Thrombospondin-2 (TSP-2) is a 150 kDa calcium-binding protein.  TSP-2 inhibits angiogenesis through direct effect on endothelial cell migration, proliferation, survival and apoptosis.  It is a potential risk factor in heart disease.

This assay is a quantitative sandwich ELISA. The immunoplate is pre-coated with a monoclonal antibody specific for human TSP-2. Standards and samples are pipetted into the wells and any human TSP-2 present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled polyclonal antibody specific for human TSP-2 is added to the wells. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STP-HRP) is added. After the last wash step, an HRP substrate solution is added and colour develops in proportion to the amount of human TSP-2 bound initially.  The assay is stopped and the optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the amount of captured human TSP-2, the unknown sample concentration can be interpolated from a reference curve included in each assay.

A. Typical representation of standard curve

The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.  

Human TSP-2 (ng/mL)	Absorbance  (450 nm)	Blanked Absorbance
0	0.094	0
0.156	0.133	0.039
0.312	0.187	0.093
0.625	0.293	0.199
1.25	0.474	0.38
2.5	0.812	0.718
5	1.317	1.223
10	2.096	2.002

 

B. Sensitivity

The lowest insulin level that can be measured by this assay is 0.156 ng/mL.

 

C. Precision

Intra-assay Precision (Precision within an assay) C.V. <4.6%.

Inter-assay Precision (Precision between assays) C.V. <7.2%.

1. Lee CH, Seto WK, Lui DT, Fong CH, Wan HY, Cheung CY, Chow WS, Woo YC, Yuen MF, Xu A, Lam KS. Circulating thrombospondin-2 as a novel fibrosis biomarker of nonalcoholic fatty liver disease in type 2 diabetes. Diabetes Care. 2021 Sep 1;44(9):2089-97.
2. Wu X, Cheung CKY, Ye D, Chakrabarti S, Mahajan H, Yan S, Song E, Yang W, Lee CH, Lam KSL, Wang C, Xu A. Serum thrombospondin-2 levels are closely associated with the severity of metabolic syndrome and metabolic associated fatty liver disease. The Journal of Clinical Endocrinology & Metabolism. 2022 Jul 14, 107(8):e3230-e3240.
3. Lee CH, Wu MZ, Lui D, Fong C, Ren QW, Yu SY, Yuen M, Chow WS, Huang JY, Xu A, Yiu KH, Lam K. Prospective associations of circulating thrombospondin-2 level with heart failure hospitalization, left ventricular remodeling and diastolic function in type 2 diabetes. Cardiovasc Diabetol. 2022 Nov 5;21(1):231.
4. Lin Z, Zhang D, Zhang X, Guo W, Wang W, Zhang Y, Liu Z, Bi Y, Wu M, Lin Z, Lu X. Extracellular status of thrombospondin-2 in type 2 diabetes mellitus and utility as a biomarker in the determination of early diabetic kidney disease. BMC Nephrol. 2023 May 31;24(1):154.
5. Lee CH, Lui DT, Cheung CY, Fong CH, Yuen MM, Chow WS, Xu A, Lam KS. Circulating thrombospondin-2 level for identifying individuals with rapidly declining kidney function trajectory in type 2 diabetes: a prospective study of the Hong Kong West Diabetes Registry. Nephrol Dial Transplant. 2023 Feb 28:gfad034.

6. Cheung JHM, Lui DTW, Fong CHY, Cheung CYY, Wong Y, Lee ACH, Hoo RLC, Xu A, Tan KCB, Lam KSL, Lee CH. Serum thrombospondin-2 level changes with liver stiffness improvement in patients with type 2 diabetes. Clinical Endocrinology. 2024 Mar;100(3):230-237.