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

Introduction: The complement system serves as a crucial defense mechanism against invading pathogens; however, dysregulation of this system can result in harmful consequences. Central to the complement cascade are the classical pathway (CP) or lectin pathway (LP) and the alternative pathway (AP) 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. Methods: In 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 CP with a substantial amplification by the AP. The combined action of 2, C5 blockers eculizumab and crovalimab let the cascade proceed up to the level of convertases but not further. In the next step, C5 inhibitors were washed away and guinea pig serum in ethylenediamine tetraacetic acid (EDTA) buffer supported the development of lytic sites on the platform of preexisting convertases. Results: The assay detects recombinant gain-of-function (GoF) 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. Conclusion: We provided a proof-of-concept of a new assay that detects convertase overactivity in individuals carrying variants of both pathogenic character or those of unknown significance in ubiquitous complement proteins such as C3.