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.
Keywords: 1,25-dihydroxyvitamin D; CLIA; LBD; automated; conformation; extraction-free; immunoassay.
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