Cd44 3961h

The BIAcore 3000 system (GE Healthcare Co. Ltd.) was used to determine the direct protein interaction between the full‐length PTX3 (#1826‐TS; R&D Systems) and the N‐terminal of CD44 (#CD44‐3961H; Creative Biomart). Full‐length PTX3 was immobilised onto a CM5 sensor chip (GE Healthcare Co. Ltd.); the immobilisation level was 4000–6000 response units using amine‐coupling chemistry. Briefly, a mixture of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide activated the surface of the chip, and then diluted full‐length PTX3 (10 μg/ml in acetate buffer pH 3) was injected into the flow cells for immobilisation. Finally, the remaining activated groups were blocked by ethanolamine. The binding parameters of the experiment were measured over a 1‐min association phase and then followed by a 2‐min dissociation phase at a fluid flow rate of 10 μl/min. Phosphate‐buffered saline (PBS, pH 7.2) was used as the analyte running buffer; 0.05% sodium dodecyl sulphate was used to regenerate the chip surface before the next analyte concentration was applied. The 1:1 Langmuir binding model (BIAevaluation 4.1; GE Healthcare Co. Ltd.) were performed to calculate its corresponding association and dissociation rate constants to determine the affinity.

The intrinsic biological functions (interactions of polymers with their corresponding antibodies) of HA and gelatin were evaluated via immunofluorescence assays (anti-CD44 and anti-gelatin antibodies, respectively) [67 (link),68 (link)]. Briefly, cryogels were saturated for 1 h at RT with 1X PBS + 0.05% Tween-20 + 1% bovine serum albumin (BSA). Next, cryogels were incubated for 3 h at RT with either 10µg/mL of recombinant human CD44 His-tagged (CD44-3961H, Creative Biomart, Shirley, NY, USA) or anti-porcine gelatin anti-serum (GELP12-S, Alpha Diagnostic International, San Antonio, TX, USA) diluted 1:100 in 1X PBS + 0.05% Tween-20 + 1% BSA. Next, cryogels were treated for 1 h at RT in the dark with Alexa Fluor 488-conjugated 6x-His Tag monoclonal antibody (Fisher Scientific) or FITC-conjugated anti-rabbit IgG (Fc-region) (Alpha Diagnostic International), respectively. Cryogels were washed 3 times in 1X PBS + 0.05% Tween-20 between each step. Methacrylated alginate (MA-alginate) cryogels polymerized as previously described [20 (link)] were used as a negative control for these experiments.