Background: The cell-surface receptor, TCblR/CD320, binds and internalizes transcobalamin-bound cobalamin. So far, no assay has been developed in order to quantify the soluble form of the receptor.
Methods: This study developed an ELISA employing a monoclonal antibody that recognized the extracellular domain of CD320 as capture antibody and a biotinylated polyclonal antibody for detection. Serum samples were analyzed from blood donors (n==153) and samples from patients referred for analysis of serum cobalamin [n=757, serum cobalamin 75-2000 pg/mL (55-1476 pmol/L)].
Results: The ELISA specifically measured soluble CD320 with a quantification limit of 1.6 arbitrary units and a total variation of 4%-8%. Size exclusion chromatography was used which showed that the immunoreactive protein observed in serum eluted in the same position as soluble CD320 in the calibrator. Male donors (n=76) showed significantly higher median concentration [18 arbitrary units (arb.u.)] than female donors (n=77, median 16 arb.u.), but the absolute difference was marginal and suggested that a common reference interval of 12-97 arb.u. can be used. A positive correlation was observed between levels of soluble CD320 and serum cobalamin with median levels increasing from 17 arb.u. [n=203, cobalamin 75-270 pg/mL (55-200 pmol/L)] to 23 arb.u. [n=350, cobalamin 814-2000 pg/mL (601-1476 pmol/L)], p<0.0001.
Conclusions: An ELISA was established for measurement of soluble CD320 and it was shown that the protein is present in human serum. The serum concentrations of soluble CD320 correlated positively with serum cobalamin levels.