Capillary zone electrophoresis with a dynamic double coating for analysis of carbohydrate-deficient transferrin in human serum. Precision performance and pattern recognition

Capillary zone electrophoresis (CZE) with a dynamic double coating permits the simultaneous, individual, quantitative determination of transferrin (Tf) isoforms in human serum and thus carbohydrate-deficient transferrin (CDT), the most specific marker available today for the detection of chronic, excessive alcohol intake. CZE of serum Tf was carefully evaluated using the P/ACE MDQ with fused-silica capillaries of 50 microm I.D. and 60.2 cm total length, the CEofix CDT kit and the instrumental conditions recommended by the kit manufacturer. The precision performance assessed over a 20-day period according to the internationally accepted NCCLS EP5-A guidelines revealed the CZE assay as being highly reproducible with within-run and total precision being dependent on the Tf isoform level and RSD values ranging between 2.2 and 17.6%. Inter-day RSD values for asialo-Tf were noted to be between 9.8 and 11.5% and for disialo-Tf between 3.8 and 8.6%, whereas those for CDT levels of 0.87 and 4.31% of total Tf were determined to be 8.6 and 3.4%, respectively. The RSD values for trisialo-Tf, tetrasialo-Tf, pentasialo-Tf and hexasialo-Tf were found to be between 0.4 and 4.1%. Tf patterns are recognized and identified via detection times of Tf isoforms (intra-day and inter-day RSD values < 1.0% and < 1.7%, respectively), immunosubtraction of Tf and enzymatic sequential cleavage of sialic acid residues. Furthermore, heterozygous Tf BC and Tf CD variants are assigned via spiking with a known mixture of Tf isoforms (e.g. the serum of a healthy Tf C homozygote). Among the non-Tf peaks monitored, the CRP peak detected shortly before disialo-Tf was identified by immunosubtraction and peak magnitudes were found to correlate well with immunochemically determined CRP serum levels. The CZE assay with dynamic double coating could thereby be shown to be sensitive enough to determine elevated CRP levels in human serum. Furthermore, unusual peaks in the gamma-region were identified by customary serum protein CZE, immunosubtraction CZE and immunofixation.