METHODS AND COMPOSITIONS FOR ASSAYING VITAMIN D

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

1) a buffer of acidic pH,

2) a specific binding partner that specifically binds to said vitamin D moiety, if present in said sample, to form a vitamin D moiety/specific binding partner complex, said binding partner being different from a natural vitamin D binding protein for said vitamin D moiety,

3) an enzyme donor (ED)-vitamin D moiety conjugate, said enzyme donor (ED) comprising a first fragment of a β-galactosidase, and

4) an enzyme acceptor (EA), said enzyme acceptor (EA) comprising a second fragment of a β-galactosidase,

wherein when said ED-vitamin D moiety conjugate is not bound to said specific binding partner, said first fragment of a β-galactosidase in said enzyme donor (ED) and said second fragment of a β-galactosidase in said enzyme acceptor (EA) are configured to reassemble to form an active β-galactosidase, said sample is contacted with said buffer of acidic pH and said specific binding partner in one or more steps, and said sample is contacted with said ED-vitamin D moiety conjugate and said enzyme acceptor (EA) in other separate one or more steps; 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 by measuring the activity of said reassembled active β-galactosidase in the presence of a β-galactosidase substrate.

2 . The method of claim 1 , wherein the vitamin D moiety is vitamin D 3 , vitamin D 2 , a vitamin D metabolite, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), or 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3 or C3-epimer).

3 . The method of claim 1 , wherein the buffer of acidic pH has a pH ranging from about 1.0 to about 5.0.

4 . 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.

5 . The method of claim 1 , wherein the vitamin D moiety to be assayed and the vitamin D moiety in the ED-vitamin D moiety conjugate have the same or a similar affinity towards the vitamin D binding partner.

6 . The method of claim 1 , wherein the β-galactosidase is E. coli wild type β-galactosidase or its mutants.

7 . The method of claim 6 , wherein the second fragment of a β-galactosidase in the enzyme acceptor (EA) comprises a deletion near the amino terminus of about 5%-10% of the β-galactosidase single subunit.

8 . The method of claim 6 , wherein the first fragment of a β-galactosidase in the enzyme donor (ED) comprises about 40 to 100 amino acid residues of the β-galactosidase that are missing from the second fragment of a β-galactosidase in the enzyme acceptor (EA).

9 . The method of claim 1 , wherein the sample is contacted with the buffer of acidic pH and the specific binding partner before the sample is contacted with the ED-vitamin D moiety conjugate and/or the enzyme acceptor (EA).

10 . The method of claim 9 , wherein the sample is contacted with the vitamin D binding partner (e.g., antibody or antibodies) and the acidic pH buffer solution, then contacted with the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate), and then contacted with the enzyme acceptor (EA).

11 . The method of claim 9 , wherein the additions of the sample and reagents follow a specific sequence or order in which:

1) the sample is first diluted with a buffer comprising a vitamin D binding partner (e.g., antibody or antibodies), and part of the diluted sample is then contacted with the acidic buffer solution comprising a β-galactosidase substrate prior to the additions of the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate) and the enzyme acceptor (EA); and

2) the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate) and the enzyme acceptor (EA) are added into the reaction mixture sequentially or in two separated steps.

12 . The method of claim 9 , wherein the additions of the sample and reagents follow a specific sequence or order in which:

1) the sample is first contacted by the acidic buffer solution comprising a specific Vitamin D binding partner (e.g., antibody or antibodies) and a β-galactosidase substrate prior to the additions of the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate) and the enzyme acceptor (EA); and

2) the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate) and the enzyme acceptor (EA) are added into the reaction mixture sequentially or in two separated steps.

13 . The method of claim 9 , which is a homogenous assay that is conducted in a single reaction container (e.g., a cuvette) comprising the steps of sample dilution with a buffer comprising the vitamin D binding partner, the acidic pH buffer solution comprising a β-galactosidase substrate, the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate), the enzyme acceptor (EA) and one or more stabilizers.

14 . The method of claim 9 , which is a homogenous assay that is conducted in two separated containers (e.g., two cuvettes) with the sample being first diluted with a buffer comprising the vitamin D binding partner in one cuvette (sample dilution cuvette) followed by mixing part of the diluted sample with the acidic pH buffer solution comprising a β-galactosidase substrate in another separated cuvette (reaction cuvette) before additions of the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate), and the enzyme acceptor (EA).

15 . The method of claim 1 , wherein the β-galactosidase substrate is o-nitrophenyl-β-D-galactoside (ONPG), chlorophenol red-β-D-galactopyranoside (CPRG), or an analogue thereof.

16 . The method of claim 1 , which has a total assay time that is about 30 minutes or shorter.

17 . The method of claim 1 , which is conducted on a general chemistry analyzer or a clinical chemistry analyzer.

18 . The method of claim 17 , wherein the general chemistry analyzer or the clinical chemistry analyzer is capable of taking at least 3 reagents in an assay.

19 . 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.

20 . A kit for assaying a vitamin D moiety in a sample, which kit comprises:

a) a buffer of acidic pH,

b) a specific binding partner that specifically binds to a vitamin D moiety, if present in said sample, to form a vitamin D moiety/specific binding partner complex, said binding partner being different from a natural vitamin D binding protein for said vitamin D moiety,

c) an enzyme donor (ED)-vitamin D moiety conjugate, said enzyme donor (ED) comprising a first fragment of a β-galactosidase, and

d) an enzyme acceptor (EA), said enzyme acceptor (EA) comprising a second fragment of a β-galactosidase,

wherein when said ED-vitamin D moiety conjugate is not bound to said specific binding partner, said first fragment of a β-galactosidase in said enzyme donor (ED) and said second fragment of a β-galactosidase in said enzyme acceptor (EA) are configured to reassemble to form an active β-galactosidase.

21 . The kit of claim 20 , 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.

22 . The kit of claim 21 , wherein the means for assessing binding between the specific binding partner and the vitamin D moiety comprises a β-galactosidase substrate or a vitamin D calibrator.

23 . The kit of claim 20 , which comprises reagents:

a) a sample dilution buffer comprising the specific vitamin D binding partner (e.g., antibody or antibodies);

b) a first assay reagent (R1) comprising a β-galactosidase substrate in the buffer of acidic pH;

c) a second assay reagent (R2) comprising the ED-vitamin D moiety conjugate (e.g., ED-25(OH)D conjugate); and

d) a third assay reagent (R3) comprising the enzyme acceptor (EA).

24 . The kit of claim 23 , which comprises the sample dilution buffer, R1, R2 and R3 in a liquid stable format.

25 . The kit of claim 24 , which further comprises a vitamin D calibrator.

26 . A kit for assaying a vitamin D moiety in a sample, which kit comprises:

a) a first assay reagent (R1) comprising an acidic pH buffer solution comprising a β-galactosidase substrate and 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;

b) a second assay reagent (R2) comprising an enzyme donor (ED)-vitamin D moiety conjugate, said enzyme donor (ED) comprising a first fragment of a β-galactosidase; and

c) a third assay reagent (R3) comprising an enzyme acceptor (EA), said enzyme acceptor (EA) comprising a second fragment of a β-galactosidase,

wherein when said ED-vitamin D moiety conjugate is not bound to said specific binding partner, said first fragment of a β-galactosidase in said enzyme donor (ED) and said second fragment of a β-galactosidase in said enzyme acceptor (EA) are configured to reassemble to form an active β-galactosidase.

27 . The kit of claim 26 , which comprises the R1, R2 and R3 in a liquid stable format or in a solid format, e.g., lyophilized format, or in a mixed format of a liquid and solid e.g., lyophilized.

28 . A method for assaying a vitamin D moiety in a sample using a kit of claim 23 , which method comprises:

a) diluting a sample with the sample dilution buffer comprising the specific vitamin D binding partner, mixing part of the diluted sample with R1, incubating the mixture for a period of time, adding R2, and after another period of incubation time, adding R3 to the reaction mixture; and

b) quantifying the amount of a vitamin D moiety (e.g., 25(OH)D) in the sample by measuring the optical change of the reaction mixture and using a set of vitamin D calibrators of known vitamin D values (e.g., 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) mixing a sample with R1 to form a reaction mixture;

b) after a period of incubation, adding R2 and R3 to the reaction mixture in two separate steps;

c) quantifying the amount of a vitamin D moiety (e.g., 25(OH)D) in the sample by measuring the optical change of the reaction mixture and using a set of vitamin D calibrators of known vitamin D values (e.g., a set of 25(OH)D calibrators).

30 . A reaction mixture for assaying a vitamin D moiety in a sample, which reaction mixture comprises, in an acidic environment:

a) a specific binding partner that specifically binds to a vitamin D moiety, if present in said sample, to form a vitamin D moiety/specific binding partner complex, said binding partner being different from a natural vitamin D binding protein for said vitamin D moiety,

b) an enzyme donor (ED)-vitamin D moiety conjugate, said enzyme donor (ED) comprising a first fragment of a β-galactosidase, and

c) an enzyme acceptor (EA), said enzyme acceptor (EA) comprising a second fragment of a β-galactosidase,

wherein when said ED-vitamin D moiety conjugate is not bound to said specific binding partner, said first fragment of a β-galactosidase in said enzyme donor (ED) and said second fragment of a β-galactosidase in said enzyme acceptor (EA) are configured to reassemble to form an active β-galactosidase.