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

•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.

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 ± 9 ng/mL, TCC = 3 ± 0.1 ng/mL and fB = 55.7 ± 0.1 ng/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 3.5 h 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.

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