Extraction and quanification of vitamins a & d in fluid samples

• US 20050079563A1
• Filed: 06/10/2003
• Published: 04/14/2005
• Est. Priority Date: 06/10/2002
• Status: Active Grant

• Current Assignee: SciMed Technologies, Inc. (Boston Scientific Corporation)
• Original Assignee: SciMed Technologies, Inc. (Boston Scientific Corporation)
• Inventors: Gupta, Rajan
• Granted Patent: US 7,776,544 B2
• Time in Patent Office: Days
• US Class Current: 435/7.920
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: G01N 33/82 involving vitamins or their receptors

Abstract

The invention discloses monoclonal antibodies for vitamins A (retinol palmitate) and D3 (cholecalciferol); a method for using monoclonal antibodies, and the monoclonal antibodies disclosed herein, in particular, to quantitate these vitamins in fluids such as dairy products, and blood, and also raw or processed agri-food and beverage products. The method involves contacting the sample with a mixture of polar and non-polar organic solvents in combination with inorganic salts to remove fat molecules into an organic fraction, and assaying the organic fraction by immunoassay involving the monoclonal antibodies. The presence of a mixture of non-polar and polar organic solvents increases the separation of vitamins from fat molecules and enables the test samples to be quantified by immunoassay without any further treatment.

Claims

1. A method of quantifying vitamin A or D3, or vitamin A and D3, in a fluid sample, comprising the steps of:

(a) extracting the fluid sample with an inorganic salt and an extraction solvent comprising a mixture of a non-polar organic solvent and a polar organic solvent, to produce an organic fraction; and

(b) determining the amount of vitamin D3 or vitamin A, or both vitamin D3 and vitamin A, in the organic fraction.

2. The method of claim 1 wherein the vitamin is quantified with an antibody which specifically binds to the vitamin.

3. The method of claim 2 wherein the antibody is a monoclonal antibody.

4. The method of claim 2 wherein the antibody is a polyclonal antibody.

5. The method of claim 1 wherein the non-polar organic solvent comprises an aliphatic hydrocarbon having from 4 to 10 carbon atoms, or a mixture thereof, and the polar organic solvent comprises a halogenated hydrocarbon having from 1 to 4 carbon atoms, or a mixture thereof.

6. The method of claim 1 wherein the inorganic salt is an alkaline metal salt.

7. The method of claim 1 wherein the inorganic salt is added to the sample in an alcoholic solution.

8. The method of claim 7 wherein the alcoholic solution comprises greater than about 50% and less than about 75% ethanol in water (v:

v).

9. The method of claim 6 wherein the alkaline metal salt comprises potassium hydroxide.

10. The method of claim 5 wherein the non-polar organic solvent comprises a mixture of hexane and petroleum ether and the polar organic solvent comprises methylene chloride

11. The method of claim 1, wherein the fluid sample is chosen from the group consisting of a dairy product, an agri-food, a beverage, blood or a biological fluid.

12. The method of claim 1 wherein the vitamin D3 or vitamin A, or vitamins D3 and vitamin A, is detected by an immunosorbent assay.

13. The method of claim 1 wherein the vitamin is quantified by direct measurement.

14. A monoclonal antibody which specifically binds to vitamin D3 and not to its metabolites or other forms of vitamin D.

15. An isolated cell line which produces the antibody of claim 14.

16. (Cancelled).

17. An isolated cell line which produces the antibody of claim 16.

19. The kit of claim 17 wherein the means for performing a quantitative assay comprises reagents and hardware for a competitive ELISA or a sandwich ELISA.

18. A kit for assaying for vitamin D3 or vitamin A, or vitamin D3 and vitamin A, in a fluid sample comprising:

(a) an inorganic salt and an extraction solvent comprising a mixture of a non-polar organic solvent and a polar organic solvent, to produce an organic fraction,

(b) a monoclonal antibody which specifically binds to vitamin D3 or vitamin A, and

(c) means for performing a quantitative assay with the antibody.