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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (4): 30-36    DOI: 10.13523/j.cb.2101031
技术与方法     
血栓调节蛋白化学发光免疫分析检测方法的建立*
李帅鹏1,任和1,安展飞2,杨艳坤1,白仲虎1,**()
1 江南大学 粮食发酵工艺与技术国家工程实验室 无锡 214122
2 江苏拜明生物技术有限公司 盐城 224005
The Development of Chemiluminescence Immunoassay Detection Method for Thrombomodulin
LI Shuai-peng1,REN He1,AN Zhan-fei2,YANG Yan-kun1,BAI Zhong-hu1,**()
1 National Engineering Laboratory of Grain Fermentation Technology, Jiangnan University, Wuxi 214122, China
2 Jiangsu Baiming Biotechnology Co., Ltd., Yancheng 224005, China
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摘要：

目的:建立并评价基于板式化学发光免疫分析(CLIA)平台的血栓调节蛋白(TM)定量检测方法。方法:以链霉亲和素包被微孔板,加入待检血浆,偶联生物素和辣根过氧化物酶的配对抗体组成分析体系,采用双抗体夹心模式,建立TM抗原定量检测方法,并对其进行条件优化和性能评价。结果:生物素化抗体和酶标抗体的工作浓度分别为0.5 μg/mL和0.75 μg/mL,加样后的孵育时间选为15 min,最低检测限为0.2 TU/mL,该检测方法的检测范围为1~200 TU/mL,批间和批内精密度(CV)均小于8%,37 ℃ 10天稳定性良好,207份临床血浆测值与希森美康测值相关性较高(R2>0.96)。结论:建立了TM板式化学发光定量检测方法,且各项性能指标良好,可满足临床检测的需要。
关键词： 血栓调节蛋白;  化学发光免疫分析;  检测方法;  性能评估    
Abstract:

Objective: To establish and evaluate a quantitative detection method for thrombomodulin (TM) based on a plate chemiluminescence immunoassay (CLIA) platform. Methods: The analysis system was composed of microplate coated with streptavidin, serum, the paired antibody coupled biotin and horseradish peroxidase. The double antibody sandwich mode was adopted to test quantitatively TM antigen. Moreover,the reaction conditions of TM were optimized and the analytical performance was evaluated. Results: The working concentration of biotinylated antibody and enzyme-labeled antibody was 0.5 μg/mL and 0.75 μg/mL, respectively. The incubation time was 15 min; the minimum detection limit was 0.2 TU/mL and the range of the detection method was 1~200 TU/mL. Inter-assay and intra-assay precision (CV) were less than 8%. The established method had good stability during 10 days at 37 ℃. 207 clinical plasma measured values had a high correlation with Sysmex measured values (R2>0.96). Conclusion: This study has successfully established a quantitative detection method of TM plate chemiluminescence, which has good performance indicators and can meet the needs of clinical detection.

Key words: Thrombomodulin    Chemiluminescence immunoassay    Detection method    Performance evaluation
收稿日期: 2021-01-25 出版日期: 2021-04-30
ZTFLH:  Q819  
基金资助: *江苏省重点研发计划资助项目(BE2018621)
通讯作者: 白仲虎     E-mail: baizhonghu@jiangnan.edu.cn
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李帅鹏
任和
安展飞
杨艳坤
白仲虎

引用本文:

李帅鹏,任和,安展飞,杨艳坤,白仲虎. 血栓调节蛋白化学发光免疫分析检测方法的建立*[J]. 中国生物工程杂志, 2021, 41(4): 30-36.

LI Shuai-peng,REN He,AN Zhan-fei,YANG Yan-kun,BAI Zhong-hu. The Development of Chemiluminescence Immunoassay Detection Method for Thrombomodulin. China Biotechnology, 2021, 41(4): 30-36.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2101031    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I4/30

Biotin-AB concentration
(μg/mL)
HRP-AB concentration
(μg/mL)		 0.5 0.75 1
0.5 0.75 1 0.5 0.75 1 0.5 0.75 1
标准品1的RLU 4 105 5788 9 814 7 654 11 037 14 438 9 352 13 767 16 156
标准品2的RLU 24 518 39 609 47 814 35 654 49 036 60 438 43 352 61 167 78 251
标准品3的RLU 92 711 155 834 192 167 136 761 205 767 282 706 173 866 259 202 380 207
标准品4的RLU 470 111 680 783 767 481 677 416 793 734 883 892 727 131 880 548 1 242 238
标准品5的RLU 992 165 1 581 074 1 917 085 1 3587 29 1 884 604 2 373 687 1 584 499 2 266 596 2 873 804
标准品6的RLU 2 405 635 3 409 013 3 983 793 2 922 509 3 665 265 4 365 232 3 325 649 3 975 402 4 574 529
表1  生物素化抗体和酶标抗体工作浓度
图1  不同孵育时间对标准品的影响
图2  孵育时间对样本影响
Theoretical concentration
(TU/mL)		 Measured concentration (TU/mL) 偏差
重复1 重复2 重复3 重复4 重复5
质控品1 5.06 4.89 4.81 4.72 4.92 4.86 -4.3%
质控品2 119.53 124.31 125.95 124.17 122.46 123.98 3.9%
表2  准确度检测
Control 1 Control 2
Batch 1 Batch 2 Batch 3 Batch 1 Batch 2 Batch 3
平均值(TU/mL) 4.94 4.96 4.99 117.64 121.52 118.73
批内CV 4.3% 3.9% 5.9% 5.2% 5.5% 6.0%
批间CV 4.7% 5.5%
表3  质控品精密度检测
时间 Control 1 Control 2
5.12 TU/mL 121.37 TU/mL
第1天 重复1 5.14 118.75
(TU/mL) 重复2 5.16 117.63
重复3 5.21 119.27
偏差 1.0% -2.3%
第3天 重复1 5.08 116.96
(TU/mL) 重复2 5.06 115.74
重复3 5.04 114.69
偏差 -1.2% -4.6%
第6天 重复1 4.92 114.32
(TU/mL) 重复2 4.94 113.98
重复3 4.88 112.17
偏差 -4.0% -6.5%
第10天 重复1 4.75 112.32
(TU/mL) 重复2 4.69 113.98
重复3 4.71 112.17
偏差 -7.9% -7.0%
表4  加速稳定性实验结果
图3  本方法与希森美康试剂检测相关性
Performance index Manufacturer
Sysmex This study
检测范围(TU/mL) 1~200 1~200
批内CV 1.95% 6.0%
批间CV 3.25% 5.5%
最低检测限(TU/mL) 0.34 0.2
检测时间(min) 14 16
37 ℃稳定性 -5.8% -7.9%
血浆对比相关性 相关系数(R2)>0.96
表5  试剂性能对比结果
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