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

Abstract

The separation of complement factors C3 and C3b by isotachophoresis in 1% agarose gel followed by immunoprecipitation and quantification is presented. Glycine was used as spacer in a nonequilibrium isotachophoresis (Acevedo, F., J. Chromatogr. 1991, 545, 391-396). Tricine, beta-alanine and Tris were the leading ion, terminating ion and counter ion, respectively. After electrophoresis the gel was incubated in rabbit anti-human complement factor C3c. The amounts of C3 and C3b in the sample were measured by optical densitometry of the Coomassie Brilliant blue-stained immunoprecipitates in the agarose gel. The correlation coefficient obtained for the logarithm of the integrated densitometric measurement vs. the logarithm of the amount of applied C3 was higher than 0.98 in calibration experiments. The extent of complement factor C3 activation is calculated as the ratio between the amount of C3b and the amount of C3b plus C3 and expressed as percent. The progress of complement activation from human blood plasma samples induced by Mg2+ and zymosan are presented as examples.