Active B12: A Rapid, Automated Assay for Holotranscobalamin on the Abbott AxSYM Analyzer

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2008-03, Vol.54 (3), p.567-573
• Main Authors: Brady, Jeff, Wilson, Lesley, McGregor, Lynda, Valente, Edward, Orning, Lars
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Anemia, Pernicious - diagnosis; Autoanalysis; Biological and medical sciences; Cell division; Expected values; Female; Fundamental and applied biological sciences. Psychology; Humans; Immunoenzyme Techniques; Investigative techniques, diagnostic techniques (general aspects); Latex; Male; Medical sciences; Monoclonal antibodies; Plasma; Reference Values; Regression Analysis; Transcobalamins - analysis; Vitamin B; Vitamin B 12 Deficiency - diagnosis
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2007.096784

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. 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 < 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. The AxSYM Active B12 assay allows rapid, precise, sensitive, specific, and automated measurement of human holoTC in serum and plasma.