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.

Figure optionsDownload as PowerPoint slide