Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling Complement cascade activation in vitro using an activated Complement serum calibration standard

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 ass...

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Bibliographic Details
Published in:Journal of immunological methods 2014-01, Vol.402 (1-2), p.50-56
Main Authors: van Vuuren, B. Jansen, Bergseth, G., Mollnes, T.E., Shaw, A.M.
Format: Article
Language:English
Subjects:
Activation
Assays
Calibration
Cascade
Complement
Complement Activation
Complement C3b - immunology
Complement Factor B - immunology
Complement Factor B - metabolism
Complement Membrane Attack Complex - immunology
Complement Membrane Attack Complex - metabolism
Electrochemical Techniques - standards
Electroluminescent
Epitope
Epitopes
Freezing
Half-Life
Humans
Immunoassay - standards
Immunoglobulin G - metabolism
Kinetics
Limit of Detection
Luminescent Measurements - standards
Peptide Fragments - blood
Peptide Fragments - immunology
Reference Standards
Zymosan - metabolism
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Summary: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. [Display omitted] •In vitro activation of the Complement cascade has been performed with HAIgG and zymosan•New antibody electroluminescent assays for fB, TCC and C3dg have been validated using the kinetic response profile to establish the location of the capture epitopes•Activated Complement serum has been used as a calibration standard.
ISSN:0022-1759
1872-7905
DOI:10.1016/j.jim.2013.11.010