A novel and sensitive functional assay for complement Factor I based on the third proteolytic clip of C3b

Area of Science:

• Immunology
• Complement System Biology

Background:

• The complement system is crucial for innate immunity.
• Complement Factor I is a key regulatory protease within the complement cascade.
• Existing assays for Factor I activity can be complex and require unstable intermediates.

Purpose of the Study:

• To describe a novel, sensitive assay for measuring the functional activity of complement Factor I.
• To provide a simpler and more robust method for assessing Factor I function compared to existing techniques.

Main Methods:

• The assay utilizes the third proteolytic clip of Factor I, which cleaves cell-bound iC3b into cell-bound C3dg and soluble C3c.
• Factor H is required as a cofactor for Factor I activity.
• The assay is performed at low ionic strength due to the low affinity of iC3b for Factor H.

Main Results:

• The described assay accurately measures Factor I functional activity.
• Cleavage of cell-bound iC3b by Factor I leads to the abolition of cell conglutination.
• This assay avoids the need for unstable intermediates like EAC142 or purified C3, simplifying the process.

Conclusions:

• A sensitive and practical assay for complement Factor I functional activity has been developed.
• This assay is based on a distinct proteolytic event (third clip) of Factor I.
• The new assay presents an easier alternative to previous methods for studying Factor I in the complement cascade.