Investigation by Isoelectric Focusing of the Initial Carbohydrate-deficient Transferrin (CDT) and non-CDT Transferrin Isoform Fractionation Step Involved in Determination of CDT by the ChronAlcoI.D. Assay

Abstract

Abstract Background: The introduction of a new set of reagents for the determination of carbohydrate-deficient transferrin (CDT) as a marker of chronic alcohol abuse requires an independent evaluation of the analytic specificity of the test. This information is needed for correct interpretation and classification of test results. Methods: Isoelectric focusing on the PhastSystemTM followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D.TM assay. Results: The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV ≤10%). Except for quantitatively unimportant traces of trisialo-Fe2-transferrin (<5% of total CDT), only asialo-, mono-, and disialo-Fe2-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe2-transferrin (during column rinsing) and disialo-Fe2-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe2-transferrin did not change from >1 (serum) to <1 (eluates) as described for the CDTect assays. The transferrin patterns in the ChronAlcoI.D. eluates were representative of those in serum. Transferrin D variants with isoelectric points close to that of trisialo-Fe2-transferrin C1 did not cause overdetermination of CDT by the ChronAlcoI.D. test. Conclusions: The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.