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

Area of Science:

• Immunology
• Complement System Biology

Background:

• The complement system is vital for pathogen defense but its dysregulation causes disease.
• Complement convertases (classical/lectin pathway and alternative pathway) are central to the cascade.
• Analyzing convertase activity is challenging due to their instability and complex interactions.

Purpose of the Study:

• To develop a novel assay for functional evaluation of complement convertase activity.
• To enable detection of gain-of-function variants in complement proteins.
• To assess the assay's utility in diagnosing complement-related diseases.

Main Methods:

• Utilized a sensitized human lymphoma cell line to activate the classical pathway (CP) and alternative pathway (AP).
• Employed C5 blockers (eculizumab, crovalimab) to halt the cascade at the convertase level.
• Washed away inhibitors and used guinea pig serum to develop lytic sites on existing convertases.

Main Results:

• The assay detects recombinant gain-of-function components of both classical/lectin pathway and alternative pathway convertases.
• Demonstrated utility in analyzing nephrological patients with C3 genetic variants.
• Successfully distinguished between patients and asymptomatic relatives with the same C3 variant.

Conclusions:

• Provided proof-of-concept for a new assay detecting convertase overactivity.
• The assay identifies individuals with pathogenic or unknown significance variants in complement proteins like C3.
• This assay can aid in diagnosing rare complement-related diseases.