# US 10,501,548 B2 · Methods for detecting 1,25-dihydroxyvitamin D and related antibodies

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- Number: US 10,501,548 B2
- Document ID: US10501548B2
- Status: Granted
- Kind: B2
- Application number: 15/606,284
- Filed: 2017-05-26
- Published: 2017-09-21
- Granted: 2019-12-10
- Earliest claimed: 2013-01-28
- Assignees: DiaSorin S.p.A.
- Applicants: Diasorin S.p.A.
- Inventors: Joshua Soldo; Gregory Olson; Michael Lutterman; John Wall; Michael New; Hector Floyd Deluca; Fabrizio Bonelli
- Web page: https://patentgrep.com/patent/US10501548B2

## CPC

- C07K 16/2869: {against hormone receptors (for antibodies against neuromediator receptors C07K16/286)}
- C07K 16/26: against hormones {; against hormone releasing or inhibiting factors}
- G01N 33/82: involving vitamins {or their receptors}
- C07K 2317/32: specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 1

A method for detecting 1,25-dihydroxyvitamin D (1,25(OH)2D) or an analog thereof selected from one or more of the group consisting of 19-nor-1α-25-dihydroxyvitamin D2, 1α-hydroxyvitamin D2, 1α-hydroxyergocalciferol and 2-methylene-19-nor-(20S)-1α,25-(OH)2D3, 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the capture moiety is immobilized on a solid support.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the biological fluid sample is whole blood, plasma, serum or urine.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

The method according to claim 1, which is a sandwich immunoassay.

### Claim 7 (depends on 6)

The method according to claim 6, wherein in step (iii) said captured complex is detected using a labeled anti-VDR-LBD detector antibody.