Semi-automatic method for determination of different isoforms of carbohydrate-deficient transferrin

Abstract

Carbohydrate deficient transferrin (CDT) has been reported to be one of the best biochemical markers of alcohol abuse. However, a need still exists for a simple and practical method for widespread laboratory use. A semi-automatic (SA) isoelectric focusing (IEF) assay for CDT (SA-IEF-CDT) by a Phast System TM is introduced here. Different isoforms of transferrin were separated by IEF on polyacrylamide gels ( pI 4.0–6.5 ) and located by immunofixation with an anti-transferrin serum. The precipitation bands were stained with Coomassie Brilliant Blue and quantitated densitometrically. The present method gave a picture of the relative amounts of 10 different transferrin isoforms. The percentage of CDT with pI ≥ 5.7 (representing dimono- and asialotransferrin) was calculated. For comparisons transferrin bands with pI ≥ 5.6 (tri-, di-, mono-, and asialotransferrin), pI ≥ 5.8 (mono- and asialotransferrin) and pI ≥ 5.9 (asialotransferrin) as well as GGT, ASAT and ALAT were calculated. The method showed good linearity and it identified different isoforms in concentrations of 10 mg/ l of transferrin. The correlation of the present method with a commercially available method employing anion exchange followed by double antibody RIA (AE-RIA-CDT) was good ( n = 38, r = 0.924). In 19/20 (95%) of healthy controls, the CDT value was below 4.4% (mean + 2 S.D.) of total transferrin, while higher values were observed in all 20 (100%) alcoholics. In conclusion, the developed semi-automatic method is a practical and reliable alternative for determination of different transferrin isoforms.