Recent studies suggest that the concentration and genotype of vitamin D binding protein are important factors that determine the bioavailability of 25OH Vit-D in blood. It has been suggested that measurement of free, non-protein bound 25OH Vit-D in serum, may provide more relevant diagnostic information than total 25OH Vit-D, for instance in chronic kidney disease, bladder cancer and pancreatic cancer, or in hemodialysis patients.
To measure free 25OH Vit-D in blood, Future Diagnostics developed a direct ELISA method. Following the first laboratory evaluation phase, a two-step enzyme-linked immunosorbent assay (ELISA) was optimized for the quantification of free 25OH Vit-D assay. Modifications were made in the protocol for the coating of the monoclonal anti-25OH Vit-D in the microtiter plates as well as in the formulation of the sample diluent and of the biotinylated vitamin D conjugate. The optimized assay was validated against the first version of the assay and showed the following performances: the calibrator range was 0.235 pg/ml. The LoB was 1.9 pg/ml; the LoD was 2.8 pg/ml. Total assay precision was 10.2% at 6.0 pg/ml, 7.6% at 10.9 pg/ml and 5.5% at 24.9 pg/ml. The cross-reactivity of the antibody towards 25OH vitamin D2 was 77% and the influence of interfering hemoglobin, bilirubin and triglycerides was also verified.
The free 25OH Vit-D assay that reproducibly determines the level of free 25OH Vit D in serum was implemented and validated on an open ELISA platform. After adaptation of the assay protocol on the instrument, manual and automated results were compared in terms of dose response curve, accuracy, precision, sensitivity and drift.
This assay can be used as a valuable tool in studies to establish the clinical relevance of free 25OH Vit-D.
14 May 2016 - 17 May 2016