The increasing evidence of the implication of the complement system in the pathogenesis of several diseases has emphasized the need for the development of specific and valid assays, optimized for quantitative detection of complement activation in vivo. In the present study, we have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C4c without interference from other products generated from the complement component C4. The C4c specific mAb was tested in different enzyme-linked immunosorbent assay (ELISA) combinations with various types of in vitro activated sera and samples from factor I deficient patients. The specificity of the mAb was further evaluated by immunoprecipitation techniques and by analysis of eluted fragments of C4 after immunoaffinity chromatography. The anti-C4c mAb was confirmed to be C4c specific, as it showed no cross-reactivity with native (un-cleaved) C4, C4b, iC4b, or C4d. Also, no reaction was observed with C4 fragments in factor I deficient plasma or serum samples. We established and validated a sandwich ELISA based on this C4c specific antibody. The normal range of C4c in EDTA/futhan plasma collected from 100 Danish blood donors was measured, with a mean of 0.85mg/L and a range of 0.19-2.21mg/L. We believe that the C4c specific antibody and the ELISA might be important tools in the future assessment of in vivo activation in situations where the classical or the lectin complement pathways are involved in the pathogenesis.
Journal of Immunological Methods, 2014, Vol 405, p. 87-96