METHODS AND COMPOSITIONS FOR ASSAYING VITAMIN D
The present invention provides methods for assaying a vitamin D moiety in a sample, using a water miscible organic solvent, a specific binding partner that specifically binds to said vitamin D moiety, the binding partner being different from a natural vitamin D binding protein for the vitamin D moiety, and a water soluble polymer that facilitates binding between the specific binding partner and the vitamin D moiety. Kits and reaction mixtures for assaying a vitamin D moiety in a sample are also provided.
1 . A method for assaying a vitamin D moiety in a sample, which method comprises:
a) contacting a sample containing or suspected of containing a vitamin D moiety with a water miscible organic solvent, a specific binding partner that specifically binds to said vitamin D moiety, if present in said sample, said binding partner being different from a natural vitamin D binding protein for said vitamin D moiety, and a water soluble polymer that facilitates binding between said specific binding partner and said vitamin D moiety; and
b) assessing binding between said specific binding partner and said vitamin D moiety to determine the presence, absence and/or amount of said vitamin D moiety in said sample.
2 . The method of claim 1 , which does not comprise a step of removing the natural vitamin D binding protein for the vitamin D moiety prior to assessing binding between the specific binding partner and the vitamin D moiety, and/or does not comprise a wash step.
3 . The method of claim 1 , which is conducted as a homogeneous assay.
4 . The method of claim 1 , wherein the sample is contacted with a single reaction mixture comprising the water miscible organic solvent, the specific binding partner and the water soluble polymer.
5 . The method of claim 1 , wherein the pH in a final reaction mixture comprising the sample, the water miscible organic solvent, the specific binding partner and the water soluble polymer ranges from about 4 to about 13.
6 . The method of claim 1 , which is used to assess status of the vitamin D moiety in a subject, and the sample is a biological sample obtained and/or derived from the subject.
7 . The method of claim 1 , wherein the vitamin D moiety is 25(OH)D2, 25(OH)D 3 or a sum of 25(OH)D2 and 25(OH)D3.
8 . The method of claim 1 , wherein the water miscible organic solvent is selected from the group consisting of dimethyl sulfoxide, dimethyl formamide, acetonitrile, methanol, ethanol, isopropyl alcohol, dimethyl acetamide, and a combination thereof.
9 . The method of claim 8 , wherein the concentration of the water miscible organic solvent(s) in the final reaction mixture ranges from about 0.25% (V/V) to about 20% (V/V).
10 . The method of claim 1 , wherein the specific binding partner that specifically binds to the vitamin D moiety is an antibody that specifically binds to the vitamin D moiety.
11 . The method of claim 10 , wherein the antibody specifically binds to 25(OH)D.
12 . The method of claim 1 , wherein the water soluble polymer is a synthetic water soluble polymer and/or a natural water soluble polymer.
13 . The method of claim 12 , wherein the synthetic water soluble polymer is selected from the group consisting of a polyoxyethylene (POE), a polyvinyl pyrrolidone (PVP), a polyvinyl alcohol (PVA), a polyacrylic acid (PAA), a polyacrylamide, a poly-N-(2-hydroxypropyl)methacrylamide (HPMA), a poly-divinyl ether-maleic anhydride (DIVEMA), a polyoxazoline, a polyphosphate, a polyphosphazene, a polyethyleneimine, a polyglycosylethyl methacrylate, and a combination thereof, and the natural water soluble polymer is selected from the group consisting of a chitosan derivative, a dextran, a carrageenan, a cellulose ether, a hyaluronic acid (HA), and a starch or starch based derivative, and a combination thereof.
14 . The method of claim 1 , wherein the water soluble polymer is a non-ionic water soluble polymer.
15 . The method of claim 1 , wherein the water soluble polymer has a molecular weight from about 1,000 to about 2,000,000.
16 . The method of claim 1 , wherein the concentration of the water soluble polymer in the final reaction mixture ranges from about 0.025% (W/V) to about 10% (W/V).
17 . The method of claim 1 , which is conducted using a particle-enhanced immunoturbidimetric method, a particle-enhanced immunonephelometric method or a magnetic particle (bead) based immunoassay method.
18 . The method of claim 17 , wherein the particle comprises polystyrene, polymethyl methacrylate, polymethyl naphthalene, poly(divinylbenzene), polyvinyl naphthalene, co-polymer of styrene, acrylic acid divinylbenzene, naphathalene, carbon 60, magnetic beads, gold, silver, silica, silicon dioxide, chromium dioxide, and/or titanium dioxide, or the particle is a nanoparticle.
19 . The method of claim 1 , which is a homogenous assay conducted in a single reaction mixture without phase separation or washing step.
20 . The method of claim 1 , which has a total assay time that is about 30 minutes or shorter.
21 . The method of claim 1 , which is conducted on a general chemistry analyzer or a clinical chemistry analyzer.
22 . A kit for assaying a vitamin D moiety in a sample, which kit comprises:
a) a water miscible organic solvent;
b) a specific binding partner that specifically binds to said vitamin D moiety, if present in said sample, said binding partner being different from a natural vitamin D binding protein for said vitamin D moiety; and
c) a water soluble polymer that facilitates binding between said specific binding partner and said vitamin D moiety.
23 . The kit of claim 22 , which further comprises means for assessing binding between the specific binding partner and the vitamin D moiety to determine the presence, absence and/or amount of the vitamin D moiety in the sample.
24 . The kit of claim 22 , which comprises reagents:
(1) a first assay reagent comprising the water miscible organic solvent and the water soluble polymer;
(2) a second assay reagent comprising a specific antibody against 25(OH)D; and
(3) a third assay reagent comprising 25(OH)D coated particles.
25 . The kit of claim 22 , which comprises reagents:
(1) a first assay reagent comprising the water miscible organic solvent, the water soluble polymer and a specific antibody against 25(OH)D; and
(2) a second assay reagent comprising 25(OH)D coated particles.
26 . The kit of claim 22 , which comprises:
(1) a first assay reagent comprising the water miscible organic solvent and the water soluble polymer, and
(2) a solid surface comprising an immobilized substance that is the specific binding partner that specifically binds to a vitamin D moiety or its analog.
27 . A method for assaying a vitamin D moiety in a sample using the kit of claim 24 , which method comprises:
a) forming a mixture of a sample, the first assay reagent and the second assay reagent and incubating the mixture for a period of time before adding the third assay reagent to the mixture; and
b) quantifying the amount of 25(OH)D in the sample by measuring the optical change of the reaction mixture and using a set of 25(OH)D calibrators.
28 . A method for assaying a vitamin D moiety in a sample using the kit of claim 25 , which method comprises:
a) forming a mixture of a sample and the first assay reagent and incubating the mixture for a period of time before adding the second assay reagent to the mixture; and
b) quantifying the amount of 25(OH)D in the sample by measuring the optical change of the reaction mixture and using a set of 25(OH)D calibrators.
29 . A method for assaying a vitamin D moiety in a sample using the kit of claim 26 , which method comprises:
a) forming a mixture of a sample and the first assay reagent and incubating the mixture for a period of time; and
b) contacting the mixture with the solid surface to determine the presence, absence and/or amount of the vitamin D moiety in the sample.
30 . A reaction mixture for assaying a vitamin D moiety in a sample, which reaction mixture comprises:
a) a water miscible organic solvent;
b) a specific binding partner that specifically binds to said vitamin D moiety, if present in said sample, said binding partner being different from a natural vitamin D binding protein for said vitamin D moiety; and
c) a water soluble polymer that facilitates binding between said specific binding partner and said vitamin D moiety.