Fractionation of human serum proteins by capillary and recycling isotachophoresis.

Abstract

The fractionation of human serum proteins using capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) in presence of low molecular mass spacer compounds is reported. Anionic CITP was performed in an instrument equipped with a Teflon capillary of 0.5 mm ID as well as in an apparatus which features an open-tubular fused-silica capillary of 75 microns ID. RITP was performed in a recycling fast flow focusing apparatus in which fluid flows rapidly through a narrow, rectangular cell and the effluent from each outlet port is reinjected into the electrophoresis chamber through the corresponding input port. Typically, 1 mL serum was processed batchwise within about 2.5 h prior to collection of 30 fractions of about 4 mL each. The fractions were analyzed separately for conductivity, pH and UV absorbance and selected fractions were characterized by an immunoassay for transferrin, as well as by gel isoelectric focusing, two-dimensional gel electrophoresis, CITP and capillary zone electrophoresis. The search for suitable electrolyte systems and spacers was executed by CITP and by computer simulation. For simple configurations, separations predicted by simulation are shown to qualitatively agree with fractionation performed by CITP and RITP. Configurations producing three protein subgroups, the first containing mainly albumin, the second transferrin and the third the globulins, are discussed.