A novel approach to the analysis of Carbohydrate-Deficient Transferrin (CDT) based on capillary electrophoresis with in-line immunosubtraction.

The objective diagnosis of alcohol abuse has a high relevance in different areas of clinical and forensic medicine, including gastroenterology, cardiology, neurology and psychiatry, occupational and traffic medicine, forensic pathology etc. In the recent years, in addition to the traditional diagnostic approach based on liver enzymes, MCV and gamma-GT, a more specific biomarker, such as Carbohydrate Deficient Transferrin (CDT), has been introduced in the laboratory practice and is now widely used worldwide. Transferrin glycoforms, collectively referred as CDT, include the asialo-Tf, monosialo-Tf and disialo-Tf isoforms. Moreover, CDT serum concentration is lower than 1,5% of total transferrin amount. That an increase of the “total CDT” (asialo-Tf + monosialo-Tf + disialo-Tf) occurs after a persistent alcohol intake was confirmed in the literature. The instrumental analysis of Carbohydrate Deficient Transferrin (CDT), is most commonly carried out by High Performance Liquid Chromatography (HPLC) or Capillary Zone Electrophoresis (CZE). Between these two techniques, CZE shows higher efficiency and productivity, but is often reported to be inferior to HPLC in terms of selectivity, because of a less specific UV detection wavelength than HPLC. On these grounds, the present work was aimed at the development of an improved CZE method for CDT determination including an on-line immunosubtraction step specifically aimed at enhancing the analytical specificity of CZE determination. The analytical conditions were as follows: uncoated fused silica capillary, 30 m X 60 cm (L= 50 cm to detector); running buffer, 100 mM borate and 6 mM DAB (1,4-Diaminobutane), pH 8.3; voltage, 30 kV; temperature, 25°C; detection, 200 nm. Under the described CZE conditions, a baseline separation between all the CDT related peaks was achieved with good analytical performances in terms of both precision and accuracy. In order to achieve unequivocal recognition of the CDT peaks, an in-capillary immunosubtraction step was included by loading a plug of anti-human transferrin antibody solution after the sample plug. This analytical approach was applied successfully to recognize CDT peaks in the presence of potential interferences.