Methods 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, 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. The assay is preferably a sandwich immunoassay.
1. A method for detecting 1,25-dihydroxyvitamin D (1,25(OH) 2 D) or an analog thereof selected from one or more of the group consisting of 19-nor-1α-25-dihydroxyvitamin D 2 , 1α-hydroxyvitamin D 2 , 1α-hydroxyergocalciferol and 2-methylene-19-nor-(20S)-1α,25-(OH) 2 D 3 , in a biological fluid sample, the method comprising the steps of:
(i) adjusting the pH of the biological fluid sample to a value comprised between 6 and 9 and simultaneously or subsequently adding to the biological fluid sample a receptor protein comprising the Ligand Binding Domain of the Rattus norvegicus Vitamin D Receptor (VDR-LBD), under conditions sufficient for forming a complex of 1,25-dihydroxyvitamin D or said analog thereof bound to the VDR-LBD of the receptor protein;
(ii) incubating said complex in the presence of a capture moiety, wherein said capture moiety is a monoclonal antibody or a fragment of the monoclonal antibody, wherein said monoclonal antibody and said monoclonal antibody fragment comprise:
heavy chain CDR1, CDR2, and CDR3 domains that respectively are the amino acid sequences of SEQ ID NOs: 1, 2 and 3; and
light chain CDRs that respectively are the amino acid sequences of SEQ ID NOs: 4, 5 and 6; and
wherein said incubation is under conditions sufficient to permit said monoclonal antibody or said monoclonal antibody fragment to bind said complex and to form a captured complex; and
(iii) detecting said captured complex.
2. The method according to claim 1 , wherein the receptor protein is the whole Vitamin D Receptor protein or the Ligand Binding Domain (LBD) thereof in an isolated or engineered form.
3. The method according to claim 1 , wherein the capture moiety is immobilized on a solid support.
4. The method according to claim 1 , wherein the biological fluid sample is whole blood, plasma, serum or urine.
5. The method according claim 1 , wherein in step (i) the pH of the biological fluid sample is adjusted to a value comprised between 7 and 8.6.
6. The method according to claim 1 , which is a sandwich immunoassay.
7. The method according to claim 6 , wherein in step (iii) said captured complex is detected using a labeled anti-VDR-LBD detector antibody.