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.
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.
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