A specific assay for quantification of human C4c by use of an anti-C4c monoclonal antibody
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Standard
A specific assay for quantification of human C4c by use of an anti-C4c monoclonal antibody. / Pilely, Katrine; Skjoedt, Mikkel-Ole; Nielsen, Christian; Andersen, Thomas Emil; Louise Aabom, Anne; Vitved, Lars; Koch, Claus; Skjødt, Karsten; Palarasah, Yaseelan.
I: Journal of Immunological Methods, Bind 405, 03.2014, s. 87-96.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - A specific assay for quantification of human C4c by use of an anti-C4c monoclonal antibody
AU - Pilely, Katrine
AU - Skjoedt, Mikkel-Ole
AU - Nielsen, Christian
AU - Andersen, Thomas Emil
AU - Louise Aabom, Anne
AU - Vitved, Lars
AU - Koch, Claus
AU - Skjødt, Karsten
AU - Palarasah, Yaseelan
N1 - Copyright © 2014 Elsevier B.V. All rights reserved.
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
KW - Animals
KW - Antibodies, Monoclonal
KW - Antibody Specificity
KW - Blood Donors
KW - Blotting, Western
KW - Complement Activation
KW - Complement C4
KW - Denmark
KW - Enzyme-Linked Immunosorbent Assay
KW - Humans
KW - Immunoglobulin G
KW - Immunoprecipitation
KW - Mice
KW - Mice, Inbred Strains
KW - Journal Article
U2 - 10.1016/j.jim.2014.01.011
DO - 10.1016/j.jim.2014.01.011
M3 - Journal article
C2 - 24472768
VL - 405
SP - 87
EP - 96
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
ER -
ID: 172399187