Simple quantification of complement factors C3 and C3b using separation byisotachophoresis

The separation of complement factors C3 and C3b by isotachophoresis in 1% a garose 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 ana counter ion, respectively. After elec trophoresis the gel was incubated in rabbit antihuman complement factor C3c . The amounts of C3 and C3b in the sample were measured by optical densitom etry of the Coomassie Brilliant blue-stained immunoprecipitates in the agar ose gel. The correlation coefficient obtained for the logarithm of the inte grated densitometric measurement vs, the logarithm of the amount of applied C3 was higher than 0.98 in calibration experiments. The extent of compleme nt factor C3 activation is calculated as the ratio between the amount of C3 b and the amount of C3b plus C3 and expressed as percent. The progress of c omplement activation from human blood plasma samples induced by Mg2+ and zy mosan are presented as examples.