Determination of vitamin D metabolite and displacement from plasma or serum binding proteins

• US 7,482,162 B2
• Filed: 12/06/2001
• Issued: 01/27/2009
• Est. Priority Date: 12/06/2000
• Status: Expired due to Fees

• Current Assignee: Immunodiagnostic Systems Ltd. (Revvity, Inc.)
• Original Assignee: Immunodiagnostic Systems Ltd. (Revvity, Inc.)
• Inventors: Laurie, David, Gardner, Michael James, Barnes, Alexander Kirkley
• Primary Examiner(s): Shibuya; Mark L
• Assistant Examiner(s): Venci; David J
• Application Number:
  US10/433,923
• Publication Number:
  US 20040096900A1
• Time in Patent Office: 2,609 Days
• Field of Search: 435/4, 435/7.1, 435/7.5, 435 77- 772, 435 792- 795, 435/287.1, 435/287.2, 435/962, 435/975, 435/7.9, 435/28, 435/188, 436/71, 436174-178, 436/817, 436/825, 436/517, 436/163, 436/18, 436/20, 436/826, 210/634, 210/635, 210/645, 210/646, 514/456, 514/822, 514/167, 514/168, 514/457, 549/400, 549/402, 549/423, 549/401, 568/327, 568/328, 424/530, 424/531, 426/425, 426/429, 516/113, 516/139, 516/141, 516/143, 516/146, 516/153, 530/402, 530/412, 530/422, 530/423, 530/424, 552/653
• US Class Current: 436/20
• CPC Class Codes: G01N 33/82 involving vitamins or their receptors
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

We disclose methods for measuring vitamin D metabolite in plasma or serum samples. The methods comprise a step of adding to the plasma or serum samples a non-competitive displacement agent comprising 8-anilino-1-naphthalenesulfonic acid ammonium salt, 3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent. The non-competitive displacement agent separates vitamin D metabolite from binding proteins in the sample, such that the displaced vitamin D metabolite is available for capture and detection in subsequent binding assays. Thus, our invention finds use in methods of separating and detecting vitamin D metabolites otherwise tightly bound to plasma or serum binding proteins.

Claims

1. A method for measuring vitamin D metabolite present in a plasma or serum sample, the method comprising:

(a) adding to a serum or plasma sample a non-competitive displacement agent comprising 8-anilino-1-napthalenesulfonic acid ammonium salt, 3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent, which separates any vitamin D metabolite in the sample from protein to which the vitamin D metabolite is bound, such that any separated vitamin D metabolite can be captured;

(b) contacting the sample with a support having immobilised thereon a binding factor, the binding factor capturing any vitamin D metabolite separated in step (a); and

(c) measuring the amount of vitamin D metabolite captured in step (b);

wherein capture of separated vitamin D metabolite occurs in step (b) without prior extraction from the sample of the protein separated from the vitamin D metabolite in step (a).

2. The method of claim 1, wherein the vitamin D metabolite is 25-hydroxy vitamin D.

5. The method of claim 1 or claim 3, wherein the water miscible solvent is methanol.

6. The method of claims 1, 2 or 3, wherein the amount of vitamin D metabolite is measured in a binding assay.

7. The method of claim 6, wherein the binding assay is a competitive binding assay.

8. The method of claim 6, wherein the binding assay is an immunoassay.

9. The method of claim 7, wherein the competitive binding assay comprises the steps of:

(a) providing a support having immobilized thereon a binding factor capable of binding a vitamin D metabolite;

(b) contacting the support with a sample comprising the vitamin D metabolite to be measured and a non-competitive displacement agent comprising 8-anilino-1-napthalenesulfonic acid ammonium salt, 3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent, which separates any vitamin D metabolite in the sample from protein to which the vitamin D metabolite is bound, so that any separated vitamin D metabolite binds to the binding factor;

(c) contacting the support with a labeled form of the vitamin D metabolite; and

(d) measuring the amount of labeled vitamin D metabolite left bound to the support,

wherein the amount of labeled vitamin D metabolite bound to the support is proportional to the amount of vitamin D metabolite in the sample.

10. The method of claim 9, wherein the binding factor immobilized on the support is vitamin D binding protein (DBP).

11. The method of claim 9, wherein the binding factor immobilized on the support is an anti-vitamin D metabolite antibody.

12. The method of claim 9, wherein the labeled vitamin D metabolite is labeled with biotin, avidin, a fluorescent molecule, or a chemiluminescent molecule.

3. A method for measuring vitamin D metabolite present in a plasma or serum sample, the method comprising:

(a) adding to a serum or plasma sample a non-competitive displacement agent comprising 8-anilino- 1-napthalenesulfonic acid ammonium salt, 3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent, which non-competitive displacement agent separates any vitamin D metabolite in the sample from protein to which the vitamin D metabolite is bound, such that any separated vitamin D metabolite can be captured;

(b) contacting the sample with a support having immobilised thereon a binding factor, which binding factor captures any vitamin D metabolite separated in step (a); and

(c) measuring the amount of vitamin D metabolite captured in step (b).

4. The method of claim 3, wherein the vitamin D metabolite is 25-hydroxy vitamin D.