Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum

Author:

Ulleland Marius1,Eilertsen Ingar1,Quadros Edward V2,Rothenberg Sheldon P2,Fedosov Sergey N3,Sundrehagen Erling1,Örning Lars1

Affiliation:

1. Axis-Shield ASA, Ulvenveien 87, PO Box 206 Økern, N-0510 Oslo, Norway

2. Division of Hematology/Oncology, State University of New York, Health Science Center, Brooklyn, NY 11203

3. Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark

Abstract

Abstract Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin. Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin. Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well (r2 = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r2 = 0.23) with total cobalamin or holo-haptocorrin. Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.

Publisher

Oxford University Press (OUP)

Subject

Biochemistry, medical,Clinical Biochemistry

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