A Direct Assay for Measuring Free 25-Hydroxyvitamin D

J AOAC Int. 2017 Sep 1;100(5):1318-1322. doi: 10.5740/jaoacint.17-0084. Epub 2017 May 11.


Recent studies suggest that the concentration and genotype of vitamin D binding protein (VDBP) are important factors that determine the bioavailability of 25-hydroxyvitamin D [25(OH)D] in blood. Accumulating data indicate that, e.g., in pregnant women, hemodialysis patients, chronic kidney disease, liver failure, and bladder and pancreatic cancers, the measurement of free 25(OH)D in serum provides more relevant diagnostic information than measurement of total 25(OH)D. The aim of this study was to develop and validate an ELISA for direct measurement of free 25(OH)D in serum. A simple and direct ELISA was developed, based on a two-step immunoassay procedure performed in a microtiter plate. The assay has been characterized in terms of precision (4-10% CV, according to concentration), sensitivity (limits of blank = 0.5-1.0 pg/mL and LODs = 1.3-1.8 pg/mL), accuracy (correlation to dialysis, ELISA = 0.99xdialysis-0.5 pg/mL, r2 = 0.74), cross-reactivity of the antibody for the D2 form (77%), and addition of both VDBP and albumin (35-38% recovery upon addition of VDBP, 53-58% upon addition of albumin). The assay has already been used in multiple studies, including its comparison with calculation methods and in studies of patients with liver failure, different ethnic groups, supplemented mice, respiratory diseases, and obesity. The free 25(OH)D ELISA can be used in studies as a valuable tool to establish the clinical relevance of free 25(OH)D.

Publication types

  • Validation Study

MeSH terms

  • Animals
  • Blood Chemical Analysis / standards*
  • Enzyme-Linked Immunosorbent Assay / standards*
  • Humans
  • Limit of Detection
  • Mice
  • Vitamin D / analogs & derivatives*
  • Vitamin D / blood


  • Vitamin D
  • 25-hydroxyvitamin D