A Cell-Based Assay to Measure the Activity of the Complement Convertases

Abstract

IntroductionThe complement system serves as a crucial defense mechanism against invading pathogens, but dysregulation of this system can result in harmful consequences. Central to the complement cascade are the classical/lectin and the alternative pathway convertases. Aberrant regulation of the convertases is often implicated in the development of rare complement-related diseases. However, analyzing convertase activity poses a significant challenge due to their labile nature and intricate interactions with serum proteins. MethodsIn this study, we propose a novel assay for the functional evaluation of these complexes. Our approach leverages a widely available human lymphoma cell line, which, when sensitized with antibodies, triggers activation of the classical pathway with a substantial amplification by the alternative pathway. The combined action of two C5 blockers eculizumab and crovalimab let the cascade proceed up to the level of convertases but not further. In the next step, C5 inhibitors are washed away and guinea pig serum in EDTA buffer supports the development of lytic sites on the platform of pre-existing convertases. ResultsThe assay detects recombinant gain-of-function components of both convertase types within human serum or plasma. Furthermore, we demonstrate the assay's practical utility in analyzing nephrological patients harboring C3 genetic variants and illustrate its capacity to distinguish between patients and asymptomatic relatives carrying the same pathogenic C3 variant. ConclusionsWe provided a proof of concept of a new assay that detects convertase overactivity in individuals carrying variants of both pathogenic character or these of unknown significance in ubiquitous complement proteins like C3.