title: 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: 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 PhastSystem(TM) 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. 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 < or = 10%). Except for quantitatively unimportant traces of trisialo-Fe(2)-transferrin (<5% of total CDT), only asialo-, mono-, and disialo-Fe(2)-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe(2)-transferrin (during column rinsing) and disialo-Fe(2)-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe(2)-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-Fe(2)-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.
authors.affiliation: Zentrum für Innere Medizin, Abteilung Kardiologie, Baldingerstrasse, Philipps-Universität, D-35033 Marburg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Hackler
authors.forename: T
authors.initials: T
authors.lastname: Arndt
authors.forename: A
authors.initials: A
authors.lastname: Helwig-Rolig
authors.forename: J
authors.initials: J
authors.lastname: Kropf
authors.forename: A
authors.initials: A
authors.lastname: Steinmetz
authors.forename: J R
authors.initials: JR
authors.lastname: Schaefer
mesh_terms: D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D006801:Humans; D007118:Immunoassay; D007501:Iron / Q000737:chemistry; D007525:Isoelectric Focusing; D009391:Nephelometry and Turbidimetry; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014168:Transferrin / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000235:genetics
chemical_list: D015415:Biomarkers; D020033:Protein Isoforms; D011933:Reagent Kits, Diagnostic; D014168:Transferrin; C056759:carbohydrate-deficient transferrin; D007501:Iron