A Direct Assay for Measuring Free 25-Hydroxyvitamin D
Nicolas Heureux1, Ernst Lindhout2, Leon Swinkels2
1DIAsource ImmunoAssays, 2 rue du Bosquet, 1348 Louvain-la-Neuve, Belgium.
Journal of AOAC International
|May 12, 2017
Summary
This study developed a direct ELISA to measure free 25-hydroxyvitamin D [25(OH)D] in serum. This assay provides more relevant diagnostic information than total 25(OH)D measurements in various patient groups.
Area of Science:
- Biochemistry
- Clinical Chemistry
- Immunology
Background:
- Vitamin D binding protein (VDBP) genotype and concentration influence 25-hydroxyvitamin D [25(OH)D] bioavailability.
- Free 25(OH)D serum levels offer more clinically relevant diagnostic information than total 25(OH)D in specific populations.
Purpose of the Study:
- To develop and validate an enzyme-linked immunosorbent assay (ELISA) for the direct measurement of free 25(OH)D in human serum.
Main Methods:
- A direct, two-step ELISA procedure was employed using a microtiter plate format.
- Assay validation included characterization of precision, sensitivity, accuracy, and cross-reactivity.
Main Results:
- The developed ELISA demonstrated good precision (4-10% CV), sensitivity (LODs 1.3-1.8 pg/mL), and accuracy (r2 = 0.74 compared to dialysis).
- The assay showed acceptable cross-reactivity for the D2 form (77%) and recovery upon addition of VDBP (35-38%) and albumin (53-58%).
Conclusions:
- The validated free 25(OH)D ELISA is a valuable tool for clinical research.
- This assay can be utilized in diverse studies to establish the clinical significance of free 25(OH)D measurements.
