Development of an enzyme-linked immunosorbent assay for thrombospondin-1 and comparison of human plasma and serum concentrations

Abstract

Background: Thrombospondin-1 (TSP-1) is a circulating matricellular glycoprotein produced from many cell types including platelets. Currently TSP-1 is measured in either plasma or serum, using expensive commercial assays. Aim: To develop and validate a cost effective in-house immunoassay for human TSP-1 suitable for quantitating levels from both plasma and serum. Methods: An in-house enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human TSP-1. Sixteen healthy volunteers (8 male and 8 female), mean age 29 years (range 21–49), body mass index (BMI) mean 23.3 kg/m2 (range 17.3–26.7) had non-fasted venous blood sampled at 0800 h and 1600 h for both plasma and serum TSP-1. Results: The assay limit of quantitation was 7.8 μg/L, inter assay CV was 17–31%, intra assay CV was 3–4% for plasma and <9% for serum. Plasma TSP-1 ranged from 133 to 478 μg/L (mean concentration 290 μg/L) in normal volunteers. Serum TSP-1 was approximately 100-fold higher, ranging from 13,700 to 44,400 μg/L (mean concentration 257,00 μg/L). There was no correlation between plasma and serum TSP-1. Conclusions: TSP-1 can be readily measured in human plasma using ELISA. Serum concentrations are 100-fold higher, reflecting documented TSP-1 release by platelets, and does not provide a meaningful measure of circulating concentrations.