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Background: Conventional tests for vitamin B(12) deficiency measure total serum vitamin B12, whereas only that portion of vitamin B12 carried by transcobalamin (holotranscobalamin) is metabolically active. Measurement of holotranscobalamin (holoTC) may be more diagnostically accurate for detecting B(12) deficiency that requires therapy. We developed an automated assay for holoTC that can be used on the Abbott AxSYM immunoassay analyzer., Methods: AxSYM Active B12 is a 2-step sandwich microparticle enzyme immunoassay. In step 1, a holoTC-specific antibody immobilized onto latex microparticles captures holoTC in samples of serum or plasma. In step 2, the captured holoTC is detected with a conjugate of alkaline phosphatase and antiTC antibody., Results: Neither apoTC nor haptocorrin exhibited detectable cross-reactivity. The detection limit was < or = 0.1 pmol/L. Within-run and total imprecision (CV ranges) were 3.4%-5.1% and 6.3%-8.5%, respectively. Assay CVs were < 20% from at least 3 pmol/L to 107 pmol/L. With diluted serum samples, measured concentrations were 104%-114% of the expected values in the working range of the assay. No interference from bilirubin, hemoglobin, triglycerides, erythrocytes, rheumatoid factor, or total protein was detected at expected (abnormal) concentrations. A comparison of the AxSYM Active B12 assay with a commercial RIA for holoTC yielded the regression equation: AxSYM = 0.98RIA + 4.7 pmol/L (S(y x), 11.4 pmol/L; n = 204). Assay throughput was 45 tests/h. A 95% reference interval of 19-134 pmol/L holoTC was established with samples from 292 healthy individuals., Conclusions: The AxSYM Active B12 assay allows rapid, precise, sensitive, specific, and automated measurement of human holoTC in serum and plasma.

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.

Transcobalamin (TC), a vitamin B12 (cobalamin, Cbl) binding protein in plasma, promotes the cellular uptake of the vitamin by receptor-mediated endocytosis. Inherited TC deficiency is an autosomal recessive disorder characterized by megaloblastic anaemia caused by cellular vitamin B12 depletion. It may be accompanied by neurological complications, including a delay in psychomotor and mental development. This report describes three sisters with inherited TC deficiency resulting from a splicing defect in the TC gene. A point mutation was identified in intron 3 splice site of the TC gene that activates a cryptic splice site in exon 3. The transcript generated has an in-frame deletion of 81 nucleotides and the resulting truncated protein is unstable and not secreted by the cells. Until now, genetic studies have been reported in only five patients with TC deficiency and the molecular defect was different in each of them, which gives evidence for a genetic heterogeneity of the disease.

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.