Complement c3 mouse elisa kit

Circulating C3 concentration was quantified using Complement C3 Mouse ELISA kit according to manufacturer's instructions (Ca ab157711, Abcam, Cambridge, UK).
Circulating C3a concentration was determined by ELISA as previously described [29 (link)]. For capture, we used a rat anti-mouse antibody with specificity for the neoepitope generated by the cleavage of C3 and not recognizing intact C3 (Ca 558250, BD Pharming, Oxford, UK). Plates were coated with antibody diluted to 2 μg/mL in borate buffered saline followed by overnight incubation at 4°C and subsequently blocked with sample buffer (PBS, 2% BSA, 0.1% tween-20, 0.02% NaN₃). Plasma was incubated overnight at 4°C in sample buffer. Purified mouse C3a protein was used as standard (Ca 558618, BD Pharming, Oxford, UK). For detection we used biotinylated rat anti-mouse C3a antibodies (0.5 μg/mL in sample buffer, Ca 558251, BD Pharming, Oxford, UK) which was incubated for 5 h at 4°C. This was followed by addition of streptavidin-alkaline phosphatase and later development with alkaline phosphatase substrate.

Supernatant of primary mouse astrocytes culture was collected and was filtered using a 0.22 μm syringe filter to remove the cellular debris. C3 secretory protein level was determined by using Mouse Complement C3 ELISA Kit (cat#ab157711, Abcam) according to the manufacturer's instructions.

Analysis of pro-inflammatory cytokines plasma levels was conducted using NHP and mouse-specific multiplex assay (K15056D and K15048D, respectively; Meso Scale Diagnostics, Rockville, MD). In addition, cynomolgus monkey MCP-1 and TNFα were analyzed using single analyte assay kits (K156UCK, K156NND, respectively; Meso Scale Diagnostics). Similarly, mouse MCP-1 was analyzed using single-plex assay kit (K152NN, Meso Scale Diagnostics).
Mouse C3 complement component levels were analyzed using Mouse Complement C3 ELISA Kit (ab157711, Abcam, Waltham, MA). C5b9 levels in cynomolgus monkey plasma were analyzed using Human C5b9 ELISA Kit (558315, BD, Franklin Lakes, NJ).

Concentration of C3 protein in the serum was measured using the mouse complement C3 ELISA kit (Abcam, ab157711) according to the manufacturer’s protocols. Briefly, serum of each mouse was diluted 1:75,000 and pipetted into designated wells of kit. The plate was incubated for 20 min at room temperature, washed, and a 1× enzyme-antibody conjugate was added. After incubation for 20 min at room temperature and additional washing, TMB substrate was added and the color alteration was determined by using a Vmax plate reader (Molecular Devices) at 450 nm.