Vitamin D assays
The disclosure relates to methods for measuring levels of 25(OH)vitamin D (250HD) in a mammalian fluid sample. The disclosure further relates to kits for measuring levels of 25(OH)vitamin D (250HD) in a mammalian fluid, the procedure comprising the step of release of the vitamin D from the serum binding protein (DBP), capturing of the released analyte by a specific antibody and competitive assay of the captured vitamin D against a conjugate between the vitamin D and a tracer. The observed signal is inversely proportional to vitamin D concentration.
1. A method of measuring 25(OH) vitamin D (25OHD) in a mammalian fluid sample comprising:
(a) contacting the sample comprising 25OHD with a dissociation buffer comprising a release reagent consisting of a mixture of 1-butyl-4-methylpyridinium chloride, 1-ethyl-3-methylpyridinium ethyl sulfate, methyl β-cyclodextrin and sodium salicylate in a well of a microtiter plate;
(b) contacting the mixture of step (a) with (i) add-in 25OHD conjugated to an enzyme and an antibody that specifically binds to 25OHD;
(c) washing the well to remove add-in 25OHD that is not specifically bound;
(d) adding a substrate of said enzyme conjugated to add-in 25OHD;
(e) quantitatively measuring a signal produced by the reaction of substrate and enzyme in the well; and
(f) comparing the measured signal to a standard curve created with known quantities of 25OHD3, 25OHD2, or a mixture thereof, to determine the amount of 25OHD in the sample.
2. The method of claim 1 , wherein the antibody is selected from a monoclonal antibody, a polyclonal antibody, or an antigen binding fragment of antibody.
3. The method of claim 1 , wherein the antibody is in solution.
4. The method of claim 1 , wherein the antibody is bound to the surface of the well.
5. The method of claim 4 , wherein the antibody is bound to said surface directly.
6. The method of claim 4 , wherein the antibody is bound to said surface indirectly.
7. The method of claim 6 , wherein the antibody is bound to said surface by a secondary antibody that specifically binds to said antibody.