Method of dissociating vitamin d from vitamin d binding protein and method of measuring vitamin d using the same

Abstract

To provide a method of dissociating vitamin D from a vitamin D-binding protein, which allows vitamin D to be dissociated from a vitamin D-binding protein more sufficiently than known techniques, and to provide a method of measuring vitamin D using the same.SOLUTION: A method for dissociating vitamin D in a sample collected from a living body from vitamin D-binding protein is provided, the method comprising treating the sample with urea and at least one surfactant selected from the group consisting of deoxycholic acid, cholic acid, and salts of the aforementioned.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for dissociating vitamin D from vitamin D-binding protein and a method for measuring vitamin D using the same. [Background technology]

[0002] Most vitamin D in the blood exists bound to vitamin D-binding protein. When measuring vitamin D in the blood using immunoassay, anti-vitamin D antibodies are used, but the complex between vitamin D and vitamin D-binding protein cannot be measured using immunoassays that use anti-vitamin D antibodies, resulting in lower vitamin D measurements than the actual value.

[0003] To solve this problem, a method for dissociating vitamin D from vitamin D-binding protein is known (Patent Document 1). Patent Document 1 describes a method for dissociating vitamin D from vitamin D-binding protein by treating a sample with a surfactant containing a steroid skeleton, such as deoxycholic acid. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6281497 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have found that the method described in Patent Document 1 does not sufficiently dissociate vitamin D from vitamin D-binding protein.

[0006] Therefore, an object of the present invention is to provide a method for dissociating vitamin D from vitamin D-binding protein, which can dissociate vitamin D from vitamin D-binding protein more efficiently than known techniques, and a method for measuring vitamin D using the same. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have discovered that treating a sample with a surfactant such as deoxycholic acid and urea can dissociate more vitamin D / vitamin D-binding protein complexes in the sample than known techniques, and have thus completed the present invention.

[0008] That is, the present invention provides the following. (1) A method for dissociating vitamin D in a sample separated from a living body from vitamin D-binding protein, the method comprising treating the sample with urea and at least one surfactant selected from the group consisting of deoxycholic acid, cholic acid, and salts thereof. (2) The method according to (1), wherein the sample is a blood sample. (3) The method according to (1), wherein the final concentration of the surfactant during the treatment is 0.05 w / v % to 1 w / v %, and the final concentration of the urea is 0.0005 M to 10 M. (4) The method according to (1), wherein the surfactant is deoxycholate. (5) A method for measuring vitamin D in a sample, comprising the steps of subjecting a sample separated from a living body to the method according to any one of (1) to (4) above, and then measuring vitamin D in the sample. (6) The method described in (5), wherein vitamin D is measured by immunoassay. (7) The method according to (6), wherein the immunoassay utilizes an antigen-antibody reaction between an anti-vitamin D antibody or an antigen-binding fragment thereof and vitamin D in a sample. (8) The method described in (7), wherein the anti-vitamin D antibody is an anti-25-hydroxyvitamin D3 antibody. [Effects of the Invention]

[0009] The present invention provides a novel method for dissociating vitamin D from vitamin D-binding protein, which can dissociate vitamin D from vitamin D-binding protein more efficiently than known techniques, and a method for measuring vitamin D using the same. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram showing measurement results in the following Examples and Comparative Examples. DETAILED DESCRIPTION OF THE INVENTION

[0011] The "sample" in the present invention is a body fluid separated from a living body and is not particularly limited as long as it contains vitamin D, but a blood sample is preferred. Here, blood samples include whole blood, serum, plasma, and dilutions thereof.

[0012] In the present invention, unless otherwise specified, "vitamin D" includes vitamin D2 and vitamin D3 as well as their metabolites (compounds in which a hydroxyl group is added to vitamin D, such as 25OH vitamin D2, 25OH vitamin D3, 1,25(OH)2 vitamin D2, and 1,25(OH)2 vitamin D3).

[0013] In the method of the present invention, a sample is treated with urea and at least one surfactant selected from the group consisting of deoxycholic acid, cholic acid, and salts thereof. The salt is preferably an alkali metal salt such as a sodium salt or a potassium salt. Furthermore, deoxycholic acid and its salts are preferred as the surfactant.

[0014] The final concentration of the surfactant during the treatment is preferably 0.05 w / v% to 1 w / v%, particularly preferably 0.1 w / v% to 0.5 w / v%. The final concentration of urea during the treatment is preferably 0.0005 M to 12 M, more preferably 0.001 M to 10 M, and even more preferably 4 M to 8 M. It is simple and preferable to prepare a vitamin D dissociation solution containing the surfactant and urea in a buffer solution in advance and mix this with the sample.

[0015] The temperature for the treatment is not particularly limited, and is usually 0°C to 37°C, but room temperature is convenient since there are no problems. The treatment time is also not particularly limited, and is usually about 1 second to 1 minute, preferably about 5 seconds to 30 seconds. It is convenient to mix the vitamin D dissociation solution with a sample and immediately subject this mixture to the next measurement, since the treatment can be carried out without any problems.

[0016] In the method for measuring vitamin D in a sample of the present invention, vitamin D contained in the sample after the dissociation method is measured. A feature of the present invention is that vitamin D is released by dissociating vitamin D / vitamin D-binding protein in the sample, so the vitamin D measurement method itself can be performed by a well-known method. While the measurement method is not particularly limited, immunoassay is preferred because it is simple. Various immunoassay methods are well-known, and any of these well-known methods can be employed. A preferred immunoassay utilizes an antigen-antibody reaction between an anti-vitamin D antibody or an antigen-binding fragment thereof and vitamin D in the sample. Examples of antigen-binding fragments include F(ab')2, Fab, Fab', and Fv. Well-known immunoassay methods, classified according to their format, include sandwich methods, competitive methods, agglutination methods, and immunochromatography. Furthermore, well-known immunoassay methods, classified according to the label used, include enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, and chemiluminescent immunoassay. Any of these methods can be employed.

[0017] The present invention will be specifically described below based on examples, although the present invention is not limited to the following examples. [Example]

[0018] 1.Material Vitamin D: 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 (MedChemExpress) Bovine serum albumin (BSA) (Boval) ·Disuccinimidyl glutarate;DSG (Thermo Fisher Scientific) Maxisorp solid-phase microtiter plate (Thermo Scientific) Sodium deoxycholate (Merck) Urea (Nacalai Tesque) Guanidine hydrochloride (Nacalai Tesque) Tris(hydroxymethyl)aminomethane hydrochloride; Tris-HCl (Nacalai Tesque) Mouse monoclonal anti-25(OH)D antibody (in-house produced) ·HRP-conjugated Goat anti Mouse IgG antibody (Dako)

[0019] 2. Preparation of Vitamin D Solid-phase Microplates 1) Vitamin D and BSA (1 mg / mL) were mixed in PBS (pH 7.2) at a ratio of 1 to 40:1. 2) 30 μL of DSG (3.26 mg / mL) was added. 3) Incubate at room temperature for 30 minutes or on ice for 2 hours. 4) 1 M Tris (pH 7.5) was added to a final concentration of 20 to 50 mM, and the mixture was incubated for 15 minutes. 5) Buffer replacement with PBS was performed to obtain vitamin D-BSA conjugate. 6) Add vitamin D-BSA conjugate (1.0 μg / mL) to one well of a microtiter plate. 100 μL was added per well. 7) Incubate at 37°C for 30 minutes. 8) The plate was washed twice with 0.01M PBS. 9) 200 μL of 10 mM PBS (pH 7.2), 1% BSA, 5% lactose, and 0.1% NaN3 was added per well. 10) The plate was left standing at 2 to 8°C for at least 12 hours. 11) The plate was washed twice with 0.01M PBS before use.

[0020] 3. Preparation of Vitamin D Dissociation Solution (Example) 1) Prepare 0.1 M Tris-HCl buffer (pH 7.6), 0.04% BSA, 0.2% sodium deoxycholate, and 0.001-6 M urea.

[0021] 2) Mouse monoclonal anti-25(OH)D antibody was added at 0.5 μg / mL to prepare a vitamin D dissociation solution (Example).

[0022] (Comparative Example 1) 1) 0.1 M Tris-HCl buffer (pH 7.6), 0.04% BSA, and 0.2% sodium deoxycholate were prepared. 2) Mouse monoclonal anti-25(OH)D antibody was added at 0.5 μg / mL to prepare a vitamin D dissociation solution (Comparative Example 1).

[0023] (Comparative Example 2) 1) Prepare 0.1 M Tris-HCl buffer (pH 7.6), 0.04% BSA, 0.2% sodium deoxycholate, and 0.001-6 M guanidine hydrochloride. 2) Mouse monoclonal anti-25(OH)D antibody was added at 0.5 μg / mL to prepare a vitamin D dissociation solution (Comparative Example 2).

[0024] 4.Immunological measurements 1) 190 μL of each vitamin D dissociation solution was added to 10 μL of serum. 2) The entire amount of the sample was added to a vitamin D solid-phase microplate. 3) Incubated at 37°C for 10 minutes. 4) The plate was washed three times with PBST. 5) HRP-labeled Goat anti-Mouse IgG antibody diluted 1:3000 in PBS was added. 6) Incubate at 37°C for 30 minutes. 7) The plate was washed three times with PBST. 8) TMB substrate reagent was added. 9) Incubate at room temperature for 10 minutes. 10) 0.5M HCl was added to stop the reaction. 11) Absorbance was measured at 450 nm / 630 nm using a plate reader (Thermo Labsystems).

[0025] 5.Measurement results The results are shown in Figure 1. When comparing the %Bias under each condition, with the signal at 25(OH)D concentrations of 0 ng / mL and above being taken as 100%, the example (dissociation solution containing deoxycholic acid and urea) had the largest bias, confirming that dissociation from vitamin D-binding protein was most efficient.

Claims

1. A method for dissociating vitamin D in a sample separated from a living body from vitamin D-binding protein, the method comprising treating the sample with urea and at least one surfactant selected from the group consisting of deoxycholic acid, cholic acid, and salts thereof.

2. The method of claim 1 , wherein the sample is a blood sample.

3. 2. The method according to claim 1, wherein the final concentration of the surfactant during the treatment is 0.05 w / v % to 1 w / v % and the final concentration of the urea is 0.0005 M to 12 M.

4. 2. The method of claim 1, wherein the surfactant is deoxycholate.

5. A method for measuring vitamin D in a sample, comprising the steps of subjecting a sample separated from a living body to the method according to any one of claims 1 to 4, and then measuring vitamin D in the sample.

6. 6. The method according to claim 5, wherein vitamin D is measured by immunoassay.

7. The method according to claim 6, wherein the immunoassay utilizes an antigen-antibody reaction between an anti-vitamin D antibody or an antigen-binding fragment thereof and vitamin D in a sample.

8. The method of claim 7, wherein the anti-vitamin D antibody is an anti-25-hydroxyvitamin D3 antibody.

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