弓形虫抗体镧系微球免疫层析试纸条方法的建立

doi:10.11843/j.issn.0366-6964.2025.05.032

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (5): 2353-2363.doi: 10.11843/j.issn.0366-6964.2025.05.032

弓形虫抗体镧系微球免疫层析试纸条方法的建立

韩阳瑞¹^,³, 夏炉明², 李瑞芳¹, 张曼玉¹, 杜晶莹¹, 孙卿¹, 李增强², 赵洪进²^,*(), 王权¹^,*()

1. 中国农业科学院上海兽医研究所, 上海 200241
2. 上海市动物疫病预防控制中心, 上海 201103
3. 郑州伊美诺生物技术有限公司, 郑州 450000

收稿日期:2024-07-25 出版日期:2025-05-23 发布日期:2025-05-27
通讯作者: 赵洪进,王权 E-mail:zhaohongjin945@163.com;wangquan@shvri.ac.cn
作者简介:韩阳瑞(1992-)，女，河南新郑人，硕士，主要从事快速检测技术研究
夏炉明(1982-)，男，江西九江人，硕士，主要从事畜间人兽共患病防控工作
第一联系人：
韩阳瑞和夏炉明为同等贡献作者
基金资助:
上海市科技兴农项目(2022-02-08-00-12-F01085); 上海自然科学基金(22ZR1476000); 上海科委项目(21N31900700)

Establishment of Lanthanide Microsphere Immunochromatographic Test Strips for the Detection of Toxoplasma gondii Antibodies

HAN Yangrui¹^,³, XIA Luming², LI Ruifang¹, ZHANG Manyu¹, DU Jingying¹, SUN Qing¹, LI Zenqiang², ZHAO Hongjin²^,*(), WANG Quan¹^,*()

1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China
2. Shanghai Animal Disease Control Center, Shanghai 201103, China
3. Zhengzhou Immuno Bio-Tech Co., Ltd, Zhengzhou 450000, China

Received:2024-07-25 Online:2025-05-23 Published:2025-05-27
Contact: ZHAO Hongjin, WANG Quan E-mail:zhaohongjin945@163.com;wangquan@shvri.ac.cn

摘要/Abstract

摘要：

近年来，人与动物感染弓形虫的风险因素增多，因此对弓形虫流行情况的调查愈加重要。鉴于镧系微球荧光寿命长、stockes位移大、光稳定性强等特点，本研究将其应用于血清中弓形虫抗体的检测。为提高检测准确性，以可溶性表达的SAG1、SAG2、GRA1的三种抗原的混合液为包被原，以荧光微球标记SPA为探针，以间接免疫侧流层析法研制检测弓形虫抗体试纸条，优化了上样方式、微球血清共孵育时间、反应时间等条件，考察了试纸条的特异性、灵敏性、重复性及可靠性。结果显示，制备的镧系微球免疫层析试纸条特异性好，与其他病原感染血清无交叉反应；灵敏度高，阳性血清稀释1 600倍仍可被检出；反应快速，最快10 min即可获得结果；稳定可靠，通过血清样本检测，Kappa分析揭示了镧系微球试纸条检测方法和ELISA之间有较好的一致性。本研究为弓形虫抗体检测提供了快速、灵敏、可定量的镧系微球免疫层析检测方法。

关键词: 镧系微球, 免疫层析, 弓形虫, 即时检测, 定量

Abstract:

In recent years, the risk factors for Toxoplasma gondii infection in humans and animals have increased, so the investigation of the prevalence of T. gondii has become more important. In view of the fluorescence characteristics of lanthanide microspheres, such as long life span, large stockes displacement and excellent photostability, this study applied the fluorescent microspheres as probes for the detection of T. gondii antibodies in serum. In order to improve the accuracy of the test, a mixture of three antigens of soluble expressed SAG1, SAG2, and GRA1 was used as the coating antigens, and fluorescent microsphere-labelled SPA was used as the probe to develop test strips for the detection of T. gondii antibodies by indirect immuno-lateral chromatography, and the conditions of the sampling method, the co-incubation time of the microsphere and sera, the reaction time were optimized, meanwhile, the specificity, sensitivity, reproducibility and reliability of the test strips were investigated. The results showed that the prepared lanthanide microsphere immunochromatographic test strips have the following characteristics: good specificity, no cross-reactivity with other pathogenic sera; high sensitivity, the positive sera can still be detected at a dilution of 1 600 times; fast detection speed, the results can be obtained in as fast as 10 minutes; stable and reliable, the Kappa analyses of serum samples show that the lanthanide microsphere test strips have good consistency with the two ELISA assays. The present study provides a rapid, sensitive and quantitative immunochromatographic method for the detection of T. gondii antibodies.

Key words: lanthanide microspheres, immunochromatography, Toxoplasma gondii, immediate detection, quantification

中图分类号:

S852.729

韩阳瑞, 夏炉明, 李瑞芳, 张曼玉, 杜晶莹, 孙卿, 李增强, 赵洪进, 王权. 弓形虫抗体镧系微球免疫层析试纸条方法的建立[J]. 畜牧兽医学报, 2025, 56(5): 2353-2363.

HAN Yangrui, XIA Luming, LI Ruifang, ZHANG Manyu, DU Jingying, SUN Qing, LI Zenqiang, ZHAO Hongjin, WANG Quan. Establishment of Lanthanide Microsphere Immunochromatographic Test Strips for the Detection of Toxoplasma gondii Antibodies[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2353-2363.

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参考文献 28

1	MONTOYA J G , LIESENFELD O . Toxoplasmosis[J]. Lancet, 2004, 363 (9425): 1965- 1976. doi: 10.1016/S0140-6736(04)16412-X
2	TENTER A M . Toxoplasma gondii in animals used for human consumption[J]. Mem Inst Oswaldo Cruz, 2009, 104 (2): 364- 369. doi: 10.1590/S0074-02762009000200033
3	DUBEY J P , SU C . Population biology of Toxoplasma gondii: what 's out and where did they come from[J]. Mem Inst Oswaldo Cruz, 2009, 104 (2): 190- 195. doi: 10.1590/S0074-02762009000200011
4	LIU X C , HE Y , HAN D G , et al. Detection of Toxoplasma gondii in chicken and soil of chicken farms in Nanjing region, China[J]. Infect Dis Poverty, 2017, 6 (1): 62. doi: 10.1186/s40249-017-0277-3
5	JIANG H H , ZHANG W B , ZHAO L , et al. Seroprevalence of Toxoplasma gondii infection in pigs in Jiangxi Province, Southeastern China[J]. Foodborne Pathog Dis, 2014, 11 (5): 362- 365. doi: 10.1089/fpd.2013.1686
6	肖祥, 沈继龙. 核酸检测技术在寄生虫病诊断中的应用[J]. 热带病与寄生虫学, 2000 (2): 114- 120.
	XIAO X , SHEN J L . Application of nucleic acid tests in the diagnosis of parasitic diseases[J]. Journal of Tropical Diseases and Parasitology, 2000 (2): 114- 120.
7	阿斯喀·夏热甫汉, 叶尔保勒, 王振宝, 等. 弓形虫病的免疫学诊断方法[J]. 今日畜牧兽医, 2023, 39 (9): 65- 67.
	ASIKA X , YEERBAOLE , WANG Z B , et al. Immunological diagnostics of toxoplasmosis[J]. Today Animal Husbandry and Veterinary Medicine, 2023, 39 (9): 65- 67.
8	王小妹. 基于镧系高敏荧光的贝类毒素、激素快速检测方法的研究[D]. 上海: 中国农业科学院, 2023.
	WANG X M. Study of rapid detection methods for shellfish poisonand hormones based on lanthanide high sensitivity fluorescence[D]. Shanghai: Chinese Academy of Agricultural Sciences, 2023. (in Chinese)
9	KOTRESHA D , NOORDIN R . Recombinant proteins in the diagnosis of toxoplasmosis[J]. Apmis, 2010, 118 (8): 529- 42. doi: 10.1111/j.1600-0463.2010.02629.x
10	代琴. 弓形虫SAG1单克隆抗体的制备及其生物传感器的初步研究[D]. 重庆: 重庆理工大学, 2023.
	DAI Q. Preparation of monoclonal antibody against Toxoplasma gondii SAG1 and preliminary study of its biosensors[D]. Chongqing: Chongqing University of Technology, 2023. (in Chinese)
11	QI T , AI J , SUN Y , et al. Application of Toxoplasma gondii-specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis[J]. Front Cell Infect Microbiol, 2022, 12, 1029768. doi: 10.3389/fcimb.2022.1029768
12	HOLEC-GĄSIOR L , FERRA B , HISZCZYŃSKA-SAWICKA E , et al. The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1) for diagnosis of ovine toxoplasmosis[J]. Vet Parasitol, 2014, 206 (3-4): 146- 152. doi: 10.1016/j.vetpar.2014.09.018
13	刘旗. 弓形虫急性感染血清标志蛋白的筛选、鉴定及其功能的研究[D]. 上海: 中国农业科学院, 2022.
	LIU Q. Screening, identification and function of SerumBiomarkers of acute Toxoplasmosis[D]. Shanghai: Chinese Academy of Agricultural Sciences, 2022. (in Chinese)
14	聂福旭, 蒋蔚, 王权, 等. 弓形虫膜表面抗原SAG2蛋白的高效表达及免疫活性分析[J]. 生物技术通报, 2010 (4): 173- 178.
	NIE F X , JIANG W , WANG Q , et al. Expression and immunological activity analysis on the surface protein SAG2 from Toxoplasma Gondii strain RH[J]. Biotechnology Bulletin, 2010 (4): 173- 178.
15	安立刚, 蒋蔚, 王权, 等. 弓形虫表面抗原SAG1截短型片段在大肠杆菌中的可溶性表达及纯化[J]. 生物技术通报, 2009 (10): 136- 141.
	AN L G , JIANG W , WANG Q , et al. Expression and purification of soluble fusion protein of Toxoplasma gondii Tsag1 in escherichia coli[J]. Biotechnology Bulletin, 2009 (10): 136- 141.
16	韩阳瑞, 蒋蔚, 陈芸, 等. 弓形虫GRA1基因的原核表达与鉴定[J]. 中国动物传染病学报, 2019, 27 (1): 70- 77.
	HAN Y R , JIANG W , CHEN Y , et al. Prokaryotic expression and characterization of GRA1 gene of Toxoplasma gondii[J]. Chinese Journal of Veterinary Parasitology, 2019, 27 (1): 70- 77.
17	HANLEY J A , MCNEIL B J . The meaning and use of the area under a receiver operating characteristic (ROC) curve[J]. Radiology, 1982, 143 (1): 29- 36. doi: 10.1148/radiology.143.1.7063747
18	ZHOU P , CHEN Z , LI H L , et al. Toxoplasma gondii infection in humans in China[J]. Parasit Vectors, 2011, 4, 165. doi: 10.1186/1756-3305-4-165
19	朱向东, 朱雅宁, 刘伟, 等. 免疫层析技术在兽医检测领域的应用[J]. 畜牧兽医科技信息, 2024 (1): 63- 65.
	ZHU X D , ZHU Y N , LIU W , et al. Application of immunochromatographic techniques in veterinary testing[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2024 (1): 63- 65.
20	郑欣瑶, 沈央红, 赵秀玲, 等. 荧光免疫层析法结合智能手机在食品危害物质快速检测中的研究进展[J]. 食品科学, 2024, 45 (19): 376- 383.
	ZHENG X Y , SHEN Y H , ZHAO X L , et al. Advance in the research of fluorescence immunochromatography combined with smartphone in the rapid detection of food hazardous substances[J]. Food Science, 2024, 45 (19): 376- 383.
21	陆廷瑾, 杨婷婷, 张玲, 等. 免疫层析荧光定量检测小麦中的重金属铅[J]. 粮食与食品工业, 2024, 31 (3): 62- 65.
	LU Y J , YANG T T , ZHANG L , et al. Quantitative detection of heavy metal lead in wheat by immunochromatographic fluorescence[J]. Cereal & Food Industry, 2024, 31 (3): 62- 65.
22	JIANG W , LIU Y , CHEN Y , et al. A novel dynamic flow immunochromatographic test (DFICT) using gold nanoparticles for the serological detection of Toxoplasma gondii infection in dogs and cats[J]. Biosens Bioelectron, 2015, 72, 133- 139.
23	刘明瑞, 殷笑丹, 杨晓帆, 等. 猪繁殖与呼吸综合征病毒荧光微球抗体检测方法的建立[J]. 中国动物检疫, 2023, 40 (10): 100- 105.
	LIU M R , YIN X D , YANG X F , et al. Establishment of a fluorescent microsphere immunoassay assay for detection of porcine reproductive and respiratory syndrome virus[J]. China Animal Health Inspection, 2023, 40 (10): 100- 105.
24	陈芷珊, 陈滢锴, 陈中婷, 等. 猪流行性腹泻病毒荧光免疫层析检测试纸条的研制和应用[J]. 畜牧与兽医, 2024, 56 (4): 95- 100.
	CHEN Z S , CHEN Y K , CHEN Z T , et al. Development and application of fluorescence immunochromatography test strips for PEDV detection[J]. Animal Husbandry & Veterinary Medicine, 2024, 56 (4): 95- 100.
25	霍炳帆, 马骉, 厉佳丽, 等. 基于荧光纳米颗粒的免疫层析技术同步快速检测药食同源药材中的3种真菌毒素[J]. 现代食品科技, 2024, 40 (7): 227- 235.
	HUO B F , MA B , LI J L , et al. Simultaneous and rapid detection of three mycotoxins in medicinal materials with homology of medicine and food by immunochromatography based on fluorescent nanoparticles[J]. Modern Food Science & Technology, 2024, 40 (7): 227- 235.
26	杨若松, 马增彬, 龚雅云, 等. 布鲁氏菌荧光微球免疫层析试纸条的研制[J]. 中国动物检疫, 2024, 41 (5): 85- 89.
	YANG R S , MA Z B , GONG Y Y , et al. Development of fluorescence microsphere immunochromatographic strips for brucellosis[J]. China Animal Health Inspection, 2024, 41 (05): 85- 89.
27	吴璞强. 金黄色葡萄球菌蛋白A亲和配基的分子改造与应用[D]. 无锡: 江南大学, 2013.
	WU P Q. Molecular modification and application of protein A as an affinity ligand from Staphylococcus aureus[D]. Wuxi: Jiangnan University, 2013. (in Chinese)
28	李淑娜, 蒋文灿, 刘义铃, 等. SPA与鸭IgG的结合性研究[J]. 黑龙江畜牧兽医, 2008 (9): 69- 70.
	LI S N , JIANG W C , LIU Y L , et al. Studies on the binding properties of SPA to duck IgG[J]. Heilongjiang Animal Science and Veterinary Medicine, 2008 (9): 69- 70.

镧系微球试纸条 LFM-ICS	酶联免疫吸附试验ELISA
镧系微球试纸条 LFM-ICS	阳性Positive	阴性Negative	合计Total
阳性Positive	14	3	17
阴性Negative	1	43	44
合计Total	15	46	61

镧系微球试纸条 LFM-ICS	商品化试剂盒Commercial ELISA kit
镧系微球试纸条 LFM-ICS	阳性Positive	阴性Negative	合计Total
阳性Positive	12	5	17
阴性Negative	2	42	44
合计Total	14	47	61

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弓形虫抗体镧系微球免疫层析试纸条方法的建立

Establishment of Lanthanide Microsphere Immunochromatographic Test Strips for the Detection of Toxoplasma gondii Antibodies

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