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 capture moiety that is capable of specifically binding the Ligand Binding Domain of Vitamin D Receptor (VDR-LBD) bound to 1,25-dihydroxy-vitamin D or analog thereof without cross-reacting with uncomplexed VDR-LBD, wherein said capture moiety comprises a monoclonal antibody or a fragment of a monoclonal antibody that comprises:
(A) heavy chain CDR1, CDR2, and CDR3 domains whose amino acid sequences are respectively the amino acid sequences of SEQ ID NOs: 1, 2 and 3; and
(B) light chain CDR1, CDR2, and CDR3 domains whose amino acid sequences are respectively the amino acid sequences of SEQ ID NOs: 4, 5 and 6.
2. The capture moiety of claim 1 , wherein said capture moiety comprises a F(ab), Fab′, F(ab′) 2 , or F(v) fragment of an antibody.
3. The capture moiety of claim 1 , wherein said capture moiety comprises a single polypeptide chain that comprises said heavy chain CDR1, CDR2, and CDR3 domains and said light chain CDR1, CDR2, and CDR3 domains.
4. The capture moiety of claim 3 , wherein said capture moiety is an scFv single chain antibody.
5. The capture moiety of claim 1 , wherein said capture moiety comprises:
(A) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 7; and
(B) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 9.
6. The capture moiety of claim 5 , wherein said capture moiety is immobilized on a solid support.
7. The capture moiety of claim 6 , wherein said solid support is a bead, particle, plate, cuvette, lateral flow device, or flow cell.
8. The capture moiety of claim 6 , wherein said solid support is a paramagnetic particle.
9. The capture moiety of claim 1 , wherein said capture moiety is immobilized on a solid support.
10. The capture moiety of claim 9 , wherein said solid support is a bead, particle, plate, cuvette, lateral flow device, or flow cell.
11. The capture moiety of claim 9 , wherein said solid support is a paramagnetic particle.
12. A kit for detecting 1,25-dihydroxyvitamin D (1,25(OH) 2 D) or an analogue 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, wherein said kit comprises:
(i) a receptor protein comprising the Ligand Binding Domain of the Rattus norvegicus Vitamin D Receptor (VDR-LBD);
(ii) a capture moiety, wherein said capture moiety comprises a monoclonal antibody or a fragment of a monoclonal antibody, wherein said monoclonal antibody and said monoclonal antibody fragment comprise:
heavy chain CDR1, CDR2, and CDR3 domains whose amino acid sequences are respectively the amino acid sequences of SEQ ID NOs: 1, 2 and 3; and
light chain CDR1, CDR2, and CDR3 domains whose amino acid sequences are respectively the amino acid sequences of SEQ ID NOs: 4, 5 and 6; and
(iii) a binding buffer which has a pH comprised between 6 and 9; and
(iv) a solid support to which said capture moiety can be passively or covalently bound.
13. The kit of claim 12 , wherein said capture moiety comprises a F(ab), Fab′, F(ab′) 2 , or F(v) fragment of an antibody.
14. The kit of claim 12 , wherein said capture moiety comprises a single polypeptide chain that comprises said heavy chain CDR1, CDR2, and CDR3 domains and said light chain CDR1, CDR2, and CDR3 domains.
15. The kit of claim 14 , wherein said capture moiety is an scFv single chain antibody.
16. The kit of claim 12 , wherein said capture moiety comprises:
(A) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 7; and
(B) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 9.
17. The kit of claim 16 , wherein said capture moiety is immobilized on a solid support.
18. The kit of claim 17 , wherein said solid support is a bead, particle, plate, cuvette, lateral flow device, or flow cell.
19. The kit of claim 17 , wherein said solid support is a paramagnetic particle.
20. The kit of claim 12 , wherein said capture moiety is immobilized on a solid support.
21. The kit of claim 20 , wherein said solid support is a bead, particle, plate, cuvette, lateral flow device, or flow cell.
22. The kit of claim 20 , wherein said solid support is a paramagnetic particle.