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

Johanna L Barclay, Sahar Keshvari, Jonathan P Whitehead, Warrick J Inder

BackgroundThrombospondin-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.AimTo develop and validate a cost effective in-house immunoassay for human TSP-1 suitable for quantitating levels from both plasma and serum. MethodsAn 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/m(2) (range 17.3 - 26.7) had non-fasted venous blood sampled at 0800h and 1600h for both plasma and serum TSP-1.ResultsThe assay limit of quantitation was 7.8 ng/mL, 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 ng/mL (mean concentration 290 ng/mL) in normal volunteers. Serum TSP-1 was approximately 100-fold higher, ranging from 13 700 to 44 400 ng/mL(mean concentration 257 00 ng/mL). There was no correlation between plasma and serum TSP-1. ConclusionsTSP-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.