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中国生物工程杂志

China Biotechnology
China Biotechnology  2021, Vol. 41 Issue (4): 30-36    DOI: 10.13523/j.cb.2101031
    
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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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 wordsThrombomodulin      Chemiluminescence immunoassay      Detection method      Performance evaluation     
Received: 25 January 2021      Published: 30 April 2021
ZTFLH:  Q819  
Corresponding Authors: Zhong-hu BAI     E-mail: baizhonghu@jiangnan.edu.cn
Cite this article:

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.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2101031     OR     https://manu60.magtech.com.cn/biotech/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
Table 1 Influence of the concentration of HRP labeled antibody and biotinylated antibody
Fig.1 Influence of the incubation time (a) Standard 2 and 3 (b) Standard 4, 5 and 6
Fig.2 Effect of incubation time on samples
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%
Table 2 Accuracy test results
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%
Table 3 Control precision test results
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%
Table 4 Accelerated stability test results
Fig.3 Linear correlation between the developed method and Sysmex kit
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
Table 5 Comparison results of reagent performance
[1]   Maruyama I, Bell C E, Majerus P W. Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta. Journal of Cell Biology, 1985,101(2):363-371.
[2]   郑娜娜. 血栓调节蛋白对肺癌细胞致瘤性和迁移的抑制作用及其机制. 苏州: 苏州大学, 2016.
[2]   Zheng N N. Thrombomodulin inhibits tumorigenesis and migration of lung cancer cells and its mechanism. Suzhou: Soochow University, 2016.
[3]   Morser J. Thrombomodulin links coagulation to inflammation and immunity. Current Drug Targets, 2012,13(3):421-431.
pmid: 22206250
[4]   Dittman W A, Majerus P W. Structure and function of thrombomodulin: a natural anticoagulant. Blood, 1990,75(2):329-336.
[5]   Conway E M. The type XIV family of C-type lectin-like domain (CTLD) containing proteins. Current Drug Targets, 2012,13(3):409-410.
pmid: 22206248
[6]   Loghmani H, Conway E M. Exploring traditional and nontraditional roles for thrombomodulin. Blood, 2018,132(2):148-158.
[7]   Owen W G, Esmon C T. Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. The Journal of Biological Chemistry, 1981,256(11):5532-5535.
[8]   Anastasiou G, Gialeraki A, Merkouri E, et al. Thrombomodulin as a regulator of the anticoagulant pathway: implication in the development of thrombosis. Blood Coagulation & Fibrinolysis, 2012,23(1):1-10.
pmid: 22036808
[9]   Nishizawa S, Kikuta J, Seno S, et al. Thrombomodulin induces anti-inflammatory effects by inhibiting the rolling adhesion of leukocytes in vivo. Journal of Pharmacological Sciences, 2020,143(1):17-22.
pmid: 32122774
[10]   王涤非, 刘国良, 吴桂萍, 等. 糖尿病血浆血栓调节蛋白水平的测定. 中国医科大学学报, 2000,29(5):368-369.
[10]   Wang D F, Liu G L, Wu G P, et al. Measurement of soluble thrombomodulin in plasma from diabetes. Journal of China Medical University, 2000,29(5):368-369.
[11]   林静, 孙志鹏, 李娟, 等. 联合检测TM、TAT、PIC、t-PAIC水平对DIC的诊断价值. 国际检验医学杂志, 2019,40(12):1413-1416.
[11]   Lin J, Sun Z P, Li J, et al. Value of combined detection of the levels of serum TM, TAT, PIC and t-PAIC for the early diagnosis of disseminated intravascular coagulation. International Journal of Laboratory Medicine, 2019,40(12):1413-1416.
[12]   Luo Y Q, Liu T, Zhu J M, et al. Label-free and sensitive detection of thrombomodulin, a marker of endothelial cell injury, using quartz crystal microbalance. Analytical Chemistry, 2015,87(22):11277-11284.
[13]   周齐洋, 黄建荣, 陈祥, 等. 血清淀粉样蛋白A化学发光免疫检测技术开发. 生物学杂志, 2019,36(4):85-88.
[13]   Zhou Q Y, Huang J R, Chen X, et al. Development of chemiluminescent immunoassay for human serum amyloid A. Journal of Biology, 2019,36(4):85-88.
[14]   Huo J Q, Barnych B, Li Z F, et al. Hapten synthesis, antibody development, and a highly sensitive indirect competitive chemiluminescent enzyme immunoassay for detection of dicamba. Journal of Agricultural and Food Chemistry, 2019,67(20):5711-5719.
doi: 10.1021/acs.jafc.8b07134 pmid: 31042038
[15]   何旭华, 徐也鲁, 王振义. 人血栓调节蛋白的分离提纯及其功能特性的初步研究. 浙江医科大学学报, 1988,17(1):1-5.
[15]   He X H, Xu Y L, Wang Z Y. Isolation and purification of human thrombomodulin and preliminary study on its functional properties. Journal of Zhejiang Medical University, 1988,17(1):1-5.
[16]   周泉生, 奚晓东, 李佩霞, 等. 一组抗人血栓调节蛋白单克隆抗体的建株. 苏州医学院学报, 1991,11(4):259-261,340.
[16]   Zhou Q S, Xi X D, Li P X, et al. Establishment of a group of monoclonal antibodies against human thrombomodulin, Suzhou University Journal of Medical Science, 1991,11(4):259-261,340.
[17]   Hosaka Y, Higuchi T, Tsumagari M, et al. Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin. Cancer Letters, 2000,161(2):231-240.
pmid: 11090974
[18]   Horowitz N A, Blevins E A, Miller W M, et al. Thrombomodulin is a determinant of metastasis through a mechanism linked to the thrombin binding domain but not the lectin-like domain. Blood, 2011,118(10):2889-2895.
[19]   Chang Y J, Cheng Y W, Lin R K, et al. Thrombomodulin influences the survival of patients with non-metastatic colorectal cancer through epithelial-to-mesenchymal transition (EMT). PLoS One, 2016,11(8):e0160550.
doi: 10.1371/journal.pone.0160550 pmid: 27512995
[20]   Kajioka H, Kagawa S, Ito A, et al. Targeting neutrophil extracellular traps with thrombomodulin prevents pancreatic cancer metastasis. Cancer Letters, 2021,497:1-13.
[21]   Karakas M, Baumert J, Herder C, et al. Soluble thrombomodulin in coronary heart disease: lack of an association in the MONICA/KORA case-cohort study. Journal of Thrombosis and Haemostasis, 2011,9(5):1078-1080.
[22]   Nakanishi K, Masuda K, Mori N, et al. Evaluation of analytical performance of HISCL TM, a chemiluminescent enzyme immunoassay and the use of thrombomodulin as a marker for endothelial dysfunction. Rinsho byori. The Japanese Journal of Clinical Pathology, 2014,62(10):942-947.
pmid: 27526539
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