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

作者：Valcour Andre^*; Zierold Claudia; Podgorski Angela L; Olson Gregory T; Wall John V; DeLuca Hector F; Bonelli Fabrizio

来源：Journal of Steroid Biochemistry and Molecular Biology, 2016, 164: 120-126.

DOI：10.1016/j.jsbmb.2015.08.005

• 1,25-dihydroxyvitamin D
• automated
• immunoassay
• extraction-free
• LBD
• conformation
• CLIA

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 = 1.07x + 3.77 (r = 0.94) for different methods from Endocrine Sciences, Laboratory Corporation of America (R) 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 mu l) than current methods.

• 出版日期2016-11