Method and kit for detecting 1,25-dihydroxyvitamin D and related antibodies
There is disclosed an assay method for selectively detecting 1,25-dihydroxy-vitamin D in a biological fluid sample. According to the method of the invention, the pH of the test sample is adjusted to 6-9 and a receptor protein comprising the Ligand Binding Domain of Vitamin D Receptor (VDR-LBD) is added to the test sample, thereby obtaining the formation of a VDR-LBD/1,25-dihydroxyvitamin D complex in which the VDR-LBD portion is conformationally changed with respect to unbound VDR-LBD. The VDR-LBD/1,25-dihydroxyvitamin D complex is then detected by means of a capture moiety which is capable of specifically binding to VDR-LBD bound to 1,25-dihydroxyvitamin D. Also disclosed are an assay kit and an antibody for carrying out the method of the invention. The assay of the invention is preferably a sandwich immunoassay.
1. A monoclonal antibody which binds to the complex formed between the Ligand Binding Domain of Vitamin D Receptor (VDR-LBD) and 1,25-dihydroxyvitamin D, wherein the amino acid sequences of the CDRs of the heavy chain variable domain of the monoclonal antibody are SEQ ID NOs: 1, 2 and 3, and the amino acid sequences of the CDRs of the light chain variable domain of the monoclonal antibody are SEQ ID NOs: 4, 5 and 6.
2. The monoclonal antibody according to claim 1 , wherein the heavy chain variable domain comprises the amino acid sequence SEQ ID NO:7 or is encoded by a nucleic acid sequence comprising SEQ ID NO: 8.
3. The monoclonal antibody according to claim 1 , wherein the light chain variable domain comprises the amino acid sequence SEQ ID NO:9 or is encoded by a nucleic acid sequence comprising SEQ ID NO:10.
4. The monoclonal antibody according to claim 2 , wherein the light chain variable domain comprises the amino acid sequence SEQ ID NO:9 or is encoded by a nucleic acid sequence comprising SEQ ID NO:10.