Hydroxycholesterol immunoassay
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.
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