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.

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Vitamin D has long been a familiar concept for any professional working in clinical biology. Nowadays it is becoming known to a large sector of the population. The great research efforts developed over the past decade have led to an explosion in the number of determinations requested of the most qualified metabolite to express the body state of vitamin D, 25-OH vitamin D (25-OHD) free, represents the small fraction not bound to transporter proteins. According to the free hormone hypothesis, it should be considered the best representation of the body state of vitamin D. Unfortunately, limited attention has been paid to this determination since, until recently, the scientific community only had tedious, indirect methods to measure it. A few years ago, a direct measurement method of free 25-OHD was already available to carry out research studies with promising results.

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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.