Isoelectric focusing/western blotting: a novel and practical method for quantitation of carbohydrate-deficient...

Carbohydrate-deficient transferrin (CDT) has been described as the single, most accurate marker of chronic alcohol consumption. Rapid, sensitive, and specific measurement of serum CDT levels can thus provide important clinical information concerning patient diagnosis and treatment. To date, however, methods used for assessing CDT concentrations [e.g., analytical isoelectric focusing combined with immunofixation and micro anion-exchange chromatography followed by radioimmunoassay (RIA)] have not been practical enough for widespread laboratory application. In the present study, we examined the use of a different technique, namely isoelectric focusing (IEF) combined with Western blotting (IEF/WB). Serum proteins (20-40 micrograms) were first focused according to isoelectric points (pI) on high-resolution agarose IEF gels (ampholyte pH range of 5-8) containing nonionic detergent. The focused proteins were transferred electrophoretically to nitrocellulose filters, and then stained immunochemically with antihuman transferrin IgG. IEF/WB completely resolved CDT (focusing at pI 5.7 and 5.9) from other serum transferring isoforms, as assessed with neuraminidase-generated CDT standards. Computerized densitometric scanning of the immunoblots allowed CDT levels to be quantitated directly rather than as a quotient. Serum CDT content determined by IEF/WB was highly correlated (r2 = 0.962; n = 17) with values determined previously by RIA. In a larger subject group, CDT levels (mg/liter) measured by IEF/WB were 139 +/- 54 in recently-drinking alcoholics (n = 58), 81 +/- 8 in abstaining alcoholics (n = 7), and 68 +/- 16 in healthy control subjects (n = 16).(ABSTRACT TRUNCATED AT 250 WORDS)