# US 8,785,603 B2 · Antibodies to 25-hydroxyvitamin D2 and D3 and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,785,603 B2 - Document ID: US8785603B2 - Status: Granted - Kind: B2 - Application number: 13/458,847 - Filed: 2012-04-27 - Published: 2013-03-07 - Granted: 2014-07-22 - Earliest claimed: 2011-05-20 - Assignees: Siemens Healthcare Diagnostics Inc. - Applicants: Niver Panosian Sahakian; Bruce A. Campbell; Spencer Hsiang-Hsi Lin; James Vincent Freeman; Qimu Liao; Ramon A. Evangelista - Inventors: Niver Panosian Sahakian; Bruce A. Campbell; Spencer Hsiang-Hsi Lin; James Vincent Freeman; Qimu Liao; Ramon A. Evangelista - Web page: https://patentgrep.com/patent/US8785603B2 ## CPC - G01N 33/82: involving vitamins {or their receptors} - A61P 3/02: Nutrients, e.g. vitamins, minerals - C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids} - G01N 2800/02: Nutritional disorders - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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) ## Abstract Provided herein are antigenic molecules that can be used to generate antibodies capable of binding to a vitamin D derivative, such as 25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such as a vitamin D-C22 immunogenic molecule or compound. Antibodies produced using these antigenic molecules, and related antigenic compounds, are also described. In addition, disclosed herein are methods for detecting vitamin D deficiency in a subject, methods for treating a subject suspected of having a vitamin D deficiency, methods for monitoring progression of vitamin D deficiency in a subject, and methods for monitoring treatment of vitamin D deficiency in a subject in need thereof. Also provided are methods and reagents for the detection or quantification of 25-hydroxyvitamin D2 and D3, methods for stabilizing vitamin D analogs, and methods for separating 25-hydroxyvitamin D2 and D3 from vitamin D binding protein in a biological sample. ## Claims ### Claim 1 An isolated antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 28, wherein the antibody binds 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. ### Claim 2 (depends on 1) The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 16. ### Claim 3 (depends on 1) The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32. ### Claim 4 (depends on 1) The antibody of claim 1, wherein the antibody is a murine antibody. ### Claim 5 (depends on 1) The antibody of claim 1, wherein the antibody is recombinant. ### Claim 6 The antibody of claim, wherein binding to 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 is with substantially similar affinity. ### Claim 7 (depends on 1) The antibody of claim 1, wherein the antibody is produced by injecting a mammal with an immunogen comprising vitamin D-C22. ### Claim 8 (depends on 7) The antibody of claim 7, wherein the immunogen further comprises a carrier protein and a linker that comprises at least 6 atoms in a linear chain. ### Claim 9 (depends on 7) The antibody of claim 7, wherein the immunogen is represented by the formula: wherein x and y are, independently, 1-12, and at least one linker-conjugated vitamin D-C22 molecule is bound to the carrier protein. ### Claim 10 (depends on 7) The antibody of claim 7, wherein the immunogen is represented by the formula: wherein at least one linker-conjugated vitamin D-C22 molecule is bound to the bovine serum albumin (BSA) carrier protein. ### Claim 11 (depends on 7) The antibody of claim 7, wherein the immunogen is represented by the formula: wherein at least one linker-conjugated vitamin D-C22 molecule is bound to the keyhole limpet hemocyanin (KLH) carrier protein. ### Claim 12 (depends on 1) An isolated cell expressing the antibody of claim 1. ### Claim 13 (depends on 12) The cell of claim 12, wherein the cell is derived from an immunized mouse. ### Claim 14 (depends on 13) The cell of claim 13, wherein the cell is a hybridoma. ### Claim 15 (depends on 12) The cell of claim 12, wherein the cell is recombinantly produced. ### Claim 16 (depends on 1) An isolated polynucleotide encoding the antibody of claim 1. ### Claim 17 (depends on 16) The isolated polynucleotide of claim 16, wherein the polynucleotide is recombinant. ### Claim 18 (depends on 16) The isolated polynucleotide of claim 16, wherein the polynucleotide is a cDNA. ### Claim 19 (depends on 1) A method for detecting vitamin D deficiency in a subject, said method comprising: contacting a biological sample derived from said subject with the antibody of claim 1 or antigen binding fragment thereof; determining the level of total 25-hydroxyvitamin D in the biological sample; wherein a decrease between the level in the biological sample and the level in a normal control or a threshold level of 30 ng/mL is indicative of a vitamin D deficiency in said subject. ### Claim 20 (depends on 1) A method for treating a subject suspected of having a vitamin D deficiency, said method comprising: contacting a biological sample derived from said subject with the antibody of claim 1 or antigen binding fragment thereof; determining the level of total 25-hydroxyvitamin D in the biological sample; and if a decrease between the level in the biological sample relative to the level in a normal control or a threshold level of 30 ng/mL is determined, administering to said subject a treatment for vitamin D deficiency. ### Claim 21 (depends on 1) A method for monitoring progression of vitamin D deficiency in a subject in need thereof, said method comprising: contacting a first biological sample derived from said subject with the antibody of claim 1 or antigen binding fragment thereof; determining the level of total 25-hydroxyvitamin D in the first biological sample derived from said subject at a first time; contacting a second biological sample derived from said subject with the antibody of claim 1 or antigen binding fragment thereof; and, determining the level of total 25-hydroxyvitamin D in the second biological sample derived from said subject at a second time later than said first time; wherein a decrease between the level in the first biological sample and the level in the second biological sample is indicative of the progression of a vitamin D deficiency in said subject, wherein little or no change between the level in the first biological sample and the level in the second biological sample is indicative of stabilization of a vitamin D deficiency in the subject, and wherein an increase between the level in the first biological sample and the level in the second biological sample is indicative of regression of a vitamin D deficiency in the subject. ### Claim 22 (depends on 1) A method for monitoring treatment of vitamin D deficiency in a subject in need thereof, said method comprising: contacting a first biological sample derived from said subject with the antibody of claim 1 or antigen binding fragment thereof; determining the level of total 25-hydroxyvitamin D in the first biological sample derived from said subject at a first time; contacting a second biological sample derived from said subject with the antibody of claim 1 or antigen binding fragment thereof; and, determining the level of total 25-hydroxyvitamin D in the second biological sample derived from said subject at a second time later than said first time and following treatment of said subject for said vitamin D deficiency; wherein an increase in or stabilization of the level in the second biological sample relative to the level in the first biological sample is indicative of efficacy of the treatment of the vitamin D deficiency in said subject, and wherein a decrease in the level in the second biological sample relative to the level in the first biological sample is indicative of inefficacy of the treatment of the vitamin D deficiency in said subject. ### Claim 23 (depends on 1) A method for detecting vitamin D deficiency in a subject by determining the level of total 25-hydroxyvitamin D in a biological sample derived from said subject, said method comprising: combining a biological sample and a displacement buffer in a vessel; adding to said vessel the antibody of claim 1 or antigen-binding fragment thereof conjugated to a first label; adding to said vessel a 25-hydroxyvitamin D analog conjugated to a carrier protein and a second label; adding to said vessel a solid phase support conjugated to an antibody that recognizes the second label; and measuring the level of total 25-hydroxyvitamin D in the biological sample; wherein a decrease between the level in the biological sample and the level in a normal control or a threshold level of 30 ng/mL is indicative of a vitamin D deficiency in said subject. ### Claim 24 (depends on 1) A hybridoma cell that produces the antibody of claim 1, or an antigen-binding fragment thereof. ### Claim 25 (depends on 1) A kit comprising the antibody of claim 1, or an antigen-binding fragment thereof. ### Claim 26 (depends on 1) A diagnostic device comprising the antibody of claim 1, or an antigen-binding fragment thereof. ### Claim 27 (depends on 25) The kit of claim 25 further comprising an alkaline phosphatase-conjugated vitamin D-C22-bovine serum albumin (BSA). ### Claim 28 (depends on 26) The diagnostic device of claim 26 further comprising an alkaline phosphatase-conjugated vitamin D-C22-bovine serum albumin (BSA). ### Claim 29 (depends on 26) The device of claim 26 wherein said device performs enhanced chemiluminescence (ECL), enzyme immunoassay (EIA), immunohistochemistry (IHC), western blot analysis, radioimmunoassay (RIA), immunofluorescence, equilibrium dialysis, immunodifferentiation, or enzyme-linked immunosorbant assay (ELISA).