Variance in the activity of the fourth component of mouse complement

Area of Science:

• Immunology
• Biochemistry

Background:

• Complement component 4 (C4) plays a crucial role in the immune system.
• Hemolytic titration is a standard method for quantifying C4 activity.
• Existing titration methods may be influenced by the sample matrix (plasma vs. serum).

Purpose of the Study:

• To compare two hemolytic titration methods for C4 (E-C and C4D methods).
• To investigate the influence of C4 molecular states in plasma and serum on assay results.
• To elucidate the role of mouse C5 in C4 hemolytic assays.

Main Methods:

• Hemolytic titration using E-C method (EAClgp, oxyC2hu, EDTA-Cgp) and C4D method (EAClgp, oxyC2hu, C4Dgp).
• Chromatographic analysis to characterize C4 molecular states in serum.
• Assay of C4 activity in plasma and serum samples from Ssh strain mice.

Main Results:

• C4 titers differed between the E-C and C4D methods depending on whether C4 was in plasma or serum.
• Serum C4 activity was higher with the E-C method, while plasma C4 activity was lower compared to the C4D method for Ssh strain.
• Chromatographic analysis revealed at least four distinct C4 molecular states in serum with varying Ss antigenicity and hemolytic activity.

Conclusions:

• The observed differences in C4 titration are attributed to the distinct molecular states of C4 present in serum versus plasma.
• Different C4 molecular forms interact uniquely with the E-C and C4D assay components.
• The study highlights the importance of considering C4 molecular heterogeneity in hemolytic assays and discusses the role of mouse C5.