Immunoassay for free vitamin D
Disclosed is the invention to conduct immuno-adsorption of free 25(OH) vitamin D from blood or blood components, notably serum or plasma, after which the absorbed material is measured. A fluoro-alkyl surfactant is used to enhance the solubility of Vitamin D and allow the measurement of free Vitamin D. The invention thus employs a binding protein to absorb the free 25(OH) vitamin D. Thereafter the binding protein comprising the 25-OH vitamin D is subjected to a competitive binding assay with a labeled vitamin D compound, preferably radiolabeled, fluorescent labeled, luminescent labeled, biotin labeled, gold labeled or enzyme labeled. Alternatively the immunocaptured 25-OH vitamin D can be quantitated by mass spectrometry.
1. A method for assaying a sample of blood or blood components for the presence of circulating, unbound vitamin D, comprising;
(a) adding an immobilized antibody for 25-OH vitamin D to the sample;
(b) mixing the sample with a diluent, said diluent comprising a fluoroalkyl surfactant wherein the fluoroalkyl surfactant is present at a level of 0.1-0.2% by weight and the fluoroalkyl surfactant is a perfluorooctanoic acid;
(c) incubating the sample for an effective amount of time to allow a desired amount of circulating, unbound Vitamin D to bind the antibody for 25-OH vitamin D;
(d) removing the non-bound sample components by washing;
(e) subjecting the immobilized antibody comprising captured vitamin D bound thereto, to competitive binding with a labeled vitamin D compound;
(f) determining the concentration of labeled vitamin D compound bound to the immobilized antibody, whereby the concentration of labeled vitamin D compound is a measure for the concentration of circulating, unbound vitamin D, the concentration being determined with reference to a calibrator concentration for vitamin D,
wherein the perfluorooctanoic acid does not dissociate vitamin D from Vitamin D Binding Protein and albumin.
2. The method of claim 1 , wherein said diluent has a buffered pH in the range of 6.0 to 8.0.
3. The method of claim 1 , wherein the sample is human serum or plasma.
4. The method of claim 1 , wherein the antibody for 25-OH vitamin D is provided in a form coated on magnetic particles.
5. The method of claim 1 , wherein the labeled vitamin D compound comprises a label selected from the group consisting of radiolabels, fluorescent labels, luminescent labels, biotin labels, gold labels and enzyme labels.
6. The method of claim 1 , wherein the circulating, unbound vitamin D is a vitamin D metabolite.
7. The method of claim 6 , wherein the vitamin D metabolite is 25-hydroxy-Vitamin D or 1,25 dihydroxy Vitamin D.