A specific assay for quantification of human C4c by use of an anti-C4c monoclonal antibody.
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.
Department of Cancer & Inflammation Research, Institute of Molecular Medicine, Faculty of Health Science, University of Southern Denmark, Odense, Denmark.
Katrine
K
Pilely
Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, Faculty of Health Sciences, University Hospital of Copenhagen, Copenhagen, Denmark.
Mikkel-Ole
MO
Skjoedt
Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
Christian
C
Nielsen
Research Unit of Clinical Microbiology, Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
Thomas Emil
TE
Andersen
Department of Dermatology and Allergy Centre, Odense University Hospital, Odense, Denmark.
Anne
A
Louise Aabom
Department of Cancer & Inflammation Research, Institute of Molecular Medicine, Faculty of Health Science, University of Southern Denmark, Odense, Denmark.
Lars
L
Vitved
Department of Cancer & Inflammation Research, Institute of Molecular Medicine, Faculty of Health Science, University of Southern Denmark, Odense, Denmark.
Claus
C
Koch
Department of Cancer & Inflammation Research, Institute of Molecular Medicine, Faculty of Health Science, University of Southern Denmark, Odense, Denmark.
Karsten
K
Skjødt
Department of Cancer & Inflammation Research, Institute of Molecular Medicine, Faculty of Health Science, University of Southern Denmark, Odense, Denmark. Electronic address: ypalarasah@health.sdu.dk.
Yaseelan
Y
Palarasah
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001782:Blood Donors; D015153:Blotting, Western; D003167:Complement Activation / Q000276:immunology*; D003181:Complement C4 / Q000032:analysis / Q000276:immunology*; D003718:Denmark; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D047468:Immunoprecipitation; D051379:Mice; D008815:Mice, Inbred Strains
D000911:Antibodies, Monoclonal; D003181:Complement C4; D007074:Immunoglobulin G
C4c; Complement; ELISA; Monoclonal antibody