High-performance affinity chromatography system for the rapid, efficient assay of glycated albumin

A high-performance affinity chromatographic system was constructed and shown to permit highly reproducible, rapid, automatic assays of serum glycated albumin (GA) by separation of albumin (Alb) on an anion-exchange column (Asahipak ES-502N) packed with a vinyl alcohol copolymer bearing diethylamino groups and consecutive separation of GA on a column packed with a vinyl alcohol copolymer bearing boronate groups. The first column selectively retained Alb free of other serum proteins and permitted at least a 95% recovery of sample Alb. The purity of the Alb peak was confirmed by two-dimensional electrophoresis. Chromatographic analyses of human serum Alb incubated with glucose on the second column showed that the peak area for GA increased in accordance with the incubation time and suggested selective adsorption of GA on the second column. Optimization of the conditions for the two-column system reduced the analysis time to 10 min. Analyses of human sera with the present system showed GA to be 16.1 +/- 1.1% (mean +/- S.D.) of total Alb in non-diabetic children and 39.9 +/- 9.1% (mean +/- S.D.) in diabetic children (0-17 years old).