Johanna L Barclay1, Sahar Keshvari2, Jonathan P Whitehead3, Warrick J Inder4. 1. Mater Research Institute, University of Queensland, Brisbane, Australia Translational Research Institute, Brisbane, Australia johanna.barclay@mater.uq.edu.au. 2. Mater Research Institute, University of Queensland, Brisbane, Australia Translational Research Institute, Brisbane, Australia School of Medicine, the University of Queensland, Brisbane, Australia. 3. Mater Research Institute, University of Queensland, Brisbane, Australia Translational Research Institute, Brisbane, Australia. 4. Translational Research Institute, Brisbane, Australia Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Brisbane, Australia School of Medicine, the University of Queensland, Brisbane, Australia.
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/m(2) (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.
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 humanTSP-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 humanTSP-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 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.
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