JP2004205504A - Enzyme immunity measurement method for endocrine-disrupting chemical - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simply, quickly, precisely, and economically measure an endocrine-disrupting chemical. <P>SOLUTION: The endocrine-disrupting chemical in a sample is made so as to immunologically react with an enzyme labeled antigen, by the competition with a solidly prepared solid antibody, and the luminous amount of the enzymatic reaction of the enzyme labeled antigen fixed to the solid phase is obtained by a solid phase antibody, arising from the addition of a luminescence reagent. The concentration of the endocrine disrupting chemical in the sample is determined indirectly, using a calibration curve. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to an enzyme immunoassay for measuring 17β-estradiol, which is an endocrine disrupting chemical.

  Endocrine disrupting chemicals (hereafter referred to as environmental hormones) have been attracting attention as one of the global environmental issues and gaining widespread interest due to the publication of "The Deprived Future" published by Shia Colborn and others in the United States. It became. Since then, there have been many scientifically unclear points in Japan regarding the problem of endocrine disrupters, and related administrative agencies and various research institutes have been investigating and conducting research from various viewpoints.

  Although there are many chemicals suspected of having endocrine disrupting effects, in Japan, the Ministry of the Environment listed 67 substances in 1998. (SPEED98: Strategic programs on Enviromental Endocrine Disruptors) And as shown in Table 1 below, 24 substances have been narrowed down.

  Environmental hormones have the same function as female hormones, and pose a problem because even a trace amount adversely affects reproductive function.

  As a conventional method for measuring environmental hormones, there is an instrumental analysis method using a high-resolution GC-MS or LC-MS / MS (a provisional manual of the Ministry of the Environment, etc.). However, high-resolution GC-MS, LC-MS / MS, and other analyzers are expensive, so there are only a few laboratories that can perform their own measurements, and complicated concentration, extraction, and cleanup operations are required. Is a problem.

  In recent years, without special equipment, it is possible to detect an object to be measured in a relatively short time with high sensitivity, and to specifically detect the target substance, and to monitor environmental substances remaining in the environment because it is economical. Environmental hormones are measured by an enzyme immunoassay suitable for monitoring.

For example, an environmental hormone is used as an antigen, and an immunoreaction with an antibody is performed by a competitive method or non-competition with a labeled antigen obtained by labeling the antigen with an enzyme. An enzyme immunoassay for measuring environmental hormones from the amount has been performed (for example, see Patent Document 1).
JP-A-2001-66311

  When measuring environmental hormones using an analyzer such as a high-resolution GC-MS or LC-MS / MS, the equipment is very expensive and requires a concentration operation, an extraction operation, a cleanup operation, etc. It requires preparation beforehand, and simple and quick measurement is difficult.

  Female hormones responsible for the development of the reproductive organs of female and female animals, maintenance of sexual characteristics, function adjustment, and the like are roughly classified into estrogen secreted from ovarian follicles and luteal hormone secreted from ovarian luteum. Estrogen is mainly responsible for the development of the reproductive organs and preservation of sexual characteristics, whereas progesterone, in cooperation with estrogen, causes the estrous cycle to continue pregnancy.

  The main three types of animal estrogens are estrone, 17β-estradiol, and estriol, of which the action of 17β-estradiol is the strongest.

  17β-estradiol has a particularly strong female hormone action among environmental hormones, and it is urgently necessary to establish a practical method for measuring 17β-estradiol.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method that can measure 17β-estradiol economically, without error, easily, and quickly.

  In order to solve the above-mentioned problem, the enzyme immunoassay for endocrine disrupting chemicals according to claim 1 comprises preparing a solid phase of an antibody, and then adding an enzyme-labeled antigen and an endocrine disrupting chemical to the solid phase. An immunological reaction is performed, and after the immunological reaction is completed, the solid phase is washed, a luminescent reagent is added, and the endocrine disrupting chemical substance is measured from the amount of luminescence by the enzymatic reaction.

  The enzyme immunoassay for endocrine disrupting chemicals according to claim 2 is characterized in that, in claim 1, the endocrine disrupting chemical is 17β-estradiol.

  The enzyme immunoassay for endocrine disrupting chemicals according to claim 3 is characterized in that, in claim 2, the antibody is prepared by binding bovine serum albumin to 17β-estradiol and immunizing rabbits. I do.

  According to a fourth aspect of the present invention, in the enzyme immunoassay for an endocrine disrupting chemical substance according to the third aspect, the concentration of the antibody is 0.75 μg / mL.

  The enzyme immunoassay for endocrine disrupting chemicals according to claim 5 is characterized in that, in claim 4, the volume of the mixed solution of the enzyme-labeled antigen and 17β-estradiol added to 1 mL of the antibody is 100 μL. And

  According to a sixth aspect of the present invention, in the enzyme immunoassay for endocrine disrupting chemicals according to the fourth aspect, the concentration of the enzyme-labeled antigen in the mixed solution is 0.125 to 0.25 μg / mL.

  An enzyme immunoassay for an endocrine disrupting chemical substance according to a seventh aspect is characterized in that, in the fifth or sixth aspect, the immune reaction is carried out while shaking at a rotation speed of 200 rpm.

  An enzyme immunoassay for an endocrine disrupting chemical substance according to claim 8 is the method according to claims 5 to 7, wherein the immune reaction is performed for 1 hour or more.

  An enzyme immunoassay for an endocrine disrupting chemical substance according to claim 9, wherein after preparing the first antibody on a solid phase, an enzyme-labeled antigen, an endocrine disrupting chemical substance, and a second antibody are added to the solid phase to perform an immune reaction. After the completion of the immune reaction, the solid phase is washed, a luminescent reagent is added, and the endocrine disrupting chemical substance is measured from the amount of light emitted by the enzymatic reaction.

  An enzyme immunoassay for an endocrine disrupting chemical substance according to a tenth aspect is characterized in that, in the ninth aspect, the endocrine disrupting chemical substance is 17β-estradiol.

  An enzyme immunoassay for an endocrine disrupting chemical substance according to claim 11 is the method according to claim 10, wherein the first antibody is an anti-rabbit IgG goat antibody, and the second antibody is an anti-17β-estradiol rabbit antibody. Features.

  In the enzyme immunoassay for endocrine disrupting chemicals according to claim 12, the concentration of the anti-rabbit IgG goat antibody is 1.0 μg / mL and the concentration of the anti-17β-estradiol rabbit antibody is 0. .75 μg / mL.

  An enzyme immunoassay for an endocrine disrupting chemical substance according to a thirteenth aspect is characterized in that, in the eleventh or twelfth aspect, the immune reaction is performed while shaking at a rotation speed of 200 rpm.

  The enzyme immunoassay is a method in which an antigen-antibody reaction is performed using an antigen or antibody labeled with an enzyme, and the amount of the antigen or antibody is determined by detecting the antigen-antibody reaction by measuring the enzyme activity.

  The invention according to claims 1 to 8 is characterized in that an endocrine-disrupting chemical substance in a sample and an enzyme-labeled antigen are competed with an antibody prepared in solid phase for immunoreaction, and immobilized on a solid phase antibody caused by adding a luminescent reagent. The amount of luminescence of the luminescence reaction of the enzyme-labeled antigen thus obtained is obtained, and the endocrine disrupting chemical substance in the sample can be indirectly measured using a calibration curve.

  Further, the invention according to claims 9 to 13 is characterized in that an endocrine disrupting chemical substance in a sample and an enzyme-labeled antigen are competed with a second antibody capable of reacting with a first antibody prepared by solid-phase preparation to cause an immunoreaction, The endocrine disrupting chemical substance in the sample is measured by measuring the enzyme-labeled antigen immobilized on the solid phase by the second antibody and the first antibody from the amount of luminescence of the luminescence reaction caused by adding the luminescence reagent. be able to.

  The labeling enzyme of enzyme-labeled 17β-estradiol is not particularly limited as long as it can catalyze a biological or chemiluminescent reaction, and examples thereof include peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, urease and the like.

  As for the antibody, either a polyclonal antibody or a monoclonal antibody may be used.

  As is clear from the above description, the present invention has the following effects.

  17β-estradiol can be easily and quickly measured by an enzyme immunoassay with an inexpensive device without using an expensive instrument analyzer such as GC-MS or LC-MS / MS.

  Since optimal values are examined for the concentration of the antibody used for the measurement of 17β-estradiol, the concentration of the enzyme-labeled antigen, the shaking effect, and the reaction time, accurate measurement is possible.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 is a schematic diagram of a measurement operation showing a measurement process of the enzyme immunoassay according to the first embodiment of the present invention. In FIG. 1, first, 17β-estradiol antibody is diluted with a dilution buffer in a polystyrene test tube, dispensed in 1 mL portions, and stored in a refrigerator overnight to prepare a solid phase antibody on the solid phase 11.

  The 17β-estradiol antibody (solid phase antibody) is prepared by binding bovine serum albumin (BSA) to 17β-estradiol and immunizing a rabbit. As a solid phase antibody dilution buffer, a 0.05 mol / L borate buffer (pH = 8.0) containing 0.9% sodium chloride and 0.1% sodium azide is used.

  Next, an enzyme-labeled antigen (enzyme-labeled 17β-estradiol) obtained by labeling 17β-estradiol with a peroxidase (enzyme) and 17β-estradiol adjusted to a desired concentration are added to a test tube, and shaken at 200 rpm. The immune reaction is performed for 1 hour while performing.

  Enzyme-labeled antigen is purchased from SIGMA, ESTRADIOL-PEROXIDAZE, and adjusted to 100 μg / mL with a 0.1 mol / L phosphate buffer (pH = 6.0). When ESTRADIOL-PEROXIDAZE is diluted to a desired concentration, a 0.05 mol / L Tris-HCl buffer (pH = 8.sup.2) containing 2% polyethylene glycol, 0.05% Tween 20, and 0.9% sodium chloride is used. Prepare and use 0).

  With respect to the antigen-antibody solution 12 obtained as described above, the test tube is washed five times with distilled water. After washing, a luminescence reagent is added to obtain a luminescence reaction solution 13, the luminescence amount of the luminescence reaction solution 13 is measured by a luminescence measuring device for 0 to 30 seconds, and the measurement data is processed in a data processor 14.

  The luminescent reagent is a reagent composed of a Tris-HCl buffer solution (pH = 8.5) containing 6 mmol / L luminol, 10 mol / L hydrogen peroxide, and 18.2 mmol / L paraiodophenol.

  The first embodiment will be specifically described with reference to the following examples.

(Example 1) Examination of solid phase antibody concentration The solid phase antibody concentration was adjusted to 0.375, 0.75, 1.5, 3.0, and 6.0 µg / mL with a buffer solution, and polystyrene was used. The solid phase tube was prepared by dispensing 1 mL at a time into a test tube made by Azuma and storing it overnight in a refrigerator.

  100 μL of the enzyme-labeled antigen prepared at 1.0 μg / mL was added to the solid-phase tube, and an immunoreaction was performed for 1 hour while shaking at a rotation speed of 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha).

  FIG. 2 shows the measurement results. FIG. 2 shows that the solid phase antibody concentration is sufficient if the solid phase antibody concentration is in the range of the enzyme-labeled antigen concentration of 1.0 μg / mL.

  Since a solid-phase antibody is prepared by immunizing an animal, it is preferable that the prepared antibody be subjected to a large number of measurements. For that purpose, the solid phase antibody concentration is preferably as low as possible, and therefore, it was determined that the solid phase antibody concentration was most preferably 0.75 μg / mL.

(Example 2) Examination of enzyme-labeled antigen concentration The solid-phase antibody concentration was adjusted to 0.75 μg / mL with a buffer solution, dispensed into a polystyrene test tube at a volume of 1 mL, and stored in a refrigerator overnight. Was prepared.

The enzyme-labeled antigen was prepared at a concentration of 0.01 μg / mL, which was divided into a plurality of samples. Further, at least 7 or more samples were prepared in a concentration range of 8 × 10 ^{−7 to} 5 × 10 ⁻⁴ g / L for 17β-estradiol. The sample of the enzyme-labeled antigen and the sample of 17β-estradiol were mixed at a ratio of 1: 1 (volume ratio), and 100 μL of each mixed solution was taken and added to the solid-phase tube.

  After the addition, the immune reaction was performed for 1 hour while shaking at a rotation speed of 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha).

  In addition, the same measurement as described above was performed for the enzyme labeled antigen concentrations of 0.1, 0.25, 0.5, 0.75, and 1.0 μg / mL.

  The measurement results are shown in FIGS. 5 to 6 confirm that the correlation between the 17β-estradiol concentration and the luminescence amount is good, and the concentration range of the enzyme-labeled antigen is 0.25 to 0.5 μg / mL. I judged it to be good.

(Example 3) Examination of enzyme labeled antigen concentration 2
In Example 2 described above, the enzyme-labeled antigen and 17β-estradiol were mixed at a ratio of 1: 1 and 100 μL was taken from the mixture and added to the solid-phase tube. Is １ ／ of the initially prepared concentration. In Example 3, it was verified whether the same result as in Example 2 was obtained when the mixing ratio of 1: 1 was changed.

  The solid phase antibody concentration was adjusted to 0.75 μg / mL with a buffer solution, dispensed 1 mL each into a polystyrene test tube, and stored in a refrigerator overnight to prepare a plurality of solid phase tubes.

The enzyme-labeled antigen was prepared at a concentration of 0.5 μg / mL, which was divided into a plurality of samples. At least 6 or more samples of 17β-estradiol were prepared in a concentration range of 8 × 10 ^{−7 to} 5 × 10 ⁻⁴ g / L.

  500 μL of a sample of the enzyme-labeled antigen prepared at a concentration of 0.5 μg / mL and 500 μL of a sample of 17β-estradiol were mixed, and 100 μL of each mixed solution was taken and added to the solid phase tube.

  After the addition, the immune reaction was performed for 1 hour while shaking at a rotation speed of 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha).

  In addition, enzyme-labeled antigens were prepared at concentrations of 1.0 and 2.0 μg / mL, respectively, and divided into at least 6 or more samples. Each mixed solution obtained by mixing a 0.5 mL sample of the enzyme-labeled antigen prepared at a concentration of 1.0 μg / mL and a 1.5 mL sample of 17β-estradiol, and an enzyme-labeled antigen prepared at a concentration of 2.0 μg / mL The same measurement as described above was performed for each mixed solution obtained by mixing 0.25 mL of the sample and 1.75 mL of the 17β-estradiol sample.

  The measurement results are shown in FIGS. In Example 3, the final concentration of the enzyme-labeled antigen was all 0.25 μg / mL. From the above results, it was confirmed that the mixing ratio does not have to be 1: 1 as long as the concentration of the enzyme-labeled antigen was obtained. . Furthermore, from the results of Example 2 and Example 3, it was determined that the final concentration of the enzyme-labeled antigen should be 0.125 to 0.25 μg / mL.

(Example 4) Examination of liquid volume The solid phase antibody concentration was adjusted to 0.75 μg / mL with a buffer solution, dispensed in 1 mL portions into polystyrene test tubes, and stored in a refrigerator overnight to prepare a plurality of solid phase antibodies. A phase tube was prepared.

The enzyme-labeled antigen was prepared at a concentration of 0.5 μg / mL, which was divided into a plurality of samples. In addition, 17β-estradiol was prepared at a concentration of 8 × 10 ⁻⁷ and 4 × 10 ⁻⁶ g / L, and a sample of the enzyme-labeled antigen and a sample of 17β-estradiol were mixed at a ratio of 1: 1. From each mixed solution, 100, 300, and 500 μL were dispensed and added to a solid phase tube.

  After the addition, the immune reaction was performed for 1 hour while shaking at a rotation speed of 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha).

  FIG. 12 shows the measurement results. From FIG. 12, it was confirmed that the amount of the mixed solution of the enzyme-labeled antigen and 17β-estradiol added to the solid-phase tube was preferably 100 μL. In addition, since the dispensing accuracy deteriorates with a liquid volume of 100 μL or less, 100 μL was determined to be an appropriate volume.

(Example 5) Examination of immune reaction sequence In Example 5, when performing an immune reaction, 100 μL of a mixed solution of an enzyme-labeled antigen and 17β-estradiol was added to a solid-phase tube to perform an immune reaction for 1 hour. A second method in which 17 β-estradiol is added to a 100 μL solid-phase tube to perform an immunoreaction for 30 minutes, and then 100 μL of an enzyme-labeled antigen is added and an immunoreaction is performed for 30 minutes. A third method in which 100 μL was added and the immune reaction was carried out for 30 minutes, and then 17β-estradiol was added in an amount of 100 μL and the immune reaction was carried out for 30 minutes, was carried out to examine which method was suitable. The mixing ratio and the addition ratio of the enzyme-labeled antigen and 17β-estradiol were all set to 1: 1.

  The solid phase antibody concentration was adjusted to 0.75 μg / mL with a buffer solution, dispensed 1 mL each into a polystyrene test tube, and stored in a refrigerator overnight to prepare a plurality of solid phase tubes.

At least 6 or more samples were prepared by diluting 17β-estradiol 5 times in a concentration range of 8 × 10 ^{−7 to} 5 × 10 ⁻⁴ g / L. Further, the enzyme-labeled antigen was prepared at a concentration of 0.5 μg / mL and divided into a plurality of samples.

  An immune reaction was performed on these samples by the first to third methods.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha).

  FIG. 13 shows the measurement results. Although the first method and the second method showed almost the same results, it was confirmed that the third method had poor sensitivity.

(Example 6) Study 1 of reaction time
The solid phase antibody concentration was adjusted to 0.75 μg / mL with a buffer solution, dispensed 1 mL each into a polystyrene test tube, and stored in a refrigerator overnight to prepare a plurality of solid phase tubes.

17β-estradiol was adjusted to a concentration of 2 × 10 ⁻⁵ g / L, enzyme-labeled antigen was adjusted to a concentration of 0.25 μg / mL, and a sample of 17β-estradiol and a sample of the enzyme-labeled antigen were paired. The mixture was mixed at a ratio of 1 and the mixed solution was dispensed in 100 μL portions and added to a solid phase tube.

  After the addition, immunoreactions were performed for 0, 15, 30, 60, 90, and 120 minutes, respectively, while shaking at 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha).

  FIG. 14 shows the measurement results. From FIG. 14, it was confirmed that there was almost no increase in the luminescence amount after the reaction was performed for 60 minutes, and it was determined that performing the immune reaction for 60 minutes or more was sufficient.

(Example 7) Study 2 of reaction time
In Example 6, the reaction time was examined for 17β-estradiol at a concentration of 2 × 10 ⁻⁵ g / L, but it is considered that the reactivity may vary depending on the concentration. Therefore, in Example 7, the immune reaction time was examined by changing the concentration of 17β-estradiol.

  The solid phase antibody concentration was adjusted to 0.75 μg / mL with a buffer solution, dispensed 1 mL each into a polystyrene test tube, and stored in a refrigerator overnight to prepare a plurality of solid phase tubes.

At least 6 or more samples were prepared by diluting 17β-estradiol 5 times in a concentration range of 8 × 10 ^{−7 to} 5 × 10 ⁻⁴ g / L. The enzyme-labeled antigen was prepared at a concentration of 0.25 μg / mL and divided into a plurality of samples. A sample of 17β-estradiol and a sample of the enzyme-labeled antigen were mixed at a ratio of 1: 1, 100 μL of the mixed solution was added, and added to the solid phase tube.

  After the addition, immunoreaction was performed for 30 minutes, 45 minutes, and 60 minutes, respectively, while shaking at a rotation speed of 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha).

  The measurement results are shown in FIGS. From the results of FIGS. 15 to 17, it was confirmed that the one having a reaction time of 60 minutes had a good correlation between the concentration and the amount of luminescence. In the range of the immune reaction time of 30 to 60 minutes, 60 minutes was the best. Was determined.

(Example 8) Investigation on the effect of shaking The difference between the case where the immune reaction was carried out while standing and the case where the immune reaction was carried out while shaking at 200 rpm was examined.

  The solid phase antibody concentration was adjusted to 0.75 μg / mL with a buffer solution, dispensed 1 mL each into a polystyrene test tube, and stored in a refrigerator overnight to prepare a plurality of solid phase tubes.

17β-estradiol was diluted 5-fold in a concentration range of 8 × 10 ^{−7 to} 5 × 10 ⁻⁴ g / L to prepare at least 6 or more samples, and the enzyme-labeled antigen was diluted to a concentration of 0.25 μg / mL. And divided into multiple samples. A sample of 17β-estradiol and a sample of the enzyme-labeled antigen were mixed at a ratio of 1: 1, 100 μL of the mixed solution was added, and added to the solid phase tube.

  After the addition, the immune reaction was carried out for 1 hour while standing, and for 1 hour while shaking at a rotation speed of 200 rpm.

  The measurement results are shown in FIGS. From the results of FIGS. 18 and 19, it was confirmed that when the immune reaction time was 1 hour, it was better to shake at a rotation speed of 200 rpm.

(Second embodiment)
FIG. 20 is a schematic diagram of a measurement operation showing a measurement process of the enzyme immunoassay according to the second embodiment of the present invention. In FIG. 20, first, an anti-rabbit IgG goat antibody (solid phase antibody), which is the first antibody, is diluted with a dilution buffer in a polystyrene test tube, dispensed in 1 mL portions, and stored in a refrigerator overnight to store the solid phase antibody. Preparation is performed on solid phase 21. The anti-rabbit IgG goat antibody was purchased and used as a commercial product from Japan Biotest Research Institute, and the dilution buffer for the solid-phase antibody was 0.05 mol / l containing 0.9% sodium chloride and 0.1% sodium azide. Prepare and use L of borate buffer (pH = 8.0).

  Next, an enzyme-labeled antigen (enzyme-labeled 17β-estradiol) obtained by labeling 17β-estradiol with peroxidase (enzyme), 17β-estradiol adjusted to a desired concentration, and an anti-17β-estradiol rabbit antibody as the second antibody were solid-phased in this order. The antibody is added to the test tube in which the antibody has been prepared, and the immune reaction is carried out for 1 hour while shaking at 200 rpm. An anti-17β-estradiol rabbit antibody is prepared by conjugated 17β-estradiol with bovine serum albumin (BSA) and immunizing a rabbit. As the enzyme-labeled antigen, ESTRADIOL-PEROXIDAZE manufactured by SIGMA is purchased and used.

  When diluting anti-17β-estradiol rabbit antibody and ESTRADIOL-PEROXIDAZE to a desired concentration, 0.05 mol / L Tris containing 2% polyethylene glycol, 0.05% Tween 20, and 0.9% sodium chloride is used. Prepare and use hydrochloric acid buffer (pH = 8.0).

  After the completion of the immune reaction between the antigen-antibody solution 22 obtained as described above and the solid-phase antibody, the test tube is washed five times with distilled water. After washing, a luminescence reagent is added to obtain a luminescence reaction solution 23, the luminescence amount of the luminescence reaction solution 23 is measured by a luminescence measuring device for 0 to 30 seconds, and the measurement data is processed by a data processor 24.

  The luminescent reagent is prepared at a concentration of 6 mmol / L, and a Tris-HCl buffer (pH = 8.5) containing 10 mol / L hydrogen peroxide and 18.2 mmol / L paraiodophenol is used.

  The second embodiment will be specifically described with reference to the following examples.

Example 9 Examination of Solid Phase Antibody Concentration The solid phase was prepared by adjusting the solid phase antibody concentration to 1.0, 2.0, and 3.0 μg / mL with a buffer solution, and using a polystyrene test tube. , And stored in a refrigerator overnight to prepare a solid phase tube.

  100 μL of the enzyme-labeled antigen prepared at 0.75 μg / mL was added to the solid phase tube. Then, anti-17β-estradiol rabbit antibody was prepared at each concentration of 0.75, 1.5, 3.0, and 6.0 μg / mL, and these were added to the solid-phase tube at 100 μL each, and the rotation speed was changed to 200 rpm. The immune reaction was carried out for 1 hour while shaking with.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 21 shows the measurement results.

  As shown in FIG. 21, it was found that the solid phase antibody concentration was 1 μg / mL.

(Example 10) Examination of anti-17β-estradiol rabbit antibody concentration The solid phase was prepared by adjusting the concentration of the solid phase antibody to 1.0 µg / mL with a buffer solution, dispensing 1 mL each into a polystyrene test tube, and refrigeration. A solid phase tube was prepared by storing overnight.

  100 μL of the enzyme-labeled antigen prepared at 0.5 μg / mL was added to the solid phase tube. Thereafter, 100 μL of anti-17β-estradiol rabbit antibody prepared at 0.75, 1.5, 3.0, and 6.0 μg / mL was added to the solid-phase tube, and the mixture was shaken at 200 rpm for 1 hour. An immunological reaction was performed for an hour.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 22 shows the measurement results.

  As shown in FIG. 22, it was found that the concentration of the anti-17β-estradiol rabbit antibody was preferably 0.75 μg / mL.

(Example 11) Examination of immune reaction time 1
The solid phase was prepared by adjusting the solid phase antibody concentration to 1.0 μg / mL with a buffer solution, dispensing 1 mL each into a polystyrene test tube, and storing it in a refrigerator overnight to prepare a solid phase tube.

  200 μL of the enzyme-labeled antigen prepared at 0.75 μg / mL was added to the solid phase tube, and 100 μL of a 17β-estradiol dilution buffer was further added. Thereafter, 100 μL of anti-17β-estradiol rabbit antibody adjusted to 0.75 μg / mL was added to the solid-phase tube.

  After the addition, immunoreaction was performed for 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 minutes while shaking at 200 rpm. .

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 23 shows the measurement results.

  From FIG. 23, it was found that the immune reaction gradually progressed immediately after the start of the reaction, and that the immune reaction did not progress so much after 90 minutes.

(Example 12) Examination of immune reaction time 2
In Example 11, although the optimal reaction time was examined using a 17β-estradiol dilution buffer, the reactivity of 17β-estradiol may vary depending on the concentration. Therefore, the concentration of 17β-estradiol was set to 6.40 × 10 −9. The dilution was adjusted by a factor of 5 into a concentration range of 1.0 to 10-4 g / L, and the optimal reaction time was examined.

  The solid phase was prepared by adjusting the solid phase antibody concentration to 1.0 μg / mL with a buffer solution, dispensing 1 mL each into a polystyrene test tube, and storing it in a refrigerator overnight to prepare a solid phase tube.

  200 μL of the enzyme-labeled antigen prepared at 0.75 μg / mL was added to the solid-phase tube, and the mixture was diluted 5-fold to a concentration range of 6.40 × 10 −9 to 1.0 × 10 −4 g / L. 100 μL of the prepared 17β-estradiol was added thereto. Thereafter, 200 μL of anti-17β-estradiol rabbit antibody adjusted to 0.75 μg / mL was added thereto.

  After the addition, immunoreaction was performed for 30, 60, and 90 minutes while shaking at a rotation speed of 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 24 shows the measurement results.

  From the results shown in FIG. 24, no significant difference was observed when the immune reaction time was in the range of 30 minutes to 90 minutes, but it was found that it was better to perform the immune reaction for 60 minutes or more in the measurement of the low concentration region.

(Example 13) Investigation of stirring effect The difference between the case where the immune reaction was carried out at rest and the case where it was carried out at 200 rpm was examined.

  The solid phase was prepared by adjusting the solid phase antibody concentration to 1.0 μg / mL with a buffer solution, dispensing 1 mL each into a polystyrene test tube, and storing it in a refrigerator overnight to prepare a solid phase tube.

  200 μL of the enzyme-labeled antigen prepared at 0.75 μg / mL was added to the solid-phase tube, and the mixture was diluted 5-fold to a concentration range of 6.40 × 10 −9 to 1.0 × 10 −4 g / L. 100 μL of the prepared 17β-estradiol was added thereto. Thereafter, 200 μL of anti-17β-estradiol rabbit antibody adjusted to 0.75 μg / mL was added thereto.

  After the addition, the immune reaction was carried out for 1 hour while standing and for 1 hour while shaking at 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 25 shows the measurement results.

  From the measurement results in FIG. 25, it was found that when the immunization time was 1 hour, it was better to perform the rotation at 200 rpm.

(Example 14) Measurement 1 of actual sample (sewage treatment water)
In order to examine whether it can be applied to the measurement of environmental hormones (17β-estradiol) contained in sewage treated water or river water mixed with various contaminants, 17β-estradiol was added to the inflow sewage and discharge water of a sewage treatment plant in Tokyo. After addition, an addition recovery test was performed.

  The solid phase was prepared by adjusting the solid phase antibody concentration to 1.0 μg / mL with a buffer solution, dispensing 1 mL each into a polystyrene test tube, and storing it in a refrigerator overnight to prepare a solid phase tube.

  200 μL of the enzyme-labeled antigen adjusted to 0.75 μg / mL was added to the solid phase tube, and 17β diluted and adjusted to a concentration range of 5.0 × 10 −8 to 5.0 × 10 −4 g / L. -Estradiol and sewage treatment water (aeration tank inlet water and discharge water of S water purification plant) were prepared so that 17β-estradiol concentration was 5.0 × 10 −7 g / L and 5.0 × 10 −5 g / L. Treated water was added. Thereafter, 200 μL of anti-17β-estradiol rabbit antibody adjusted to 0.75 μg / mL was added.

  After the addition, an immunoreaction was performed for 1 hour while shaking at 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 26 shows the measurement results.

  From the measurement results in FIG. 26, it can be seen that the enzyme immunoassay of the present invention is a very specific reaction. It was found that it could be measured.

(Example 15) Measurement 2 of actual sample (sewage treatment water)
The quality of treated water at sewage treatment plants varies depending on the sewage treatment plant. For example, on a process having a sludge treatment facility, there is return water from sludge treatment, and the return water contains a relatively large amount of sulfide. In that sense, 17β-estradiol was added to the inflow sewage and the effluent of a sewage treatment plant in Tokyo different from that in Example 14, and an addition recovery test was performed.

  The solid phase was prepared by adjusting the solid phase antibody concentration to 1.0 μg / mL with a buffer solution, dispensing 1 mL each into a polystyrene test tube, and storing it in a refrigerator overnight to prepare a solid phase tube.

  200 μL of the enzyme-labeled antigen adjusted to 0.75 μg / mL was added to the solid phase tube, and 17β diluted and adjusted to a concentration range of 5.0 × 10 −8 to 5.0 × 10 −4 g / L. -Estradiol and sewage treatment water (one sedimentation effluent and discharge water of C treatment plant) were prepared so that the 17β-estradiol concentration was 5.0 × 10 −7 g / L and 5.0 × 10 −5 g / L. Treated water was added. Thereafter, 200 μL of anti-17β-estradiol rabbit antibody adjusted to 0.75 μg / mL was added.

  After the addition, an immunoreaction was performed for 1 hour while shaking at 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 27 shows the measurement results.

  From the measurement results shown in FIG. 27, it can be seen that the enzyme immunoassay of the present invention is a very specific reaction, and therefore, even in the case of sewage treated water mixed with various contaminants, there is little influence of cross-reactivity. It was found that the measurement could be performed without the need.

(Example 16) Measurement 3 of actual sample (river water)
In order to examine whether it can be applied to the measurement of 17β-estradiol contained in sewage treated water or river water containing various contaminants, 17β-estradiol was added to river water in Tokyo, and an addition recovery test was performed. .

  The solid phase was prepared by adjusting the solid phase antibody concentration to 1.0 μg / mL with a buffer solution, dispensing 1 mL each into a polystyrene test tube, and storing it in a refrigerator overnight to prepare a solid phase tube.

  200 μL of the enzyme-labeled antigen adjusted to 0.75 μg / mL was added to the solid phase tube, and 17β diluted and adjusted to a concentration range of 5.0 × 10 −8 to 5.0 × 10 −4 g / L. -Estradiol and treated water prepared so that the 17β-estradiol concentration became 5.0 × 10 −7 g / L and 5.0 × 10 −5 g / L to river water (M river). Thereafter, 200 μL of anti-17β-estradiol rabbit antibody adjusted to 0.75 μg / mL was added.

  After the addition, an immunoreaction was performed for 1 hour while shaking at 200 rpm.

  After the completion of the immune reaction, the cells were washed five times with distilled water.

  After washing, a luminescence reagent containing 6 mM luminol as a main component was added, and the luminescence amount for 0 to 30 seconds was measured with a luminescence measurement device (Luminometer UPD-4000 manufactured by Meidensha). FIG. 28 shows the measurement results.

  From the measurement results in FIG. 28, since the enzyme immunoassay of the present invention is a very specific reaction, even in the case of river water mixed with various contaminants, there is little influence of cross-reactivity. It turned out to be measurable.

It is a schematic diagram of the measurement operation of the first embodiment of the present invention. FIG. 4 is a characteristic diagram showing the results of Example 1 of the present invention. FIG. 4 is a characteristic diagram (concentration of enzyme-labeled antigen: 0.01 μg / ml) showing the results of Example 2 of the present invention. FIG. 4 is a characteristic diagram (concentration of enzyme-labeled antigen: 0.1 μg / ml) showing the results of Example 2 of the present invention. FIG. 4 is a characteristic diagram (concentration of enzyme-labeled antigen: 0.25 μg / ml) showing the results of Example 2 of the present invention. FIG. 4 is a characteristic diagram showing the results of Example 2 of the present invention (concentration of enzyme-labeled antigen is 0.5 μg / ml). FIG. 5 is a characteristic diagram (concentration of enzyme-labeled antigen: 0.75 μg / ml) showing the results of Example 2 of the present invention. FIG. 4 is a characteristic diagram (concentration of enzyme-labeled antigen: 1.0 μg / ml) showing the results of Example 2 of the present invention. FIG. 5 is a characteristic diagram (concentration of enzyme-labeled antigen: 0.5 μg / mL) showing the results of Example 3 of the present invention. FIG. 4 is a characteristic diagram showing the results of Example 3 of the present invention (concentration of enzyme-labeled antigen is 1.0 μg / mL). FIG. 4 is a characteristic diagram (concentration of enzyme-labeled antigen: 2.0 μg / mL) showing the results of Example 3 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 4 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 5 of the present invention. FIG. 13 is a characteristic diagram showing the results of Example 6 of the present invention. FIG. 11 is a characteristic diagram (immune reaction time: 30 minutes) showing the results of Example 7 of the present invention. FIG. 10 is a characteristic diagram (immune reaction time: 45 minutes) showing the results of Example 7 of the present invention. FIG. 14 is a characteristic diagram (immune reaction time: 60 minutes) showing the results of Example 7 of the present invention. FIG. 10 is a characteristic diagram (reacted for 1 hour after standing still) showing the results of Example 8 of the present invention. FIG. 11 is a characteristic diagram (reaction for 1 hour with shaking at 200 rpm) showing the results of Example 8 of the present invention. It is a schematic diagram of the measurement operation of the second embodiment of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 9 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 10 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 11 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 12 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 13 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 14 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 15 of the present invention. FIG. 14 is a characteristic diagram showing the results of Example 16 of the present invention.

Explanation of reference numerals

11, 21 ... solid phase 12, 22 ... antigen-antibody solution 13, 23 ... luminescence reaction solution 14, 24 ... data processing

Claims (13)

After preparing the antibody on a solid phase, an enzyme-labeled antigen is added to the solid phase, and an endocrine disrupting chemical substance is added to cause an immune reaction,
After the end of the immune reaction, the solid phase is washed, a luminescent reagent is added, and the endocrine disrupting chemical is measured from the amount of light emitted by the enzymatic reaction.   The method of claim 1, wherein the endocrine disrupting chemical is 17β-estradiol.   The method according to claim 2, wherein the antibody is prepared by immunizing rabbits by binding bovine serum albumin to 17β-estradiol.   The method according to claim 3, wherein the concentration of the antibody is 0.75 µg / mL.   5. The method according to claim 4, wherein a volume of the mixed solution of the enzyme-labeled antigen and 17β-estradiol added to 1 mL of the antibody is 100 μL.   The method according to claim 4, wherein the concentration of the enzyme-labeled antigen in the mixed solution is 0.125 to 0.25 µg / mL.   The method according to claim 5 or 6, wherein the immune reaction is performed while shaking at a rotation speed of 200 rpm.   8. The method according to claim 5, wherein the immune reaction is performed for 1 hour or more. After preparing the first antibody on a solid phase, an enzyme-labeled antigen, an endocrine disrupting chemical substance, and a second antibody are added to the solid phase to cause an immune reaction,
After the end of the immune reaction, the solid phase is washed, a luminescent reagent is added, and the endocrine disrupting chemical substance is measured from the amount of light emitted by the enzymatic reaction.   The method of claim 9, wherein the endocrine disrupting chemical is 17β-estradiol.   The method according to claim 10, wherein the first antibody is an anti-rabbit IgG goat antibody, and the second antibody is an anti-17β-estradiol rabbit antibody.   The endocrine disrupting chemical substance according to claim 11, wherein the concentration of the anti-rabbit IgG goat antibody is 1.0 µg / mL, and the concentration of the anti-17β-estradiol rabbit antibody is 0.75 µg / mL. Enzyme immunoassay.   13. The method according to claim 11, wherein the immune reaction is carried out while shaking at a rotation speed of 200 rpm.

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