Mouse recombinant c3a

All animal experiments were performed in compliance with federal laws and in strict accordance with the guidelines established by the Division of Comparative Medicine at Washington University. The animal protocol is subjected to annual review and approval by The Animal Studies Committee of Washington University. Mice (n=33, ≥ 5/treatment group) were injected i.v. with PBS (negative control) or nanoparticles at 5 μl/g of body weight and plasma was obtained at 30 min for C3a ELISA. ELISA plates were coated overnight at 4°C with anti-mouse C3a monoclonal antibody (4 μg/ml; BD Pharmingen). After blocking with 1% BSA, the plates were washed and incubated with samples (100 μl of fresh plasma diluted 1:100 in PBS) for 2 h at room temperature, followed by biotinylated anti-mouse C3a monoclonal antibody (250 ng/ml; BD Pharmingen, San Jose, CA). Following a 20 min incubation with streptavidin-peroxidase (400 ng/ml; Sigma), 100 μl of peroxide-chromogen solution (R&D Systems, Minneapolis, MN) was added to each well, and color development was read at 450 nm with a SpectraMax Plus reader (Molecular Devices, Sunnyvale, CA). Mouse recombinant C3a (BD Pharmingen) was used to establish the standard curve.

Adult male C57BL/6 mice (8–12 weeks) were immunized i.p. on day 0 and day 7 with 20 µg OVA suspended in 2.25 mg of aluminum hydroxide (total volume 100 µL). On days 14, 15, 16 mice were challenged intranasally (i.n.) with 30 µg OVA in PBS (total volume 30 µL) following anesthesia with isoflurane. Mice were sacrificed on day 17 and their bronchoalveolar lavage fluid (BALF) and lungs were harvested for analysis and histology. In some experiments mice were also administered heat inactivated or active Salp20 (9 µg i.n.) concomitantly with OVA (total volume 45 µL).
IL-13 level in the BALF was measured with an ELISA kit (cat#DY413, R&D Systems). For C3a ELISA, BALF (100 µL) was applied to plates coated with anti-mouse C3a monoclonal antibody (4 µg/mL, cat#558250, BD Pharmingen) and incubated for 2 h at RT, followed by biotinylated anti-mouse C3a monoclonal antibody (250 ng/mL, cat#558251, BD Pharmingen). After washing and incubation with streptavidinperoxidase (400 ng/mL, cat#890803, R&D Systems), 100 µL of peroxide-chromogen solution (cat#DY999, R&D Systems) was added to each well and color development was read at 450 nm with a SpectraMax Plus Reader (Molecular Devices). Mouse recombinant C3a (Cat# 558618 BD Pharmingen) was used to establish the standard curve.

The extent of complement activation following nanoparticle injection was determined by C3a ELISA as reported previously^{39 (link),40} . Briefly, wild type C57BL/6J mice were injected with 30 μL of nanoparticles or phosphate-buffered saline (PBS, negative control). After 30 minutes, their plasma was collected in EDTA containing tubes to inhibit further ex vivo complement activation. For the ELISA, plates were coated overnight at 4°C with anti-mouse C3a (4 μg/mL) monoclonal antibody (BD Pharmingen, San Jose, CA). After being blocked with 1% BSA, the plates were washed and incubated with samples (100 μL of fresh plasma diluted 1:100 in 1% BSA in PBS) for 2 hours at room temperature, followed by biotinylated anti-mouse C3a (250 ng/mL) monoclonal antibody (BD Pharmingen). After washes and incubation with streptavidin-peroxidase (400 ng/mL; Sigma-Aldrich, St. Louis, MO), 100 μL of peroxide-chromogen solution (R&D Systems, Minneapolis, MN) was added to each well, and color development was read at 450 nm with a SpectraMax Plus reader (Molecular Devices, Sunnyvale, CA). Mouse recombinant C3a (BD Pharmingen) was used to establish the standard curve. 50 mol% N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium methyl-sulfate (DOTAP) nanoparticles served as positive controls for robust complement activation.