Towards harmonization of directly measured free 25-hydroxyvitamin D using an enzyme-linked immunosorbent assay

Anal Bioanal Chem. 2022 Nov;414(27):7793-7803. doi: 10.1007/s00216-022-04313-y. Epub 2022 Sep 16.


The majority of circulating 25-hydroxyvitamin D (25(OH)D) is protein bound and perhaps less available than the free fraction of 25(OH)D; therefore, researchers have proposed that the measurement of free 25(OH)D in human serum may be a better indicator of vitamin D health status than total 25(OH)D. The availability of a new enzyme-linked immunosorbent assay (ELISA) for the determination of free 25(OH)D provides a method for direct measurement of the low levels of non-protein bound 25(OH)D. As an initial step towards harmonization of measurements of free 25(OH)D, the ELISA was used to measure free 25(OH)D in three existing Standard Reference Materials (SRMs): SRM 972a Vitamin D Metabolites in Frozen Human Serum, SRM 2973 Vitamin D Metabolites in Frozen Human Serum (High Level), and SRM 1949 Frozen Prenatal Human Serum. Target values for free 25(OH)D in the nine SRM serum pools, obtained by combining the results from two laboratories, ranged from 3.76 ± 0.36 to 10.0 ± 0.58 pg/mL. Of particular significance is the assignment of free 25(OH)D target values to SRM 1949, which consists of four serum pools from non-pregnant female donors of reproductive age and pregnant women in each of the three trimesters and which also has values assigned for vitamin D binding protein, which increases during pregnancy. The availability of target values for free 25(OH)D in these SRMs will allow researchers to validate new analytical methods and to compare their results with other researchers as an initial step towards harmonization of measurements among different studies and laboratories.

Keywords: Enzyme-linked immunosorbent assay (ELISA); Free 25-hydroxyvitamin D; Standard Reference Materials (SRMs); Total 25-hydroxyvitamin D; Vitamin D binding protein.

MeSH terms

  • 25-Hydroxyvitamin D 2
  • Calcifediol
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Humans
  • Pregnancy
  • Vitamin D* / analogs & derivatives
  • Vitamin D* / metabolism
  • Vitamin D-Binding Protein* / metabolism
  • Vitamins


  • Vitamin D-Binding Protein
  • Vitamins
  • Vitamin D
  • 25-Hydroxyvitamin D 2
  • 25-hydroxyvitamin D
  • Calcifediol