Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in...

Area of Science:

• Immunology
• Biochemistry

Background:

• The first complement component (C1) plays a crucial role in the innate and adaptive immune responses.
• Accurate quantification of activated C1 is essential for understanding complement system function and related pathologies.
• Existing functional assays for C1 often lack specificity or are technically demanding.

Purpose of the Study:

• To develop and validate a novel functional assay for quantifying free activated C1 in human plasma.
• To establish an assay that is specific for activated C1 and independent of C1-esterase inhibitor (C1-inh) levels.
• To compare the performance characteristics of the new assay with conventional hemolytic assays.

Main Methods:

• Plasma C1 was maximally activated by adding monospecific antiserum to C1-inh.
• Activated C1 concentration was determined using an amidolytic rate assay with a chromogenic substrate.
• Assay conditions including incubation time, antiserum concentration, ionic strength, and pH were optimized.
• Assay specificity was confirmed by its independence from C1-inh concentration in the sample.

Main Results:

• The developed assay specifically measures free activated C1, not C1-C1-inh complexes.
• Optimized assay conditions ensured reliable and reproducible measurements.
• Plasma C1 concentrations determined by the new assay showed significant correlation with results from a hemolytic assay (r = 0.55, P < 0.001).
• The assay demonstrated high precision with an interassay coefficient of variation (CV) of 5% and an intra-assay CV of 1%.

Conclusions:

• A novel, sensitive, and precise functional assay for activated C1 in plasma has been developed.
• This assay offers significant advantages over conventional hemolytic assays in terms of performance and practicality.
• The assay provides a reliable tool for clinical and research applications involving the complement system.