Simple quantitative haemolytic microassay for determination of complement alternative pathway activation (AP50).

Area of Science:

• Immunology
• Complement System Biology

Background:

• The alternative pathway (AP) of complement activation plays a crucial role in innate immunity.
• Accurate measurement of AP activity is essential for understanding immune responses and diagnosing deficiencies.

Purpose of the Study:

• To develop a simple, reliable, and quantitative method for measuring alternative pathway-dependent complement activation.
• To establish a functional assay (AP50) for assessing AP activity using rabbit erythrocytes.

Main Methods:

• Quantification of alternative pathway activity via lysis of rabbit erythrocytes.
• Definition of AP50 as the reciprocal of plasma volume required for 50% erythrocyte lysis.
• Inhibition of the classical pathway using EGTA and MgCl2.
• Verification of assay specificity using C4 and Factor B depleted plasmas and heat inactivation.

Main Results:

• The AP50 assay demonstrated high reproducibility (CV < 5%) and satisfactory sensitivity.
• Assay specificity was confirmed by depleted plasmas and heat treatments, showing dependence on Factor B.
• The method allows for semi-automation using standard laboratory equipment.

Conclusions:

• The developed AP50 assay provides a robust and precise method for measuring alternative pathway complement activation.
• This assay is suitable for clinical and research settings requiring accurate assessment of AP function.
• The assay's simplicity and potential for automation enhance its utility in complement research.