title: Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.
abstract: Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.
authors.affiliation: College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4PB, UK.
authors.forename: B Jansen
authors.initials: BJ
authors.lastname: van Vuuren
authors.affiliation: Research Laboratory, Bodø Hospital, Bodø and University of Tromsø, Norway.
authors.forename: G
authors.initials: G
authors.lastname: Bergseth
authors.affiliation: Research Laboratory, Bodø Hospital, Bodø and University of Tromsø, Norway.
authors.forename: T E
authors.initials: TE
authors.lastname: Mollnes
authors.affiliation: College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4PB, UK. Electronic address: Andrew.m.shaw@exeter.ac.uk.
authors.forename: A M
authors.initials: AM
authors.lastname: Shaw
mesh_terms: D002138:Calibration; D003167:Complement Activation; D003179:Complement C3b / Q000276:immunology; D011415:Complement Factor B / Q000276:immunology* / Q000378:metabolism; D015938:Complement Membrane Attack Complex / Q000276:immunology* / Q000378:metabolism; D055664:Electrochemical Techniques / Q000592:standards*; D000939:Epitopes; D005615:Freezing; D006207:Half-Life; D006801:Humans; D007118:Immunoassay / Q000592:standards*; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D057230:Limit of Detection; D008163:Luminescent Measurements / Q000592:standards; D010446:Peptide Fragments / Q000097:blood / Q000276:immunology; D012015:Reference Standards; D015054:Zymosan / Q000378:metabolism
chemical_list: D015938:Complement Membrane Attack Complex; D000939:Epitopes; D007074:Immunoglobulin G; D010446:Peptide Fragments; C045477:complement C3d,g; D003179:Complement C3b; D015054:Zymosan; D011415:Complement Factor B
keywords: Activation; Assays; Cascade; Complement; Electroluminescent; Epitope