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.
Determination of the SLAMF1 self-association affinity constant with sedimentation velocity ultracentrifugation
