A simple alternative pathway for hemolytic assay of human complement component C3 using methylamine-treated plasma

Area of Science:

• Immunology
• Complement System Biology

Background:

• The complement system is crucial for innate immunity.
• Accurate quantification of complement component C3 is vital for diagnosing immune disorders.
• Existing assays may be costly, time-consuming, or affected by C3 degradation products.

Purpose of the Study:

• To develop and validate a novel, quantitative assay for human complement component C3 using the alternative pathway (AP).
• To establish a rapid, reproducible, and cost-effective method for C3 measurement.

Main Methods:

• Developed a hemolytic assay utilizing rabbit erythrocytes as complement activators.
• Employed methylamine-treated plasma, depleted of C3 and C4, as the complement source.
• Optimized assay conditions for stability and insensitivity to variations in reagents and C3 degradation products.

Main Results:

• The AP-C3 assay is inexpensive, rapid (20 min), simple, and reproducible.
• The assay is insensitive to C3 degradation products and shows optimal stability within specific reagent concentrations.
• Demonstrated specificity for functional C3, with minimal deviation (≤2%) in relative C3 determinations across plasma samples.

Conclusions:

• The developed alternative pathway C3 (AP-C3) assay provides a reliable and efficient method for quantifying functional C3.
• This assay is well-suited for C3 determination in plasma samples and during C3 purification processes.
• The assay's robustness and cost-effectiveness offer significant advantages for both clinical diagnostics and research.