JP2011117779A - Method for measuring steroid hormones or vitamins in blood - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for implementing immunoassay of an object to be measured, requiring measurement accuracy in a low concentration range using a simple operation, in an immunoassay method of steroid hormones or vitamins. <P>SOLUTION: The immunoassay of steroid hormones or vitamins can be accurately implemented in low concentration, by making one or more types selected from among the group including a 2-hydroxy-3-naphthoic acid, 2-amino naphthalene sulfonic acid, aniline-2,5-disulfonic acid, 3-amino-2,7-naphthalene disulfonic acid, and their salts to coexist. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for immunologically measuring steroid hormones or vitamins in blood (hereinafter, both may be collectively referred to as “small molecules”), and 2-hydroxy-3-naphthoate as a reaction system. At least one selected from the group consisting of acid, 2-aminonaphthalenesulfonic acid, aniline-2,5-disulfonic acid, 3-amino-2,7-naphthalenedisulfonic acid and salts thereof (hereinafter referred to as “protein binding inhibitor”) The concentration of steroid hormones or vitamins in the blood can be accurately measured by coexisting with the blood.

  Most of the small molecules in blood are specifically or non-specifically bound to proteins and lipids. Therefore, in order to accurately and accurately measure the concentration of these small molecules, it is necessary to release the small molecules from the protein or the like. Each small molecule has a specific binding protein, which contributes to the improvement of stability in blood. For example, sex hormone / binding globulin and vitamin D binding protein (DBP) are well known.

  Conventionally known specific binding inhibitors of steroid hormones include glutamic acid solution in cortisol immunoassay (Patent Document 1), and 8-anilino-1-naphthalenesulfonic acid or salt thereof in Cortisol analysis method (Patent Document 1) 2) has been reported. Specific binding inhibitors of vitamin D metabolites include 8-anilino-1-naphthalenesulfonic acid ammonium salt, 3- (acetonylbenzyl) -4-hydroxycoumarin and hydrating solvent in the detection method of vitamin D metabolites A non-competitive displacement agent (Patent 3787121), 0.01-5% cyclodextrin, 0.01-5% sodium salicylate, and 0.1-1.0 M NaOH in a vitamin D assay. Patent Document 3) has been reported.

JP-A-53-101521 JP 61-12547 A Japanese Patent No. 4130958

  Even when the aforementioned glutamic acid solution or the like is used, it is difficult to obtain sufficient sensitivity only by adding these when the measurement accuracy in a low concentration range is required as in the estradiol measuring reagent. Although it is possible to increase the sensitivity by adding a glutamic acid solution or the like to a high concentration, there is a possibility that the reaction (immune reaction) for immunoassay of a small molecule may occur, It is also difficult to add so that the concentration is generally high.

  As a result of intensive studies in view of the above situation, the present inventors have determined the concentration of a small molecule by allowing a protein binding inhibitor to coexist in a reaction system in a method for immunologically measuring a small molecule. The inventors have found that it can be measured accurately and have completed the present invention. That is, the present invention is selected from the group consisting of 2-hydroxy-3-naphthoic acid, 2-aminonaphthalenesulfonic acid, aniline-2,5-disulfonic acid, 3-amino-2,7-naphthalenedisulfonic acid and salts thereof It is an immunoassay method for steroid hormones or vitamins, characterized in that one or more of them are allowed to coexist. Hereinafter, the present invention will be described in detail.

  The small molecule referred to in the present invention is a measurement target having a low molecular weight, such as steroid hormones or vitamins. Examples of steroid hormones include estradiol, testosterone, progesterone, cortisol, androstenedione, and dehydroepiandrosterone sulfate. Examples of vitamins include vitamin D metabolites such as 25-hydroxyvitamin D, and vitamins. Examples thereof include B12 and folic acid.

  The immunoassay method applied to the measurement of a small molecule 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 them, a sandwich method or a competitive method using an enzyme, a fluorescent substance, or a chemiluminescent substance as a label, which has high measurement sensitivity in principle and does not require the use of a reagent that requires special handling in implementation, is preferable.

  The coexisting protein binding inhibitor consists of 2-hydroxy-3-naphthoic acid, 2-aminonaphthalenesulfonic acid, aniline-2,5-disulfonic acid, 3-amino-2,7-naphthalenedisulfonic acid and their salts It may be at least one selected from the group. That is, one of these may be used alone, or two or more may be used in combination. In particular, when two or more types are used, a synergistic effect of individual protein binding inhibitors can be expected.

  The protein binding inhibitor may be added to a reaction solution that coexists in the reaction system, that is, causes an immune reaction, when the above-described immunoassay is performed. The concentration to be coexisted is 0.1 to 20%, preferably 0.5 to 10% in terms of weight / volume (w / v). When two or more kinds are mixed and used, the total amount is within this range. What should I do?

  There is no particular restriction on the coexistence of the protein binding inhibitor, and the small molecule and the immune reaction component that are first contacted with the component for immune reaction (antigen, antibody, etc.) that is first contacted with the small molecule Any of contacting simultaneously may be sufficient.

  The immune reaction in the presence of the protein binding inhibitor can be performed in the same manner as the immune reaction in the absence of the inhibitor, that is, at the same reaction temperature and time.

  According to the present invention, it is possible to accurately measure the concentration of a small molecule only by coexisting a protein binding inhibitor. For example, most of the small molecules measured by EIA and RIA, which are very effective means in clinical tests, bind to proteins and lipids in a specific and non-specific manner. It is not easy to measure molecules accurately. It is very useful to measure accurate small molecule concentration in such a measurement system, and it easily solves many requests for high precision measurement, shortening of measurement time, and development of automation. The application range is wide.

  Example

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.

  Using an AIA-1800 automatic immunoassay device (trade name, manufactured by Tosoh Corporation) as an immunoassay device, dehydroepiandrosterone sulfate was measured by a one-step competition method. The reagents necessary for the measurement were prepared as described later.

  First, a test sample (serum) was added to a reaction cup containing a carrier immobilized with goat anti-rabbit antibody and dehydroepiandrosterone sulfate labeled with alkaline phosphatase and rabbit anti-dehydroepiandrosterone sulfate antibody at 37 ° C. The mixture was kept warm with stirring. The reaction cup is prepared by first dispensing a carrier with immobilized goat anti-rabbit antibody and an alkaline phosphatase-labeled dehydroepiandrosterone sulfate solution in order to compete with the labeled dehydroepiandrosterone sulfate when the test sample is added. After freezing, a rabbit anti-dehydroepiandrosterone sulfate antibody solution was dispensed, frozen, and lyophilized.

  Next, unreacted substances are removed by B / F separation, and alkaline phosphatase-labeled dehydroepiandrosterone sulfate bound via dehydroepiandrosterone sulfate is added to 4-methylumbelliferyl phosphate, and the amount per unit time is added. The production of 4 methylumbelliferone (nM / sec) was measured by fluorescence. This degree of production is proportional to the amount of alkaline phosphatase.

  The reaction reagent for dehydroepiandrosterone sulfate measurement was prepared as follows. After adding a solution containing alkaline phosphatase-labeled dehydroepiandrosterone sulfate to a carrier on which a goat anti-rabbit antibody is immobilized and freezing, a solution containing rabbit anti-dehydroepiandrosterone sulfate antibody is added, lyophilized and dehydrohydrated. An epiandrosterone sulfate measurement reagent (reagent A) was prepared. In addition, a dehydroepiandrosterone sulfate measurement reagent (reagent B) to which 0.67% (w / v) of sodium 2-hydroxy-3-naphthoate was added under the same conditions was prepared. Similarly, a dehydroepiandrosterone sulfate measurement reagent (reagent C) containing 2.67% (w / v) sodium 2-aminonaphthalenesulfonate was prepared.

  The dehydroepiandrosterone sulfate standard solution was prepared by adding commercially available dehydroepiandrosterone sulfate (manufactured by Sigma) to desteroidal human serum at 0, 5, 12, 60, 300, and 1200 μg / dl.

  Next, using the reagents A, B, and C prepared as described above, the standard solution prepared as described above is measured with the automatic immunoassay apparatus, and 4-methylumbelliferone, which is a fluorescent substrate for alkaline phosphatase, is measured. The increase rate of the fluorescence intensity was measured (nM / sec). Each standard solution was measured three times.

At each standard solution concentration (μg / dl), the binding rate (B / B ₀ ) of labeled dehydroepiandrosterone sulfate to the antibody is expressed as “B / B ₀ = (fluorescence increase rate of each standard solution / standard of concentration 0) Liquid fluorescence increase rate) × 100 ”. The results are shown in Table 1.

As is clear from Table 1, when the binding ratios B / B ₀ of the reagents A, B, and C are compared when the standard solution is 5 μg / dl, sodium 2-hydroxy-3-naphthoate or sodium 2-aminonaphthalenesulfonate Reagents B and C containing benzene are much lower, so even when a standard solution of the same concentration is added, the amount of free dehydroepiandrosterone sulfate is higher in reagents B and C, and labeled dehydroepi It can be seen that it competed with androsterone sulfate. Therefore, dehydroepiandrosterone sulfate can be measured with high sensitivity by the coexistence of sodium 2-hydroxy-3-naphthoate or sodium 2-aminonaphthalenesulfonate.

  The value obtained by converting “standard deviation of fluorescence increase rate at concentration 0−2 × standard deviation of fluorescence increase rate at concentration 0” into a concentration (μg / dl) using a calibration curve created from the measurement results of each standard solution described above is minimized. It was set as the detection limit (MDC). The results are shown in Table 2.

  It can be seen that MDC is much lower for reagents B and C containing sodium 2-hydroxy-3-naphthoate and sodium 2-aminonaphthalene sulfonate. Therefore, by coexisting sodium 2-hydroxy-3-naphthoate or sodium 2-aminonaphthalenesulfonate, a low concentration of dehydroepiandrosterone sulfate can be accurately measured.

  Using an AIA-1800 automatic immunoassay device (trade name, manufactured by Tosoh Corporation) as an immunoassay device, estradiol was measured by a one-step competition method. The reagents necessary for the measurement were prepared as described later.

  First, a test sample (serum) was added to a reaction cup containing estradiol labeled with a carrier on which a goat anti-rabbit antibody was immobilized and alkaline phosphatase and a rabbit anti-estradiol antibody, and the mixture was stirred and kept at 37 ° C. In addition, the reaction cup was first prepared by dispensing and freezing an alkaline phosphatase-labeled estradiol solution and a carrier on which a goat anti-rabbit antibody was immobilized in order to react with the labeled estradiol when the test sample was added. The antibody solution was dispensed, frozen, and lyophilized.

  Next, unreacted substances were removed by B / F separation, and alkaline phosphatase-labeled estradiol bound via estradiol was added to 4 methylumbelliferyl phosphate to produce 4 methylumbelliferone per unit time ( nM / sec) was measured by fluorescence. This degree of production is proportional to the amount of alkaline phosphatase.

  The reagent for measuring estradiol measurement was prepared as follows. A solution containing alkaline phosphatase-labeled estradiol is added to a carrier on which a goat anti-rabbit antibody is immobilized and frozen, and then a solution containing a rabbit anti-estradiol antibody is added and lyophilized to prepare an estradiol measurement reagent (reagent D) did. In addition, a reagent for measuring estradiol (reagent E) to which 1.33% (w / v) of monosodium aniline-2,5-disulfonate was added under the same conditions was prepared. Similarly, an estradiol measuring reagent (reagent F) to which 0.67% (w / v) of 3-sodium 3-amino-2,7-naphthalenedisulfonate was added was prepared.

  The estradiol standard solution was prepared by adding commercially available estradiol (SIGMA) to deestradiol human serum at 0, 42, 105, 516, 1030, 3270 pg / ml.

  Next, using the reagents D, E, and F prepared as described above, the standard solution prepared as described above is measured with the automatic immunoassay device, and 4-methylumbelliferone, which is a fluorescent substrate for alkaline phosphatase, is measured. The increase rate of the fluorescence intensity was measured (nM / sec). Each standard solution was measured three times.

The binding rate (B / B ₀ ) of labeled estradiol to the antibody at each standard solution concentration (pg / ml) was calculated as described above. The results are shown in Table 3.

As is apparent from Table 3, when the binding ratios B / B ₀ of the reagents D, E, and F are compared at the standard solution of 42 pg / ml, aniline-2,5-disulfonic acid monosodium and 3-amino-2 Reagents E and F containing monosodium 7-naphthalenedisulfonate are lower, so even when standard solutions of the same concentration are added, the amount of free estradiol is greater in reagents E and F, and labeled estradiol It turns out that it competed with. Therefore, estradiol can be measured with high sensitivity by the coexistence of monosodium aniline-2,5-disulfonate and monosodium 3-amino-2,7-naphthalenedisulfonate.

  Next, the minimum detection limit (MDC) was calculated as described above. The results are shown in Table 4.

  It can be seen that MDC is lower in reagents E and F containing monosodium aniline-2,5-disulfonate and monosodium 3-amino-2,7-naphthalenedisulfonate. Therefore, low concentrations of estradiol can be accurately measured by the coexistence of monosodium aniline-2,5-disulfonate and monosodium 3-amino-2,7-naphthalenedisulfonate.

Claims (3)

Coexist with one or more selected from the group consisting of 2-hydroxy-3-naphthoic acid, 2-aminonaphthalenesulfonic acid, aniline-2,5-disulfonic acid, 3-amino-2,7-naphthalenedisulfonic acid and salts thereof A method for immunoassay of steroid hormones or vitamins, characterized by comprising: The immunoassay method according to claim 1, wherein the steroid hormone is estradiol, testosterone, progesterone, cortisol, androstenedione, or dehydroepiandrosterone sulfate. The immunoassay method according to claim 1, wherein the vitamin is a vitamin D metabolite, vitamin B12, or folic acid.