以钩端螺旋体重组蛋白为包被抗原的间接ELISA检测试剂盒的研制与性能评测

doi:10.3969/j.issn.1674-8115.2021.04.007

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (4): 459-466.doi: 10.3969/j.issn.1674-8115.2021.04.007

以钩端螺旋体重组蛋白为包被抗原的间接ELISA检测试剂盒的研制与性能评测

钱杰¹(), 杨坚², 张玥², 张蓉², 张湘燕³(), 郭晓奎¹^,³()

^1.上海交通大学医学院免疫学与微生物学系，上海 200025
^2.上海科新生物技术股份有限公司，上海 201203
^3.上海交通大学医学院-国家热带病研究中心全球健康学院，卫生部寄生虫病原与媒介生物学重点实验室，上海 200025

收稿日期:2020-11-05 出版日期:2021-04-28 发布日期:2021-05-14
通讯作者: 张湘燕,郭晓奎 E-mail:changeqj@163.com;zhangxy@shsmu.edu.cn;xkguo@shsmu.edu.cn
作者简介:钱杰（1985—），男，硕士生；电子信箱：changeqj@163.com。
基金资助:
美国中华医学基金会基金(20-365);上海交通大学科技创新专项资金融合集成创新基金(2020JCPT01)

Development and performance evaluation of indirect ELISA kit with recombinant leptospira protein as coating antigen

Jie QIAN¹(), Jian YANG², Yue ZHANG², Rong ZHANG², Xiang-yan ZHANG³(), Xiao-kui GUO¹^,³()

^1.Department of Microbiology and Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Shanghai Kexin Biotech Co. , Ltd, Shanghai 201203, China
^3.Key Laboratory of Parasite and Vector Biology, Ministry of Health, School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2020-11-05 Online:2021-04-28 Published:2021-05-14
Contact: Xiang-yan ZHANG,Xiao-kui GUO E-mail:changeqj@163.com;zhangxy@shsmu.edu.cn;xkguo@shsmu.edu.cn
Supported by:
The China Medical Board (CMB) fund(20-365);Shanghai Jiao Tong University Science and Technology Innovation Special Fund Fusion Integrated Innovation Fund(2020JCPT01)

摘要/Abstract

摘要：

目的·研制适用于检测钩端螺旋体（钩体）特异性抗体的间接酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）检测试剂盒，并对其性能进行评估。方法·选择我国主要致病性钩体流行株中高度保守的外膜蛋白进行原核表达，将获得的重组蛋白纯化后作为包被抗原行质谱鉴定，并对其抗原性进行检测。选用40例经确诊的钩体病患者血清、50例其他感染类疾病患者血清和391例健康对照者的血清样本作为检测样本，用钩体病确诊患者血清制备试剂盒内控阳性对照品和参考品、高效价混合阳性血清，用健康对照者血清制备试剂盒内控阴性对照品和参考品，用高效价混合阳性血清制备试剂盒内控弱阳性对照品（临界参考品）及灵敏度参考品，从钩体病确诊患者血清中选择强阳性、弱阳性血清（各1份）分别作为试剂盒内控强阳性、弱阳性精密度参考品。建立间接ELISA试剂盒检测体系，优化其制备工艺及反应条件，并对该试剂盒的精密度、敏感性、特异性和稳定性等性能进行评估。结果·钩体重组外膜蛋白纯化后经质谱鉴定为目标蛋白，其作为包被抗原具有较强的抗原性。经性能评估显示，间接ELISA试剂盒的批内精密度<15%、批间精密度<20%，其敏感性为95.0%、特异性为95.0%，且在2~8 ℃下储存12个月后性能仍然稳定。结论·以钩体重组蛋白为包被抗原的间接ELISA钩体抗体检测试剂盒的特异性、敏感性及其他性能指标均符合体外检测试剂盒性能要求，适用于临床辅助诊断、钩体病哨点监测及流行病学调查。

关键词: 钩端螺旋体, 重组蛋白, 间接酶联免疫吸附测定, IgG抗体, 敏感度/特异度

Abstract:

Objective·To develop an indirect enzyme-linked immunosorbent assay (ELISA) kit for the detection of leptospira-specific antibodies and evaluate its performance.

Methods·The highly conserved outer membrane protein in the epidemic strains of the main pathogenic leptospira in China was selected and expressed by prokaryotic expression system. The recombinant protein was purified and used as coating antigen for mass spectrometry identification, and its antigenicity was detected. Serum samples from 40 patients with leptospirosis, 50 patients with other infectious diseases and 391 healthy controls were selected as test samples. The positive control, positive reference and high-titer mixed positive serum for internal control of the kit were prepared from the serum of patients with leptospirosis, the negative control and reference for internal control of the kit were prepared from the serum of healthy controls, and the weak positive control (critical reference) and sensitivity reference for internal control of the kit were prepared from high-titer mixed positive serum. One strong positive serum and one weak positive serum selected from the serum of patients with leptospirosis were used as strong positive and weak positive precision reference for internal control of the kit. The detection system of the indirect ELISA kit was established, its preparation process and reaction conditions were optimized, and its precision, sensitivity, specificity and stability were evaluated.

Results·The purified recombinant outer membrane protein of leptospira was identified as the target protein by mass spectrometry. The results of performance evaluation showed that the intra assay precision and inter assay precision of the indirect ELISA kit were less than 15% and 20%, respectively, and the sensitivity and specificity were 95.0% and 95.0%, respectively. The performance of indirect ELISA kit was still stable after 12 months' storage at 2?8 ℃.

Conclusion·The specificity, sensitivity and other performance indicators of the leptospira antibody detection kit meet the requirements of the in vitro test kit, which may be suitable for clinical auxiliary diagnosis, sentinel monitoring of leptospirosis and epidemiological investigation.

Key words: leptospira, recombinant protein, indirect enzyme-linked immunosorbent assay, IgG antibody, sensitivity/specificity

中图分类号:

R377⁺5

钱杰, 杨坚, 张玥, 张蓉, 张湘燕, 郭晓奎. 以钩端螺旋体重组蛋白为包被抗原的间接ELISA检测试剂盒的研制与性能评测[J]. 上海交通大学学报(医学版), 2021, 41(4): 459-466.

Jie QIAN, Jian YANG, Yue ZHANG, Rong ZHANG, Xiang-yan ZHANG, Xiao-kui GUO. Development and performance evaluation of indirect ELISA kit with recombinant leptospira protein as coating antigen[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 459-466.

图/表 11

参考文献 15

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Reference for internal control of the kit	Concentration of coating antigen
Reference for internal control of the kit	1 μg/mL	2 μg/ mL	5 μg/ mL	10 μg/ mL
Positive Reference
Strong positive reference	1.709	1.744	1.798	1.810
Weak positive reference	0.518	0.531	0.547	0.556
Negative reference	0.117	0.126	0.136	0.149

Reference for internal control of the kit	Concentration of coating antigen
Reference for internal control of the kit	1 μg/mL	2 μg/ mL	5 μg/ mL	10 μg/ mL
Positive Reference
Strong positive reference	1.709	1.744	1.798	1.810
Weak positive reference	0.518	0.531	0.547	0.556
Negative reference	0.117	0.126	0.136	0.149

Reference for internal control of the kit	Intra batch precision			Inter batch precision
Reference for internal control of the kit	Average D (450 nm/630 nm)	SD	CV/%	Average D (450 nm/630 nm)	SD	CV/%
Weak positive reference	0.349	0.032	9.18	0.396	0.053	13.38
Strong positive reference	1.518	0.053	3.49	1.614	0.081	5.02

Reference for internal control of the kit	Intra batch precision			Inter batch precision
Reference for internal control of the kit	Average D (450 nm/630 nm)	SD	CV/%	Average D (450 nm/630 nm)	SD	CV/%
Weak positive reference	0.349	0.032	9.18	0.396	0.053	13.38
Strong positive reference	1.518	0.053	3.49	1.614	0.081	5.02

Serum sample	Positive		Negative
Serum sample	True Positve	Faulse Positve	True Negative	Faulse Negative
Leptospirosis patients/n	38	?	?	2
Other infectious diseases patients/n	?	3	47	?
Healthy controls/n	?	2	48	?

以钩端螺旋体重组蛋白为包被抗原的间接ELISA检测试剂盒的研制与性能评测

Development and performance evaluation of indirect ELISA kit with recombinant leptospira protein as coating antigen

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图/表 11

参考文献 15

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Metrics

Kit test result	MAT test result			Total/n
Kit test result	Positive/n		Negative/n	Total/n
Positive/n		41	2	43
Negative/n		4	93	97
Total/n		45	95	140

Internal control of the kit and its detection index	D (450 nm/630 nm)
Internal control of the kit and its detection index	The first batch	The second batch	The third batch
Control
Positive control	0.647	0.612	0.682
Weak positive control	0.195	0.204	0.212
Negative control	0.062	0.082	0.054
Detection Index
Positive/Negative	10.44	7.46	12.63
Weak positive/Negative	3.15	2.49	3.93

Reference for internal control of the kit	The first batch		The second batch		The third batch
Reference for internal control of the kit	D (450 nm/630 nm)	Negative or positive	D (450 nm/630 nm)	Negative or positive	D (450 nm/630 nm)	Negative or positive
Positive reference 1	0.245	＋	0.229	＋	0.263	＋
Positive reference 2	1.295	＋	1.324	＋	1.378	＋
Positive reference 3	0.327	＋	0.307	＋	0.396	＋
Positive reference 4	0.601	＋	0.549	＋	0.587	＋
Positive reference 5	0.738	＋	0.695	＋	0.785	＋
Negative reference 1	0.105	－	0.085	－	0.115	－
Negative reference 2	0.112	－	0.122	－	0.096	－
Negative reference 3	0.117	－	0.109	－	0.118	－
Negative reference 4	0.085	－	0.106	－	0.096	－
Negative reference 5	0.067	－	0.075	－	0.086	－
Critical reference	0.195	N/A	0.204	N/A	0.212	N/A

Kit batch	Precision reference for internal control of the kit	Intra batch precision
Kit batch	Precision reference for internal control of the kit	Average D(450 nm/630 nm)	SD	CV/%
The first batch	Weak positive precision reference	0.255	0.025	9.79
	Positive precision reference	0.731	0.047	6.47
	Strong positive precision reference	1.317	0.050	3.77
The second batch	Weak positive precision reference	0.238	0.026	11.08
	Positive precision reference	0.695	0.050	7.18
	Strong positive precision reference	1.183	0.065	5.47
The third batch	Weak positive precision reference	0.265	0.027	10.08
	Positive precision reference	0.753	0.058	7.74
	Strong positive precision reference	1.366	0.088	6.46

Reference for internal control of the kit	Before accelerated experiment		6 d after accelerated experiment		7 d after accelerated experiment		8 d after accelerated experiment
Reference for internal control of the kit	D(450 nm/630 nm)	Negative or positive	D(450 nm/630 nm)	Negative or positive	D(450 nm/630 nm)	Negative or positive	D(450 nm/630 nm)	Negative or positive
Positive reference 1	0.279	＋	0.231	＋	0.222	＋	0.139	－
Positive reference 2	1.519	＋	1.324	＋	1.015	＋	0.459	＋
Positive reference 3	0.458	＋	0.349	＋	0.243	＋	0.204	＋
Positive reference 4	0.792	＋	0.527	＋	0.412	＋	0.319	＋
Positive reference 5	0.963	＋	0.719	＋	0.623	＋	0.510	＋
Negative reference 1	0.102	－	0.092	－	0.076	－	0.098	－
Negative reference 2	0.154	－	0.124	－	0.098	－	0.010	－
Negative reference 3	0.062	－	0.085	－	0.065	－	0.076	－
Negative reference 4	0.134	－	0.106	－	0.09	－	0.123	－
Negative reference 5	0.057	－	0.057	－	0.032	－	0.045	－
Critical reference	0.225	N/A	0.186	N/A	0.178	N/A	0.172	N/A

Time after accelerated experiment	Precision reference for internal control of the kit	Intra batch precision
Time after accelerated experiment	Precision reference for internal control of the kit	Average D (450 nm/630 nm)	SD	CV/%
6 d	Weak positive precision reference	0.254	0.027	10.54
	Positive precision reference	0.691	0.052	7.58
	Strong positive precision reference	1.243	0.057	4.58
7 d	Weak positive precision reference	0.234	0.034	14.52
	Positive precision reference	0.495	0.049	9.89
	Strong positive precision reference	1.034	0.067	6.48
8 d	Weak positive precision reference	0.198	0.037	18.69
	Positive precision reference	0.375	0.032	8.53
	Strong positive precision reference	0.843	0.045	5.34

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