title: A novel, fully-automated, chemiluminescent assay for the detection of 1,25-dihydroxyvitamin D in biological samples.
abstract: 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.
  
  
  
  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.
  
  
  
  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).
  
  
  
  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.
authors: Center for Esoteric Testing, Laboratory Corporation of America(®) Holdings, Burlington, NC 27215, United States. Electronic address: ValcouA@labcorp.com.
authors: Andre
authors: A
authors: Valcour
authors: DiaSorin Inc, 1951 Northwestern Avenue, Stillwater, MN 55082, United States.
authors: Claudia
authors: C
authors: Zierold
authors: DiaSorin Inc, 1951 Northwestern Avenue, Stillwater, MN 55082, United States.
authors: Angela L
authors: AL
authors: Podgorski
authors: DiaSorin Inc, 1951 Northwestern Avenue, Stillwater, MN 55082, United States.
authors: Gregory T
authors: GT
authors: Olson
authors: DiaSorin Inc, 1951 Northwestern Avenue, Stillwater, MN 55082, United States.
authors: John V
authors: JV
authors: Wall
authors: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, United States.
authors: Hector F
authors: HF
authors: DeLuca
authors: DiaSorin Inc, 1951 Northwestern Avenue, Stillwater, MN 55082, United States.
authors: Fabrizio
authors: F
authors: Bonelli
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D001331:Automation; D002853:Chromatography, Liquid; D006801:Humans; D007368:Interferometry; D008163:Luminescent Measurements / Q000379:methods*; D012016:Reference Values; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D000911:Antibodies, Monoclonal; D014807:Vitamin D; C097949:1,25-dihydroxyvitamin D
keywords: 1,25-dihydroxyvitamin D; CLIA; LBD; automated; conformation; extraction-free; immunoassay