Simple quantification of complement factors C3 and C3b using separation by isotachophoresis

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• The complement system is crucial for innate immunity.
• Accurate quantification of complement factors is essential for understanding immune responses and diagnosing diseases.
• Existing methods for complement factor analysis can be complex and time-consuming.

Purpose of the Study:

• To develop and validate a method for separating and quantifying complement factor C3 and its activated form, C3b.
• To assess the extent of complement factor C3 activation in biological samples.

Main Methods:

• Non-equilibrium isotachophoresis in 1% agarose gel using glycine as a spacer.
• Immunoprecipitation with rabbit anti-human complement factor C3c.
• Quantification of C3 and C3b via optical densitometry of Coomassie Brilliant blue-stained immunoprecipitates.
• Calculation of complement activation as the ratio of C3b to total C3 (C3b + C3).

Main Results:

• Successful separation and quantification of C3 and C3b.
• High correlation coefficient (>0.98) between logarithmic densitometric measurements and applied C3 amounts in calibration experiments.
• Demonstrated application in measuring complement activation in human plasma induced by Mg2+ and zymosan.

Conclusions:

• The developed isotachophoresis-based method provides a reliable and accurate approach for quantifying complement factor C3 and C3b.
• This method allows for precise measurement of complement activation extent.
• The technique is applicable to studying complement system dynamics in biological samples.