Heparin sodium salt from porcine intestinal mucosa grade 1 a

Heparin sodium salt from porcine intestinal mucosa Grade I-A (Sigma-Aldrich) or hyaluronic acid sodium salt from bovine vitreous humor (Sigma-Aldrich) were diluted in PBS and added at a concentration of 25 μg/ml on BD heparin-binding plates (BD Biosciences, San Jose, CA) and incubated overnight at room temperature [55 (link)]. Plates coated with heparin or hyaluronic acid were washed with PBS and blocked with 3% BSA in PBS blocking solution for 1 h at 37°C, followed by washing with PBS. The antibody-cytokine fusion proteins or equivalent molar amount of control IgG3 were incubated in triplicate using serial four-fold dilutions ranging from 0.06 μg/mL to 4 μg/mL for 2 h at 37°C. For the competition study, wild type IgG3-(IL-12) was also incubated in triplicates in the presence of 5.5 μg/ml of heparin or equivalent molar amount of hyaluronic acid. The plates were then washed and incubated with a goat anti-human κ AP-conjugated antibody (Sigma-Aldrich) 1:30,000 in 1% BSA in PBS for 1 h at 37°C. After washing, 1 mg/ml of AP substrate (p-nitrophenyl phosphate disodium, Sigma-Aldrich) dissolved in diethanolamine buffer [9.6% diethanolamine (v/v), 0.24 mM MgCl₂, in water (pH 9.8)] was added and incubated for 60 min at room temperature before measuring the absorption at 405 nm using a FilterMax F5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA).

MB (98%) and PBS tablets were purchased from Fisher. Dissolving one PBS tablet in 200 mL deionized water should result in 0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M NaCl, and pH 7.4 at 25 °C. Heparin sodium salt from porcine intestinal mucosa (grade I-A, ≥ 180 U/mg) and protamine sulfate salt from salmon were purchased from Sigma. Heparin (sodium injection, 1000 United States Pharmacopeia (USP) U/mL) was purchased from SAGENT pharmaceuticals. Deuterium oxide (D, 99.9%) was purchased from Cambrige Isotope Laboratories, Inc. Enoxaparin sodium (LMWH) injection was purchased from Winthrop. Laboratory polyethylene tubing (OD: 1.27 mm, ID: 0.85 mm) was purchased from Harvard Apparatus.

Fibronectin was purified from fresh frozen rat plasma by gelatin-Sepharose affinity chromatography [47 (link)]. Heparin sodium salt from porcine intestinal mucosa (Grade I-A, ≥180 USP units/mg) (unfractionated heparin) was obtained from Sigma (St. Louis, MO). Different sized heparin GAG chains, H016 (average molecular weight 4650) and H030 (average molecular weight >9000), were obtained from Iduron (Galen Laboratory Supplies, Middletown, CT).