Methods and composition for measuring the amount of Vitamin D derivatives

A method for measuring the amount or concentration of a vitamin D derivative in a sample comprising:

• a) contacting a sample with a polypeptide comprising a modified VDR ligand-binding domain (VDR-LBD) and a donor-acceptor pair of fluorophores;
• and
• b) measuring the amount of fluorescence, wherein said amount of fluorescence is detectably higher or lower in the presence of the vitamin D derivative relative to the amount of fluorescence in the absence of the vitamin D derivative;
• wherein the modified VDR comprises at least one mutation causing said modified VDR to have a higher binding affinity to 25-hydroxyvitamin D relative to calcitriol, and wherein said at least one mutation comprises a substitution at one or more amino acid positions selected from the group consisting of 274, 147, 150, 227, 230, 233, 234, 271, 275, 286, 288, 300, 309, 313, 418, 237, 143, 278, 305 and 397 of SEQ ID NO:
• 1.