Pdgf bb duoset elisa

ELISA plates (Medium binding, Greiner Bio-one) were coated with 100 nM of ECM proteins in 50 μl of PBS for 1 h at 37 °C. Then, wells were washed with PBS-T (PBS with 0.05% Tween-20) and blocked with 300 μl PBS-T containing 1% BSA for 1 h at room temperature. ECM and control wells (no ECM and blocked with BSA) were further incubated 1 h at room temperature with solutions of PDGF-BB variants or IL-1Ra variants at increasing concentrations (50 μl in PBS-T containing 0.1% BSA; PROKEEP tubes, Watson bio lab). Then, wells were washed three times with PBS-T and bound PDGF-BB and IL-1Ra variants were detected using biotinylated antibodies in PBS-T containing 0.1% BSA. Detection antibodies were from PDGF-BB DuoSet ELISA (R&D Systems, DY8464) for PDGF-BB and IL-1Ra/IL-1F3 DuoSet ELISA (R&D Systems, DY480) for IL-1Ra. To calculate the dissociation constants (K_D) specific-binding values were fitted with a one site specific binding model using A₄₅₀ nm = B_max*[PDGF-BB or IL-1Ra variants]/(K_D + [PDGF-BB or IL-1Ra variants]).

The release of bone-related growth factors from HPS-F and NS-F was evaluated over 7 days. The growth factors examined included vascular endothelial growth factor-A (VEGF-A), OPG, OPN, platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF), and sRANKL. For the experimental setup, HPS-F and NS-F were prepared as described in Section 4.3 for 1 mL of HPS and NS, respectively, and transferred into a 5 mL tube (Eppendorf, Hamburg, Germany). After a gelation time of 1 min, 1 mL of PBS was added, and the tubes were incubated on a shaker plate at room temperature. The supernatants were collected after 12 h and after 1, 2, 3, 4, 5, 6, and 7 days. Samples were stored at −80 °C until ELISA measurements were taken. ELISAs were carried out according to the manufacturing protocol (VEGF DuoSet ELISA (Catalog # DY293B); OPG DuoSet ELISA (Catalog # DY805); OPN DuoSet ELISA (Catalog #: DY1433) or PDGF-BB DuoSet ELISA (Catalog #: DY220); bFGF DuoSet ELISA (Catalog #: DY233) R&D Systems, Minneapolis, MN, USA). Readout was performed through measurement of the optical density using a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).

ELISA plates (Medium binding, Greiner Bio-one) were coated with 100 nM of ECM proteins in 50 μl of PBS for 1 h at 37 °C; human plasma fibronectin (Sigma), human vitronectin (Peprotech), human tenascin C (R&D Systems), human fibrinogen (Enzyme Research Laboratories). Then, wells were washed with PBS-T (PBS with 0.05% Tween-20) and blocked with 300 μl PBS-T containing 1% BSA for 1 h at room temperature. ECM and control wells (no ECM and blocked with BSA) were further incubated 1 h at room temperature with solutions of murine PDGF-BB (Peprotech), IL-1Ra (R&D Systems) or PlGF_123–141-fused proteins at concentrations ranging from 0 to 100 nM (50 μl in PBS-T containing 0.1% BSA; PROKEEP tubes, Watson bio lab). Then, wells were washed three times with PBS-T and bound PDGF-BB and IL-1Ra variants were detected using biotinylated antibodies in PBS-T containing 0.1% BSA. Antibodies used were from PDGF-BB DuoSet ELISA (R&D Systems, DY8464) for PDGF-BB and IL-1Ra/IL-1F3 DuoSet ELISA (R&D Systems, DY480) for IL-1Ra. To calculate the dissociation constants (K_d) specific-binding values were fitted with a one site-specific binding model using A₄₅₀ nm = B_max*[growth factors or IL-1Ra variants]/(K_d + [growth factors or IL-1Ra variants]).