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

Abstract Background 1,25-Dihydroxyvitamin D (1,25-(OH) 2 D), 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) 2 D is described. Method The novel 1,25-(OH) 2 D 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) 2 D 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) 2 D 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  = 1.07 x  + 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 (1.57 pg/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) 2 D method exhibited excellent correlation with well validated LC–MS/MS assays from two laboratories. Significantly, its 65 min turn-around time is quicker, and sample volume smaller (75 μl) than current methods.