Quantitation of C3 subcomponents on red cells coated with complement in vitro

Area of Science:

• Immunology
• Complement System
• Red Blood Cell Analysis

Background:

• Characterizing human red blood cells coated with complement is crucial for understanding immune responses.
• Reproducibility of complement component quantification on red cells requires detailed investigation.

Purpose of the Study:

• To characterize and evaluate the reproducibility of human red blood cells coated with complement in vitro.
• To quantify the number of C3 subcomponent molecules per red blood cell using Scatchard analysis.

Main Methods:

• Scatchard analysis of equilibrium concentrations of bound and free antibody using (125)I-labelled goat anti-rabbit IgG.
• Determination of C3b and C3d molecules on red cells coated via classical and alternative complement pathways.
• Assays using different anti-C3d sera and treatments (trypsinisation, KAF) to assess antigen detection and stability.

Main Results:

• Classical pathway-coated cells had twice as much bound C3b and C3d as alternative pathway-coated cells.
• Different anti-C3d sera yielded varying antigen amounts, but results were reproducible for a given antiserum and cell type.
• Trypsinisation and KAF treatment significantly reduced C3b on classical pathway-coated cells, with trypsinisation also reducing C3b on alternative pathway-coated cells.

Conclusions:

• The study provides a reproducible method for quantifying complement components on red blood cells.
• Differences in C3 subcomponent deposition and detection highlight the distinct roles of classical and alternative complement pathways.
• The minimal detectable levels of C3d and C3b by agglutination were established, offering insights into diagnostic assay sensitivity.