-6964.2025.03.034

非洲猪瘟病毒p72蛋白抗体全自动化学发光酶免疫检测方法的建立

马晓莉¹(), 李段¹^,³(), 曾道平³, 刘燕玲¹, 王晓敏¹, 彭国良², 宋长绪¹, 王磊¹^,²^,*(), 徐铮¹^,*()

1. 华南农业大学实验动物中心/动物科学学院/猪禽种业全国重点实验室/国家生猪种业工程技术研究中心, 广州 510642
2. 韶关学院英东生物与农业学院, 韶关 512005
3. 温氏食品集团股份有限公司/广东标允生物科技有限公司, 云浮 527400

收稿日期:2024-04-03 出版日期:2025-03-23 发布日期:2025-04-02
通讯作者: 王磊,徐铮 E-mail:maxl@scau.edu.cn;517341252@qq.com;9286980098@qq.com;stonezen@scau.edu.cn
作者简介:马晓莉(1981-)，女，广东深圳人，实验师，硕士，主要从事预防兽医及实验动物模型研究，E-mail: maxl@scau.edu.cn
李段(1987-)，女，河南上蔡人，兽医师，硕士，主要从事动物疫病诊断技术研究，E-mail: 517341252@qq.com
第一联系人：
马晓莉和李段为同等贡献作者
基金资助:
国家重点研发计划(2021YFD2001205)；广东省基础与应用基础研究基金(2023A1515010234)

Establishment of a Fully Automated Chemiluminescent Enzyme Immunoassay for Detecting Antibodies against African Swine Fever Virus p72

MA Xiaoli¹(), LI Duan¹^,³(), ZENG Daoping³, LIU Yanling¹, WANG Xiaomin¹, PENG Guoliang², SONG Changxu¹, WANG Lei¹^,²^,*(), XU Zheng¹^,*()

1. Laboratory Animal Center and College of Animal Science, National Engineering Center for Swine Breeding Industry, State Key Laboratory of Swine and Poultry Breeding Industry, South China Agricultural University, Guangzhou 510642, China
2. Henry Fok School of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China
3. Wens Foodstuff Group Co., Ltd, Guangdong Biaoyun Biotechnology Co., Ltd, Yunfu 527400, China

Received:2024-04-03 Online:2025-03-23 Published:2025-04-02
Contact: WANG Lei, XU Zheng E-mail:maxl@scau.edu.cn;517341252@qq.com;9286980098@qq.com;stonezen@scau.edu.cn

摘要/Abstract

摘要：

抗体检测对于非洲猪瘟病毒(African swine fever virus，ASFV)感染诊断和疫苗研究至关重要，因此建立一种基于ASFV p72蛋白抗体的全自动化学发光酶免疫分析方法(chemiluminescent enzyme immunoassay，CLEIA)具有重要意义。本研究以重组p72蛋白偶联的磁微粒(magnetic particles，MPs)捕获p72蛋白抗体，再结合碱性磷酸酶(alkaline phosphates, AP)标记的p72蛋白，借助全自动化学发光分析仪，分析猪血清中p72蛋白特异性抗体水平。经反应条件优化，建立了一种ASFV p72蛋白抗体全自动化学发光酶免疫检测方法。结果显示，建立的CLEIA阴性、阳性临界值为9.505 U·mL^-1，诊断特异性和灵敏性分别为98.33% 和100%，与其他猪病原阳性血清无交叉反应，批内和批间变异系数分别小于5% 和10%，各种试剂组分可以保存6个月以上，对ASFV阳性标准血清，分析敏感性为1∶12 800，与国产商品化试剂盒的总符合率达95.90%。综上，本研究建立的化学发光方法具有高灵敏性和可重复性，有自动化检测、操作步骤简单的优势，可为ASFV的检测提供新的技术支持。

关键词: 非洲猪瘟病毒, p72, 化学发光酶免疫, 抗体检测

Abstract:

Antibody detection is important for African swine fever virus (ASFV) infection diagnosis and vaccine research. Therefore, it is of great significance to establish a fully automatic chemiluminescent enzyme immunoassay (CLEIA) based on ASFV p72 protein antibody. For the detection, antibodies against p72 were captured by magnetic particles (MPs) coupled with purified p72 protein, then combined with alkaline phosphates (AP)-labelled p72, and ASFV p72 antibodies can be determined using a fully automated chemiluminescence analyzer. Under optimal working conditions, the cut-off value of the established CLEIA was 9.505 U·mL^-1 with a diagnostic specificity and sensitivity of 98.33% and 100%, respectively, and no cross-reactivity with other porcine pathogen-positive sera were found. The intra- and inter-batch coefficients of variation were less than 5% and 10%, respectively, and all components can be stored for more than 6 months at 4 ℃. The lowest detection limit for ASFV-positive standard serum was 1∶12 800. The total coincidence rate with the commercial kit was up to 95.90%, indicating a good applicability. In conclusion, the established CLIEA has the advantages of high sensitivity and reproducibility, combining with automated detection and simple operation steps, which can provide an important tool for the clinical diagnosis of ASF.

Key words: African swine fever virus, p72, chemiluminescent enzyme immunoassay, antibody detection

中图分类号:

S852.659.1

马晓莉, 李段, 曾道平, 刘燕玲, 王晓敏, 彭国良, 宋长绪, 王磊, 徐铮. 非洲猪瘟病毒p72蛋白抗体全自动化学发光酶免疫检测方法的建立[J]. 畜牧兽医学报, 2025, 56(3): 1355-1365.

MA Xiaoli, LI Duan, ZENG Daoping, LIU Yanling, WANG Xiaomin, PENG Guoliang, SONG Changxu, WANG Lei, XU Zheng. Establishment of a Fully Automated Chemiluminescent Enzyme Immunoassay for Detecting Antibodies against African Swine Fever Virus p72[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1355-1365.

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参考文献 31

1	WANG N , ZHAO D M , WANG J L , et al. Architecture of African swine fever virus and implications for viral assembly[J]. Science, 2019, 366 (6465): 640- 644.
2	GAUDREAULT N N , MADDEN D W , WILSON W C , et al. African swine fever virus: an emerging DNA arbovirus[J]. Front Vet Sci, 2020, 7, 215.
3	GE S Q , LI J M , FAN X X , et al. Molecular characterization of African swine fever virus, China, 2018[J]. Emerg Infect Dis, 2018, 24 (11): 2131- 2133.
4	向国庆, 连聪, 孙栋, 等. 化学发光免疫分析法及在动物生产领域检测中的应用进展[J]. 中国兽医科学, 2023, 53 (10): 1320- 1325.
	XIANG G Q , LIANG C , SUN D , et al. Progress of chemiluminescence immunoassay and its application in the detection of animal production[J]. Chinese Veterinary Science, 2023, 53 (10): 1320- 1325.
5	赵协, 安利民, 高沙沙, 等. 化学发光免疫分析技术在动物疫病检测中的应用[J]. 中国动物检疫, 2020, 37 (8): 82- 87.
	ZHAO X , AN L M , GAO S S , et al. Application of chemiluminescence immunoassay in detection of animal diseases[J]. China Animal Health Inspection, 2020, 37 (8): 82- 87.
6	LIU B J , SU X B , YU G , et al. An automated chemiluminescent immunoassay (CLIA) detects SARS-CoV-2 neutralizing antibody levels in COVID-19 patients and vaccinees[J]. Int J Infect Dis, 2022, 115, 116- 125.
7	MOELLER M E , ENGSIG F N , BADE M , et al. Rapid quantitative point-of-care diagnostic test for post COVID-19 vaccination antibody monitoring[J]. Microbiol Spectr, 2022, 10 (2): e0039622. doi: 10.1128/spectrum.00396-22
8	孙雨, 宋晓晖, 王睿男, 等. A型口蹄疫病毒抗体化学发光免疫分析检测方法的建立[J]. 动物医学进展, 2020, 41 (8): 29- 36.
	SUN Y , SONG X H , WANG R N , et al. Establishment of chemiluminescent immunoassay for detection of type A FMDV antibody[J]. Progress in Veterinary Medicine, 2020, 41 (8): 29- 36.
9	马震原, 王淑娟, 闫若潜, 等. 猪伪狂犬病病毒gB蛋白抗体竞争化学发光酶联免疫检测方法的建立[J]. 畜牧兽医学报, 2020, 51 (3): 574- 583. doi: 10.11843/j.issn.0366-6964.2020.03.017
	MA Z Y , WANG S J , YAN R Q , et al. Establishment of competitive chemiluminescent enzyme immunoassay for detecting antibodies against gB protein of pseudorabies virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51 (3): 574- 583. doi: 10.11843/j.issn.0366-6964.2020.03.017
10	ALONSO C , BORCA M , DIXON L , et al. ICTV virus taxonomy profile: Asfarviridae[J]. J Gen Virol, 2018, 99 (5): 613- 614.
11	DIXON L K , CHAPMAN D A G , NETHERTON C L , et al. African swine fever virus replication and genomics[J]. Virus Res, 2013, 173 (1): 3- 14.
12	LIU Q , MA B T , QIAN N C , et al. Structure of the African swine fever virus major capsid protein p72[J]. Cell Res, 2019, 29 (11): 953- 955.
13	ALEJO A , MATAMOROS T , GUERRA M , et al. A proteomic atlas of the African swine fever virus particle[J]. J Virol, 2018, 92 (23): e01293- 18.
14	LIU W M , LI H C , LIU B , et al. A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine[J]. Emerg Microbes Infect, 2023, 12 (2): 2233643.
15	CHEN X X , YANG J F , JI Y H , et al. Recombinant Newcastle disease virus expressing African swine fever virus protein 72 is safe and immunogenic in mice[J]. Virol Sin, 2016, 31 (2): 150- 159.
16	WANG L , LI D , LIU Y L , et al. Development of an effective one-step double-antigen sandwich ELISA based on p72 to detect antibodies against African swine fever virus[J]. Front Vet Sci, 2023, 10, 1160583.
17	LIU F F , ZOU J J , LUO X X , et al. A point-of-care chemiluminescence immunoassay for pepsinogen I enables large-scale community health screening[J]. Anal Bioanal Chem, 2021, 413 (17): 4493- 4500.
18	WANG W W , OUYANG H , YANG S J , et al. Multiplexed detection of two proteins by a reaction kinetics-resolved chemiluminescence immunoassay strategy[J]. Analyst, 2015, 140 (4): 1215- 1220.
19	柳方方. 胃蛋白酶原I磁微粒化学发光检测方法的建立与应用评价[D]. 广州: 华南理工大学, 2021.
	LIU F F. Establishment and evaluation of the magnetic particle-based chemiluminescence immunoassay for pepsinogen Ⅰ[D]. Guangzhou: South China University of Technology, 2021. (in Chinese)
20	KHRAMTSOV P , BARKINA I , KROPANEVA M , et al. Magnetic nanoclusters coated with albumin, casein, and gelatin: size tuning, relaxivity, stability, protein corona, and application in nuclear magnetic resonance immunoassay[J]. Nanomaterials (Basel), 2019, 9 (9): 1345.
21	LIU W , SHAO J J , ZHANG G L , et al. Development of an indirect chemiluminescence immunoassay using a multiepitope recombinant protein to specifically detect antibodies against foot-and-mouth disease virus serotype O in swine[J]. J Clin Microbiol, 2021, 59 (3): e02464- 20.
22	LIU Z Z , SHAO J J , ZHAO F R , et al. Chemiluminescence immunoassay for the detection of antibodies against the 2C and 3ABC nonstructural proteins induced by infecting pigs with foot-and-mouth disease virus[J]. Clin Vaccine Immunol, 2017, 24 (8): e00153- 17.
23	ZHOU Y , ZHOU T , ZHOU R , et al. Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using horseradish peroxidase/detection antibody-coated gold nanoparticles as nanoprobes[J]. Luminescence, 2014, 29 (4): 338- 343.
24	YE K , SHI D W , ZHANG Z G , et al. A chemiluminescence immunoassay for precise automatic quality control of glycoprotein in human rabies vaccine[J]. Vaccine, 2021, 39 (51): 7470- 7476.
25	QIN Y J , SHA R C , FENG Y C , et al. Comparison of double antigen sandwich and indirect enzyme-linked immunosorbent assay for the diagnosis of hepatitis C virus antibodies[J]. J Clin Lab Anal, 2020, 34 (11): e23481.
26	YANG Y , LV C J , FAN J Q , et al. Development of a chemiluminescence immunoassay to accurately detect African swine fever virus antibodies in serum[J]. J Virol Methods, 2021, 298, 114269.
27	SHI Z W , CAO L Y , LUO J C , et al. A chemiluminescent magnetic microparticle immunoassay for the detection of antibody against African swine fever virus[J]. Appl Microbiol Biotechnol, 2023, 107 (11): 3779- 3788. doi: 10.1007/s00253-023-12518-z
28	CINQUANTA L , FONTANA D E , BIZZARO N . Chemiluminescent immunoassay technology: what does it change in autoantibody detection?[J]. Autoimmun Highlights, 2017, 8 (1): 9.
29	银凤桂, 侯力丹, 魏艳秋, 等. 鸭甲型肝炎病毒1型抗体CLEIA检测方法的建立[J]. 中国预防兽医学报, 2017, 39 (6): 451-455, 460.
	YIN F G , HOU L D , WEI Y Q , et al. Development of chemiluminescent enzyme immunoassay for detection of antibodies against duck hepatitis A virus type 1[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39 (6): 451-455, 460.
30	PEGHIN M , BONTEMPO G , DE MARTINO M , et al. Evaluation of qualitative and semi-quantitative cut offs for rapid diagnostic lateral flow test in relation to serology for the detection of SARS-CoV-2 antibodies: findings of a prospective study[J]. BMC Infect Dis, 2022, 22 (1): 810.
31	TANNOUS B A , VERHAEGEN M , CHRISTOPOULOS T K , et al. Combined flash- and glow-type chemiluminescent reactions for high-throughput genotyping of biallelic polymorphisms[J]. Anal Biochem, 2003, 320 (2): 266- 272.

项目 Item	p72-MP用量/μL p72-MP volume
项目 Item	50	100	150	200
阳性血清RLU值(P) RLU value of positive serum (P)	167 220	238 945	225 090	240 024
阴性血清RLU值(N) RLU value of negative serum (N)	45 620	46 805	57 009	63 726
P/N值P/N value	3.66	5.11	3.95	3.77

项目 Item	p72-AP用量/μL p72-AP volume
项目 Item	50	100	150	200
阳性血清RLU值(P) RLU value of positive serum (P)	17 200	240 050	250 740	260 575
阴性血清RLU值(N) RLU value of negative serum (N)	41 201	42 575	55 099	59 366
P/N值P/N value	4.18	5.64	4.55	4.39

项目 Item	反应时间/min Reaction time
项目 Item	1	3	5	8
阳性血清RLU值(P) RLU value of positive serum (P)	120 056	175 578	273 667	269 900
阴性血清RLU值(N) RLU value of Negative serum (N)	30 093	41 173	45 552	45 892
P/N值P/N value	3.99	4.26	6.01	5.88

样品 Sample	稀释度 Dilution ratios	CLEIA		英吉钠ELISA		普泰ELISA
样品 Sample	稀释度 Dilution ratios	浓度	结果 Result	X/%	结果 Result	S/P	结果 Result
ASFV阳性标准血清 ASFV standard serum	1∶400	320.00	+	76	+	2.80	+
	1∶800	160.0	+	53	+	2.10	+
	1∶1 600	80.0	+	27	－	1.55	+
	1∶3 200	40.0	+	10	－	1.01	+
	1∶6 400	20.0	+	-7	－	0.59	+
	1∶12 800	10.0	+	-19	－	0.33	－
	1∶25 600	5.0	－	-21	－	0.21	－
临床样品 Clinical sample	1∶2	18.2	+	39	－	1.76	+
	1∶4	11.2	+	18	－	0.58	+
	1∶8	7.9	－	7	－	0.25	－
	1∶16	4.82	－	-2	－	0.19	－

血清编号 Serum number	批内变异系数Intrabatch coefficient of variation		批间变异系数Interbatch coefficient of variation
血清编号 Serum number	${\bar x}$±s	CV/%	${\bar x}$±s	CV/%
1	4.36±0.33	0.76	4.70±0.24	5.10
2	4.66±0.10	2.15	4.59±0.16	3.49
3	11.14±0.54	4.74	11.89±0.59	9.75
4	16.57±0.48	2.90	16.94±1.04	6.13
5	67.17±0.87	1.30	64.07±1.59	2.49
6	102.02±5.0	4.90	11.85±0.15	1.27

非洲猪瘟病毒p72蛋白抗体全自动化学发光酶免疫检测方法的建立

Establishment of a Fully Automated Chemiluminescent Enzyme Immunoassay for Detecting Antibodies against African Swine Fever Virus p72

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参考文献 31

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方法 Methods		商品化ELISA Commercial ELISA			符合率/% Coincidence rate
方法 Methods		阳性样品数 Positive serum numbers	阴性样品数 Negative serum numbers	总数 Total	符合率/% Coincidence rate
CLEIA	阳性样品数Positive serum numbers	15	3	18	83.33
	阴性样品数Negative serum numbers	2	102	104	98.10
	总数Total	17	105	122	95.90

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