Arendt, Johan Frederik Berg4; Quadros, Edward V.2; Christensen, Anna Lisa3; Nexø, Ebba4
1 Department of Clinical Medicine - Clinical Biochemistry, Department of Clinical Medicine, Health, Aarhus University2 2 Departments of Medicine and Cell Biology, SUNY Downstate Medical Center, Brooklyn, New York, USA3 Klinisk Biokemisk Afdeling, Aarhus Universitetshospital4 Department of Clinical Medicine - Clinical Biochemistry, Department of Clinical Medicine, Health, Aarhus University
Background: Recently, the cell-surface receptor involved in the internalisation of the cobalamin(vitamin B12, Cbl) transporting protein, transcobalamin(TC), was described, and was found to be CD320(1). So far, it remains unsolved whether CD320 is present in a soluble form (sCD320) in serum. Our aim was to establish an ELISA for sCD320 and to explore the occurrence of sCD320 in human serum. Methods: In order to develop an ELISA we used an in-house monoclonal antibody as capture antibody, and for detection, a biotin labelled polyclonal antibody (R&D Systems) combined with an avidin based detection system. A medium containing recombinant CD320 (produced in-house) and commercially available CD320 (Abnova) were explored as suitable calibrators. Standard Western blot was performed to ensure the specificity of the capture antibody. Serum from patients referred for measurement of plasma Cbl(measured on Cobas 6000, Roche Diagnostics) and patients with extreme values of serum TC was analysed. Total TC was measured employing an in-house ELISA (2). Results: The Western blot showed a highly specific capture antibody. The ELISA assay showed a curvilinear response for dilutions of the medium/recombinant CD320 0.4-4 nmol/L. For practical reasons medium was used as calibrator. Four serum samples showed dilutions curves comparable to that of the calibrator. Preliminary analysis of patient samples with different Cbl levels showed that sCD320 was present in nine out of 28 (32%) patients with Cbl<200 pmol/L (lower level of the interval of reference) and 16 out of 37 (43%) patients with Cbl≥200 pmol/L. Interestingly, a high level of sCD320 was observed in four of seven (57%) patients suffering from inherited TC deficiency, and also in three of seven (43%) patients with high levels of TC(>2000 pmol/L). Conclusions: We have developed an ELISA for measurement of sCD320. Preliminary results show no relation between low plasma cobalamins and the presence of sCD320, but an increased amount of the protein was observed in patients with low or high levels of TC. The possible clinical utility of sCD320 remains to be established .