Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum.
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 >10(10) 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 (r(2) = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r(2) = 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.
Ulleland
Marius
M
Axis-Shield ASA, Ulvenveien 87, PO Box 206 Økern, N-0510 Oslo, Norway.
Eilertsen
Ingar
I
Quadros
Edward V
EV
Rothenberg
Sheldon P
SP
Fedosov
Sergey N
SN
Sundrehagen
Erling
E
Orning
Lars
L
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D008875:Middle Aged; D012016:Reference Values; D020349:Surface Plasmon Resonance; D014155:Transcobalamins / Q000378:metabolism*; D014805:Vitamin B 12 / Q000032:analysis* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D014155:Transcobalamins; D014805:Vitamin B 12