Hydroxycholesterol immunoassay

Patent metadata

Field	Value
Publication number	US-11971419-B2
Application number	US-202117229939-A
Country	US
Kind code	B2
Filing date	Apr 14, 2021
Priority date	Nov 9, 2010
Publication date	Apr 30, 2024
Grant date	Apr 30, 2024

Abstract

Provided is an antibody composition comprising antibodies that specifically bind to 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol. Also provided is a method of making antibodies that specifically bind to 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. Further provided is a method of assaying for 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. Still further provided is a kit for detecting 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol.

First claim

What is claimed is:

1. A method of assaying for 24(S)-hydroxycholesterol in a fluid or tissue sample from a mammal, the method comprising
combining the sample with antibodies that specifically bind to 24(S)-hydroxycholesterol,
wherein the antibodies are generated by (a) preparing a 24(S)-hydroxycholesterol immunogen by conjugating 3-O-succinoyl-24(S)-hydroxycholesterol to a carrier protein for immunization of a rabbit and the carrier protein is attached through a linkage at the 3-position; (b) immunizing the rabbit with the 24(S)-hydroxycholesterol immunogen under conditions such that the immune system of the rabbit makes antibodies to 24(S)-hydroxycholesterol; and (c) collecting the anti-24(S)-hydroxycholesterol polyclonal antibodies from the rabbit; and
detecting whether the antibodies specifically bind to 24(S)-hydroxycholesterol from the sample;

wherein specific antibody binding to 24(S)-hydroxycholesterol from the sample indicates that 24(S)-hydroxycholesterol is present in the sample.

2. The method of claim 1 , wherein the carrier protein is keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin.

3. The method of claim 1 , wherein the sample is from cerebrospinal fluid.

4. The method of claim 1 , wherein the sample is from blood.

5. The method of claim 1 , wherein the sample is brain tissue.

6. The method of claim 1 , wherein the sample is from a human that has cognitive impairment, Huntington's disease, Alzheimer's disease, or multiple sclerosis.

7. The method of claim 1 , performed on a solid phase.

8. The method of claim 7 , wherein the solid phase is a bead or microplate.

9. The method of claim 1 , wherein the immunoassay is an enzyme linked immunosorbent assay (ELISA).

10. The method of claim 9 , wherein the ELISA is an indirect competitive ELISA.

11. The method of claim 10 , wherein the indirect competitive ELISA comprises binding the anti-24(S)-hydroxycholesterol polyclonal antibodies to a solid phase.

12. The method of claim 11 , wherein the anti-24(S)-hydroxycholesterol polyclonal antibodies are bound to second antibodies that are directly bound to the solid phase.

13. The method of claim 1 , comprising a microarray assay.

14. A method of assaying for 24(S)-hydroxycholesterol in a fluid or tissue sample from a mammal; wherein the method employs anti-24(S)-hydroxycholesterol polyclonal antibodies specific to 24(S)-hydroxycholesterol that are obtained by immunizing a rabbit with 3-O-succinoyl-24(S)-hydroxycholesterol conjugated to a carrier protein through a linkage at the 3-position; the method comprising the steps of
a. noncovalently binding second antibodies to a solid phase;
b. adding the anti-24(S)-hydroxycholesterol polyclonal antibodies to the solid phase under conditions such that the second antibodies specifically bind to the anti-24(S)-hydroxycholesterol polyclonal antibodies;
c. adding the sample and a biotinylated 24(S)-hydroxycholesterol to the solid phase under conditions such that the biotinylated 24(S)-hydroxycholesterol binds to binding sites on the anti-24(S)-hydroxycholesterol polyclonal antibodies in competition with 24(S)-hydroxycholesterol in the sample;
d. adding an enzyme conjugate that is an avidin-enzyme conjugate or a streptavidin-enzyme conjugate to the solid phase under conditions such that the enzyme conjugate specifically binds to the biotinylated 24(S)-hydroxycholesterol that bound in step c;
e. adding a substrate to the enzyme in the enzyme conjugate, wherein the substrate is converted to a colored product that absorbs light at a specified wavelength in proportion to the amount of the enzyme bound to the solid phase; and
f. measuring the absorbance of light at the specified wavelength, wherein the absorbance is inversely proportional to the amount of 24(S)-hydroxycholesterol present in the sample.

15. The method of claim 14 , wherein, in step c, the sample and the biotinylated 24(S)-hydroxycholesterol are incubated with the solid phase at the same time such that 24(S)-hydroxycholesterol in the sample competes with the biotinylated 24(S)-hydroxycholesterol for anti-24(S)-hydroxycholesterol polyclonal antibody binding sites.

16. The method of claim 14 , wherein the anti-24(S)-hydroxycholesterol polyclonal antibodies specific to 24(S)-hydroxycholesterol are obtained by immunizing a rabbit with 3-O-succinoyl-24(S)-hydroxycholesterol conjugated to the carrier protein selected from the group consisting of keyhole limpet hemocyanin, bovine serum albumin, and ovalbumin.

17. The method of claim 14 , wherein the biotinylated 24(S)-hydroxycholesterol has the chemical structure of

18. The method of claim 14 , wherein the biotinylated 24(S)-hydroxycholesterol has the chemical structure of

19. The method of claim 14 , wherein the biotinylated 24(S)-hydroxycholesterol is a biotin derivative of 3-O-succinoyl-24(S)-hydroxycholesterol N-hydroxysuccinimide ester having the chemical structure of

Assignees

• Enzo Life Sciences Inc

Inventors

• Mullenix Michael C
• Zipkin Robert Elliot
• Adams Jeffrey Kroll
• Patton Wayne Forrest
• Donegan James J

Classifications

• G01N33/92 Primary

  involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} · CPC title

• C07J9/00

  Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane · CPC title

• C07J43/003

  not condensed · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• C07K16/44

  against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids} · CPC title