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Conventional tests for vitamin B12 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 B12 deficiency that requires therapy. We developed an automated assay for holoTC that can be used on the Abbott AxSYM immunoassay analyzer. 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. Neither apoTC nor haptocorrin exhibited detectable cross-reactivity. The detection limit was ≤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^sub 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. The AxSYM Active B12 assay allows rapid, precise, sensitive, specific, and automated measurement of human holoTC in serum and plasma.

REFERENCE INTERVAL AND EXPECTED VALUES

An ANOVA of the log-transformed data revealed holoTC concentration to be weakly dependent on age but not on sex. Further analysis revealed that the positive correlation with age was confined to women. The 95% reference interval was 19-134 pmol/L (mean, 50 pmol/L), which is similar to the reference interval (24157 pmol/L) previously obtained with the commercial holoTC assay, HoloTC RIA (B).

Serum samples were obtained from 5 patients who had received a clinical diagnosis of pernicious anemia but had not yet been treated. HoloTC and serum vitamin B12 were measured with the AxSYM Active B12 and Abbott AxSYM B12 assays, respectively. The holoTC and serum vitamin B,2 concentrations for the 5 patients were <3,3,4,3, and 4 pmol/L, and 62,92,62, 87, and 49 pmol/L, respectively.

In conclusion, AxSYM Active B12 is a sensitive and highly specific high-throughput assay for quantitative measurement of the biologically active fraction of vitamin B12, holoTC, in serum and plasma.