Anti mouse c3a monoclonal antibody

A C3a sandwich ELISA was performed to measure complement activation in mouse serum following incubation with NPs. EIA plates (Corning 9018) were coated with an anti-mouse C3a monoclonal antibody (BD Biosciences, clone I87-1162) diluted 1:250 in coating buffer (eBioscience) overnight at 4 °C. Mouse serum was incubated 1:1 with either PBS or NPs at 37 °C for 50 min. Serial dilutions of purified mouse C3a protein (BD Biosciences) were included in each ELISA plate to establish a standard curve. Serum samples were added to wells in duplicate (50 μL total volume) and incubated for 3 h. Anti-C3a-biotinylated detection antibody (BD Biosciences, clone I87-419) was used at a 1:500 dilution in 1× assay diluent, and incubated for 40 min. Streptavidin-HRP (BD) was diluted 1:250 in 1× assay diluent for 30 min. 1× TMB substrate solution (eBioscience) was added to develop color. The reaction was stopped with 0.2 N H₂SO₄, and absorbance was read at 450 nm with a reference wavelength of 570 nm on a SpectraMax (Molecular Devices) plate reader.

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.