Microvue ic3b eia kit

Human blood containing sodium citrate as the anti-coagulant was subjected to centrifugation at 2500g for 10 minutes and plasma was collected. In a microcentrifuge tube, 100 μL each of Veronal buffer (Boston Bioproducts, Ashland, MA), plasma and test sample were mixed and incubated at 37 °C for 30 minutes. Appearance of iC3b was measured using the MicroVue iC3b EIA kit (Quidel Corp., San Diego, CA) following manufacturer’s instructions. As a positive control, the vehicular composition of Taxol® was prepared by mixing Cremophor-EL (Milliore Sigma, Burlington, MA) 1:1 with ethanol containing 2 mg/mL citric acid.

Effects of SELP nanogels on the human complement system were studied by enzyme immunoassay (EIA) via a commercial MicroVue iC3b EIA kit (Quidel Corporation, San Diego, CA, USA). Pooled human plasma was obtained from four healthy donors following collection, anticoagulation with sodium citrate, and centrifugation (10 min, 2500 g). Samples were immediately mixed with plasma and veronal buffer at a 1:1:1 ratio, incubated at 37 °C for 30 min, immediately frozen, and stored in a −80 °C freezer until use in EIA. The EIA was performed following manufacturer’s protocol. Tested sample concentrations during plasma incubation included SELP 815K (0.667 mg mL⁻¹) in PBS, DOX (52.0 μg mL⁻¹) in PBS, SELP 815K + DOX (SELP at 0.667 mg mL⁻¹, DOX at 52.0 μg mL⁻¹, sample created following loading protocol in 2.7), and SELP 815K-RS1 and SELP 815K-RS2 (both at 0.667 mg mL⁻¹). All samples were additionally tested at 5x, 25x, and 125x dilution concentrations. Positive controls (Cremophor EL), negative controls (PBS), vehicle controls (DMSO at 0.1% in PBS), and inhibition/enhancement controls were also tested.

Purified C3, C3b, and iC3b were obtained from Complement Technologies (Tyler, TX). Monoclonal antibodies to C3 fragments were obtained from Quidel (San Diego, CA). Polyclonal C3 antibodies were obtained from Dako (Carpinteria, CA) and MP Biomedicals (Solon, OH). The MicroVue iC3b EIA Kit was obtained from Quidel.