JP6331413B2 - Sample preparation method and immunoassay method for vitamins - Google Patents

Description

  The present invention relates to a method for immunologically measuring vitamins in blood, and a method for accurately measuring the concentration of vitamins in blood by mixing a surfactant in an alkali-treated blood sample. It is.

The vitamins that are measured by immunoassay there are small molecules such as vitamin D metabolites, vitamin B ₁₂ and folic acid. Most of these small molecules in the blood are bound specifically or non-specifically to proteins, lipids and the like. Therefore, in order to measure those small molecules, it is necessary to release the small molecules from the protein or the like (hereinafter sometimes referred to as pretreatment). Each vitamin has a specific binding protein, which contributes to stability in blood. For example, vitamin D binding protein (DBP), haptocholine, intrinsic factor and the like are well known.

  Alkaline treatment has been reported as a conventionally known method for releasing vitamin-binding proteins, and in particular, an aqueous sodium hydroxide solution is often used as an alkaline agent. Patent Document 1 reports a sodium hydroxide aqueous solution containing β-random methylated cyclodextrin and sodium salicylate, and Patent Document 2 reports a sodium hydroxide aqueous solution containing EDTA, DTT, and ethylene carbonate.

  However, alkaline treatment denatures proteins in the blood and generates insoluble matter. Therefore, when using the processed sample for immune reaction after neutralization, there is a problem that measurement accuracy deteriorates.

Japanese Patent No. 4130958 International Publication No. 2011/144661 Pamphlet

  In the field of clinical examination, the measurement of vitamins in blood is often performed in the order of alkali treatment and immune reaction, and is required to be rapid and fully automatic including alkali treatment. Alkali treatment denatures proteins in blood and generates insoluble matter. Therefore, when using the processed sample for immune reaction after neutralization, there is a problem that measurement accuracy deteriorates. Separation and extraction are commonly used to remove denatured proteins, but it is difficult to automate them while achieving rapid measurement.

  Accordingly, an object of the present invention is to provide a method for preparing a measurement sample by mixing a surfactant in an alkali-treated blood sample, and accurately measuring the concentration of vitamins in the blood.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have obtained a measurement sample by mixing a surfactant into an alkali-treated blood sample in a method for immunologically measuring vitamins. It has been found that the concentration of vitamins in the blood can be accurately measured by preparing the present invention, and the present invention has been completed.

That is, the present invention is as follows.
(1) A method for preparing a blood sample mixture for measuring vitamins, which comprises mixing a surfactant with an alkali-treated blood sample.
(2) The preparation method according to (1), wherein the surfactant is a nonionic surfactant.
(3) The preparation method according to (1) or (2), wherein the vitamins are vitamin D metabolites, vitamin B ₁₂ or folic acid.
(4) An immunoassay method for vitamins, wherein vitamins in a blood sample mixture obtained by the method according to any one of (1) to (3) are measured by an immunological method.

  Hereinafter, the present invention will be described in detail.

In the present invention, it refers to a vitamin D metabolites, vitamin B ₁₂ or folic acid and vitamins. As vitamin D metabolites, vitamin D ₃ (cholecalciferol), vitamin D ₂ (ergocalciferol), 25-hydroxyvitamin D ₃ , 25-hydroxyvitamin D ₂ , 1,25-dihydroxyvitamin D ₃ , 1,25 - dihydroxyvitamin _D 2, 24,25-dihydroxyvitamin _D 3, there can be mentioned 24,25-dihydroxyvitamin _{D 2.} Among these, it is preferable to apply the present invention to 25-hydroxyvitamin D ₃ , 25-hydroxyvitamin D ₂ , vitamin B ₁₂ and folic acid, which are attracting attention as measurement items for clinical tests.

  Although it does not specifically limit as an alkali agent used for the alkali treatment of a blood sample, Sodium hydroxide, potassium hydroxide, ammonia, etc. can be used, Especially, sodium hydroxide is preferable. The concentration of the alkali agent during the alkali treatment is preferably 0.01 to 10M. The temperature and time at that time are not particularly limited, but it is preferable to perform the treatment at 34 to 40 ° C. for 5 to 15 minutes.

  In addition, other various chemicals (such as a water-soluble solvent, a reducing agent, and a separating agent) may be used as a pretreatment agent used by mixing with an alkaline agent.

As the surfactant, a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant is used. Preferably a nonionic surfactant is used.
Nonionic surfactants include, for example, Nonided P-40, TWEEN20, TWEEN80, TritonX-100, etc., anionic surfactants such as alkylbenzene sulfonate and SDS, and cationic surfactants such as dodecyl Examples of amphoteric surfactants such as trimethylammonium chloride and didodecyldimethylammonium chloride include CHAPS and CHAPSO.

  In the present invention, a nonionic surfactant is preferably used, and particularly preferably non-dead P-40 is used.

  The concentration of the surfactant to be mixed with the alkali-treated blood sample is preferably 0.001 to 1% by weight, more preferably 0.001 to 0.1% by weight, especially 0.001 to 0. 0%. Although it is preferably 05% by weight, it is preferable to determine the optimum concentration according to the measurement object and the surfactant used. There are no particular limitations on the temperature and time at this time, but it is preferable to perform the treatment at 20 to 40 ° C. for 1 to 30 seconds.

  The blood sample mixture thus prepared can be used to measure vitamins by immunoassay without separation or extraction.

  The immunoassay method applied to the measurement of vitamins is not limited to the principle or the type of label for detection. That is, as a principle, a sandwich method, a competition method, an aggregation method, or the like can be used, and as a label, a radioisotope, an enzyme, chemiluminescence, biochemiluminescence, or the like can be used. Among these, a competitive method using an enzyme, a fluorescent substance, or a chemiluminescent substrate as a label is preferable.

  According to the present invention, a sample mixture for measuring vitamins can be prepared by a simple operation of mixing a surfactant with an alkali-treated blood sample, and the sample mixture for measurement can be separated or extracted. Even if it is not performed, it is possible to accurately measure the concentration of vitamins by an immunological measurement method. For example, most of the vitamins measured by EIA and RIA, which are effective means in clinical tests, are specifically or non-specifically bound to proteins, lipids, etc., so there is an example of alkali treatment. Occupy a large number. In that case, since it is not easy to accurately measure the concentration of vitamins, the present invention simply solves many demands for development of high-precision measurement and automation, and its application range is wide.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by a present Example.

Example 1
A fully automated enzyme immunoassay device (AIA-2000, manufactured by Tosoh Corporation) is used as an immunoassay device, and a 25-hydroxyvitamin D immunoreaction reagent for the device is used as a reagent for immunoassay. D was measured. Other reagents necessary for the measurement were prepared as described later.

  As an alkali agent used for alkali treatment of a blood sample, a 0.3 M sodium hydroxide aqueous solution was prepared.

  A solution containing a surfactant used as a neutralizing solution is a 0.2 M phosphate buffer solution containing a preservative, and 0 wt% (reagent A) and 0.001 wt% (reagent B) of non-dead P-40, respectively. 0.005 wt% (reagent C), 0.01 wt% (reagent D), 0.02 wt% (reagent E), 0.05 wt% (reagent F), 0.1 wt% (reagent G) 7 types were prepared by adding.

Serum 25-hydroxyvitamin _{D 3} were added 80,160ng / mL was prepared the 2 types of serum samples as a blood sample for measurement.

  Next, the reagent A, B, C, D, E, F, and G are used as the alkaline agent and neutralizing solution prepared as described above, and the automatic immunoassay device and the 25-hydroxyvitamin D immune reaction reagent for the device are used. Two types of serum samples were measured and the increase rate [nM / s] of the fluorescence intensity of 4-methylumbelliferone, which is a fluorescent substrate for alkaline phosphatase, was measured. Each serum sample was measured three times with each reagent (neutralizing solution). At this time, serum samples were mixed with an alkaline agent and alkali-treated, and then neutralized solution containing a surfactant was mixed, and then immunoassay was performed without separation and extraction.

  The results are shown in Tables 1 and 2. Note that CV (Coefficient of Variation), which is an index of measurement accuracy shown in Tables 1 and 2, is calculated based on the following equation.

  CV [%] = [(standard deviation of fluorescence intensity increase rate measured three times) ÷ (average value of fluorescence intensity increase rate measured three times)] × 100

ノニデッドＰ−４０が未添加（０重量％：試薬Ａ）の場合は２サンプルのＣＶがそれぞれ９．８％、６．９％であったが、ノニデッドＰ−４０を添加した場合（試薬Ｂ〜Ｇ）は、ＣＶは１．０〜３．８％であり、明らかにＣＶが低くなる傾向が見られた。これはノニデッドＰ−４０を添加したことで測定精度が向上したことを示している。但し、ノニデッドＰ−４０を０．１重量％添加すると蛍光強度増加速度が低下し、ＣＶもわずかながら高くなったため、０．００１〜０．０５重量％が最も好ましいと考えられる。このことからノニデッドＰ−４０の濃度を上げすぎると免疫測定に影響を与えると推測される。つまり、ノニデッドＰ−４０を添加する場合、免疫測定に影響を与えるような高濃度は好ましくないが、適当な濃度を添加することで血液試料中に含まれるビタミン類を精度よく測定することができることが確認された。

When non-dead P-40 was not added (0 wt%: reagent A), the CVs of the two samples were 9.8% and 6.9%, respectively, but when non-dead P-40 was added (reagent B- As for G), CV was 1.0 to 3.8%, and the tendency for CV to become low clearly was seen. This indicates that the measurement accuracy has been improved by adding non-dead P-40. However, when 0.1% by weight of nonided P-40 was added, the rate of increase in fluorescence intensity decreased and the CV also increased slightly, so 0.001 to 0.05% by weight is considered most preferable. From this, it is presumed that if the concentration of nonided P-40 is increased too much, immunoassay is affected. That is, when adding noned P-40, a high concentration that affects immunoassay is not preferable, but vitamins contained in blood samples can be accurately measured by adding an appropriate concentration. Was confirmed.

Claims (4)

A method for preparing a blood sample mixture for measuring vitamins, comprising mixing a surfactant with an alkali-treated blood sample by treatment at 20 to 40 ° C. for 1 to 30 seconds .   The preparation method according to claim 1, wherein the surfactant is a nonionic surfactant.   The preparation method according to claim 1 or 2, wherein the vitamins are vitamin D metabolites, vitamin B12 or folic acid.   A method for immunoassay of vitamins, characterized in that vitamins in the blood sample mixture obtained by the method according to any one of claims 1 to 3 are measured by an immunological method.