Complement activation in semi-solid medium: Insolubilization of properdin and the third component of complement (C3)...

Area of Science:

• Immunology
• Complement System Biology

Background:

• The precise function of properdin in the alternative pathway of complement activation is not fully understood.
• Prior evidence suggested properdin forms complexes with complement components like C3.

Purpose of the Study:

• To directly visualize and characterize the formation of complexes between properdin and other serum components.
• To investigate the conditions and requirements for properdin-mediated complex formation.

Main Methods:

• Ouchterlony double diffusion assay in agar gel using normal human serum and purified properdin.
• Analysis of precipitate formation under varying pH, tonicity, and ionic conditions.
• Radiolabeling (125I) of complement components (C3, Factor B) and properdin to identify incorporated proteins.

Main Results:

• A precipitation line formed between properdin and normal human serum at pH 8.6 in Veronal buffer, indicating complex formation.
• The reaction was magnesium-dependent and occurred with serum deficient in C2, C5, or immunoglobulins, but not with aged, heat-inactivated, or C3b inactivator-deficient serum.
• 125I-labeled C3 and properdin, but not Factor B, were found in the precipitate.
• Sera with C3 nephritic factor did not precipitate with properdin, but showed identity with the properdin-normal serum precipitate.

Conclusions:

• The study provides direct visual evidence for the formation of insoluble complexes between properdin and C3 in normal human serum.
• These complexes form under specific laboratory conditions (pH 8.6, Mg++ dependent) and involve C3 and properdin.
• The findings contribute to understanding the role of properdin in the alternative complement pathway and suggest interactions with agar may facilitate complex visualization.