A novel, fully-automated, chemiluminescent assay for the detection of 1,25-dihydroxyvitamin D in biological samples.

BACKGROUND: 1,25-Dihydroxyvitamin D (1,25-(OH)2D), the hormonal form of vitamin D, is difficult to measure because of its low circulating levels (pg/mL), and similarity to more abundant metabolites. Here a fully-automated chemiluminescent assay that accurately and precisely measures 1,25-(OH)2D is described. METHOD: The novel 1,25-(OH)2D assay was conceived based on four pillars: (1) the VDR's ligand binding domain (LBD) as a capture molecule; (2) reaction conditions wherein 1,25-(OH)2D favors binding to LBD vs. the vitamin D binding protein; (3) exploitation of liganded-LBD's conformational change; (4) a monoclonal antibody specific to liganded-LBD. This specific, conformational, sandwich approach, unique for automated measurement of haptens, is superior to more cumbersome, conventional competitive formats. RESULTS: Accuracy of the 1,25-(OH)2D assay was corroborated by its alignment against LC-MS/MS with fit Deming regression equations of y=0.98x + 1.93 (r=0.92), and y=1.07x+3.77 (r=0.94) for different methods from Endocrine Sciences, Laboratory Corporation of America® and the University of Washington, respectively. Good analytical precision was manifested by its low estimated limit of quantitation (1.57pg/mL), average intra-assay imprecision (3.5%CV; range 1.1-4.7%), and average inter-assay imprecision (4.5%CV; range 3.4-7.2%). Expected and measured recovery values were congruent (93.4% mean). CONCLUSIONS: The novel 1,25-(OH)2D method exhibited excellent correlation with well validated LC-MS/MS assays from two laboratories. Significantly, its 65min turn-around time is quicker, and sample volume smaller (75µl) than current methods.

Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON automated analyzer.

OBJECTIVE: Methods to assess serum 25 OH vitamin D have improved in accuracy, precision, and ease of use. We describe the analytical and clinical validation of an automated, antibody- and microparticle-based, chemiluminescent immunoassay method for the determination of 25 OH vitamin D. DESIGN AND METHODS: The LIAISON 25 OH Vitamin D assay is a rapid automated method with first results available in 40 min, and a subsequent throughput of 180 samples per hour. Assay performance characteristics of precision and recovery were determined according to the National Committee for Clinical Laboratory Standards (NCCLS) protocols. Analytical and functional sensitivity were determined according to standard protocols. Samples for method comparison studies were obtained from routine clinical samples submitted for 25 OH Vitamin D determination or from apparently healthy normal volunteers. RESULTS: The detection limit for this assay was <2.0 nmol/L across three lots of materials. Functional sensitivity (inter-assay imprecision <20%) was 17.5 nmol/L. Total imprecision (CV) was <15% at 42.5-137.5 nmol/L. Mean (SD) recovery was 101% (13%). The assay was linear on dilution. Comparison with radioimmunoassay (RIA) yielded acceptable correlation (r = 0.88) and clinical equivalence in the range from 37.5 to 150 nmol/L. CONCLUSIONS: The LIAISON 25 OH Vitamin D assay is a rapid, accurate, and precise tool for the measurement of 25 OH vitamin D.