JP2010169507A - Method for measuring immunity of equol in sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem that pretreatment of a sample, which is essential, takes a long time in a method for measuring equol in the sample. <P>SOLUTION: An antibody recognizing specifically equol with strong affinity and low cross-reactivity with an allied substance is obtained by performing immunogen synthesis properly in production of an antibody recognizing equol. An immunity measuring method capable of measuring equol without de-conjugation treatment is established by building an immunity measuring method using this antibody. Consequently, equal can be measured in a short time without performing the pretreatment of a sample. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is an immunoassay for equol in a sample using an antigen-antibody reaction.

  Isoflavones contained in soybean have a preventive effect (antiestrogenic effect) against breast cancer, prostate cancer, etc., and an improvement effect on menopause, postmenopausal osteoporosis, hyperlipidemia, hypertension, etc. (estrogenic effect) ) Is widely known.

  Recently, the direct clinical effect of soy isoflavone has been questioned, and it has been reported that equol, an active metabolite of soy isoflavone, is the key to its effectiveness in clinical applications instead of soy isoflavone. In other words, various reports have shown that equol, a metabolite of soy isoflavone, is more effective than soy isoflavone for breast cancer, prostate cancer, menopause and postmenopausal osteoporosis. It is supported to be a sex body.

  In addition, since equol is produced by intestinal bacteria, it has been reported that the amount of equol produced varies depending on the intestinal flora, and there are individual differences. Therefore, it cannot be denied that there are humans who cannot expect the desired anti-estrogen effect and estrogen-like effect even if they consume processed soybean products.

  Thereafter, lactic acid bacteria that efficiently produce equol were isolated and identified (Patent Document 1), and the lactic acid bacteria were orally ingested to establish the lactic acid bacteria in the intestine, thereby efficiently incorporating equol into the body. Be expected. At this time, for example, by measuring equol in urine, it is possible to confirm the presence or absence of colonization of the lactic acid bacteria in the intestine.

  As a method for measuring this equol, a method using a liquid chromatograph mass spectrometer or a gas chromatograph mass spectrometer for blood samples and a method using instrumental analysis such as high performance liquid chromatography for urine samples are known. A promising measurement system is expected.

  In addition, as a method for measuring equol by immunoassay, Labmaster TR-FIA Research Reagents for the Measurement of Equal (Labmaster Master Diagnostics) using time-resolved fluorescence immunoassay is commercially available (Non-patent Document 1). The sample pre-treatment process by the method is essential, which is an obstacle to the measurement of multiple samples in a short time.

JP 2006-296434 A

E. Browers et al., Journal of Steroid Biochemistry & Molecular Biology, 2003, 84, p. 577-588

  Using an immunoassay method which is a simple measurement system, equol can be measured in a short time.

  In the equol measurement, the most time-consuming process is a pretreatment process using a sample enzyme or the like. Therefore, the composition of the measurement reagent that does not require this step must be considered. In view of simplicity of measurement, an immunoassay method is desirable.

  In addition, antibodies have a significant influence on the performance of immunoassay methods. As the nature of this antibody, in addition to affinity, which is the strength of binding to an antigen, specificity is known which serves as an index of cross-reactivity with similar substances.

  Then, when the production method of the antibody used for the measurement by the immunoassay method of equol was examined, it became possible to measure equol equally regardless of the presence or absence of the binding to the clathrate.

That is, the present invention has the following configuration.
(1) A method for measuring equol in a sample by immunization, wherein the equol is measured without deconjugation from the conjugate.
(2) The immunoassay method for equol in a sample according to (1), wherein the conjugate is a sulfate conjugate or a glucuronic acid conjugate.
(3) The immunoassay method for equol in a sample according to (1) or (2), wherein the immunoassay method is an ELISA method.
(4) The immunoassay method for equol in a sample according to (1) or (2), wherein the immunoassay method is an RIA method.
(5) The immunoassay method for equol in a sample according to (1) or (2), wherein the immunoassay method is an immunochromatography method.
(6) The immunoassay method for equol in a sample according to (1) or (2), wherein the immunoassay method is a latex agglutination method.
(7) The immunoassay method according to any one of (1) to (6), wherein the antibody used in the method for measuring equol in a sample by an immunoassay is a polyclonal antibody and / or a monoclonal antibody.
(8) The immunoassay method according to any one of (1) to (7), wherein the sample is a blood sample or a urine sample.

  Since equol, which is a measurement target of the present invention, is a low molecular weight hapten antigen, it does not have immunogenicity by itself. Therefore, in order to obtain an antibody for use in an immunoassay, a conjugate with an antigen-supporting substance must be synthesized to produce an immune antigen.

  As the antigen-supporting substance, a polymer substance (molecular weight of 10,000 or more, preferably 50,000 to 1,000,000) is preferable, and among them, protein, lipid and the like are preferable. Examples of preferred proteins include antibodies, bovine serum albumin (BSA), casein, gelatin, ferritin and the like, and examples of lipids include gloposide glycolipids, ganglioside glycolipids, phospholipids and the like. However, it is not limited to these.

  In order to bind the hapten antigen and the antigen-supporting substance, covalent bonding is preferable, and a known technique can be appropriately used. For example, when a protein is used as the antigen support substance, the succinimide group introduced into the hapten antigen is reacted with the amino group of the protein or glycoprotein by the N-hydroxysuccinimide activated ester method, and the hapten antigen and the antigen support substance are reacted. Can be combined.

  Further, in order to increase the distance between the hapten antigen and the antigen support material when binding the hapten antigen and the antigen support material, a spacer compound is introduced into the hapten antigen and then bound to the antigen support material, thereby Changes in affinity and antibody specificity can be given.

  A labeled hapten antigen that detects the presence of an antibody can be prepared in the same manner by using a label instead of the antigen-supporting substance, and by introducing a spacer compound between the hapten antigen and the label. The affinity with the antibody to be detected can be adjusted.

  In the present invention, as a result of examining the suitability of equol, spacer, and antigen-supporting substance or labeling substance, an immunoassay that does not require deconjugation from the conjugate has become possible.

  Polyclonal antibodies and monoclonal antibodies used in the immunoassay can be prepared by carrying out by known methods. That is, a polyclonal antibody is prepared by mixing an immune antigen in which equol and an antigen-supporting substance are combined with Freund's complete adjuvant to prepare an immunizing agent, and the prepared immunizing agent is periodically added to rabbit, goat, horse, sheep, guinea pig. It can be prepared by purifying or diluting the antiserum obtained after multiple injections subcutaneously and / or intradermally, such as chickens.

  A monoclonal antibody is prepared by mixing an immune antigen in which equol and an antigen-supporting substance are combined with Freund's complete adjuvant to prepare an immunizing agent, and the prepared immunizing agent is periodically subcutaneously and / or intradermally in mice, rats, etc. After multiple injections, the immunizing antigen is injected intravenously or intraperitoneally, and the fused cells are selectively cultured by fusing spleen cells prepared from the spleen removed several days later and myeloma cells.

  The proliferated fused cells are cloned and cultured, and a cell line producing an IgG antibody that specifically binds to equol is selected. It can be prepared by purifying or diluting the culture supernatant obtained by culturing the selected cell line.

  Next, examples of equol immunoassay are shown below, but the essence of the present invention is not limited to these examples.

  For the measurement of equol in urine by the ELISA method, a diluted urine sample solution is prepared by adding a sample diluent to a urine sample, and prepared in a well of a microplate in which an anti-rabbit goat antibody is immobilized in advance. The solution, horseradish peroxidase-labeled equol and diluted anti-equol rabbit antiserum are added and allowed to react for a certain period of time. After the reaction, the wells are washed, a chromogenic substrate solution is added and allowed to react for a certain period of time, the reaction is stopped after a certain period of time, the absorbance is measured, and the equol concentration in the sample is calculated from the calibration curve measured in the same manner.

For the measurement of equol in urine by the RIA method, a diluted urine sample solution is prepared by adding a sample diluent to a urine sample, and prepared in a well of a microplate in which an anti-rabbit goat antibody is immobilized in advance. The solution, ¹²⁵ I-labeled equol and diluted anti-equol antibody-producing cell culture supernatant are added and allowed to react for a certain period of time. After the reaction, after washing the well, the radioactivity of the well of the microplate is measured, and the equol concentration in the sample is calculated from the calibration curve measured in the same manner.

  For the measurement of equol in blood by immunochromatography, prepare a diluted serum sample solution by adding colloidal gold-labeled anti-equol mouse monoclonal antibody to the serum sample, and add the diluted serum sample solution dropwise to the sample pad of the immunochromatography strip. Let react for hours. After the reaction, the coloration of the capturing part to which equol is immobilized by the colloidal gold labeling substance is measured, and the concentration of equol in the sample is calculated from the calibration curve similarly measured.

  For the measurement of equol in blood by the latex agglutination method, an aggregated antigen that reacts and aggregates with a serum sample and an anti-equol rabbit polyclonal antibody is added to the absorbance measurement cell and reacted for a certain period of time. After the reaction, an anti-equol rabbit polyclonal antibody-immobilized latex solution is added, the absorbance is measured twice after a certain period of time to determine the difference in absorbance twice, and the equol concentration in the sample is calculated from the calibration curve obtained in the same manner.

  Samples for measuring equol include plasma samples, stool samples, tissue extracts, cell extracts, foods, etc., in addition to the urine samples and serum samples.

  By carrying out the present invention, the equol in the sample is not only measured in a short time, but the pretreated specimen is diluted in order to suppress inhibition of the immune reaction caused by the compound contained in the pretreatment reagent. It is possible to avoid lowering the sensitivity.

FIG. 1 is a correlation diagram of the results measured by the direct method and the enzyme treatment method of the ELISA method. (Example 5) FIG. 2 is a correlation diagram of the results measured by the direct method of the ELISA method and the HPLC method. (Example 6)

The quality of antibodies used when measuring low molecular equol by immunization depends on the synthetic immunogen. Furthermore, it is important to produce an antibody that measures equol equally, regardless of whether it is bound to a conjugate.
Hereinafter, preparation and evaluation of the antibody and measurement using the antibody will be described.

Synthesis of immunogen 100 mg equol (Equol) is dissolved in 400 μL DMSO, 58 μL Methyl Bromoacetate and 140 mg K ₂ CO ₃ are added and reacted at 25 ° C. for 4 hours. After completion of the reaction, the reaction mixture was acidified with hydrochloric acid, extracted with ethyl acetate, dehydrated and evaporated to dryness.

  Subsequently, using chloroform methanol (19: 1) as a developing solvent, it was developed and separated on thin layer chromatography (1.05717, Merck Japan Ltd.), and one molecule of CME (carboxymethylether) -methylester was converted into one molecule of Equol. Only the introduced fraction was collected.

  8N sodium hydroxide is added to the collected equol-CME-methylester bonded to each molecule and heated at 50 ° C. for 20 minutes. After heating, the mixture was acidified with hydrochloric acid, extracted with ethyl acetate, and evaporated to dryness to obtain Equol-CME-acid.

  To 2 mg of the obtained Equol-CME-acid, 1.5 mg of N-hydroxysuccinimide (NHS) and 1.5 mg of water-soluble carbodiimide (WSC) are added and dissolved in 20 μL of DMSO and reacted at 25 ° C. for 1 hour. , Equol-CME-NHS was obtained.

  To 250 μL of 20 mg / mL BSA solution (20 mg of bovine serum albumin dissolved in 1 mL of 50 mM carbonate buffer (pH 9.7)), 10 μL of Equol-CME-NHS (10 mg / mL DMF solution) was added and reacted at 25 ° C. for 30 minutes. Let After the reaction was completed, the unreacted product was removed by gel filtration to obtain a stock solution of Equol-CME-BSA antigen.

Preparation of antiserum Synthesized immunogen Equol-CME-BSA (2.5 mg / mL) and Freund's complete adjuvant were mixed in equal amounts to prepare an immunizing agent. Using this immunizing agent, 1 mL / dose was subcutaneously injected at several sites on the back of the rabbit every 3 weeks. Blood collection was started every 3 weeks from the 12th week after the start of immunization, and after 5 partial blood collections, whole blood collection was performed. Table 1 shows the antibody titer of the collected antiserum.

Preparation of equol measurement ELISA kit
1. Preparation of anti-rabbit goat antibody-immobilized plate 100 μL of 10 μg / mL anti-rabbit goat antibody solution is added to each well of a 96-well microplate. After standing at 4 ° C. for 2 nights, the anti-rabbit goat antibody solution is removed by suction.

  Add 300 μL of 0.5 mg / mL bovine serum albumin solution to each well of the 96-well microplate that has been removed by suction. After standing at 4 ° C. for 18 hours, the bovine serum albumin solution is removed by suction and vacuum dried to obtain an anti-rabbit goat antibody-immobilized plate.

2. Preparation of horseradish peroxidase-labeled equol 100 mg of equol (Equol) was dissolved in 400 μL of DMSO, 58 μL of 4-Bromo-n-butyric Acid and 140 mg of K ₂ CO ₃ were added and reacted at 25 ° C. for 4 hours. . After completion of the reaction, the reaction mixture was acidified with hydrochloric acid, extracted with ethyl acetate, dehydrated and evaporated to dryness.

  Subsequently, using chloroform methanol (19: 1) as a developing solvent, it was developed and separated on thin layer chromatography (1.05717, Merck Japan), and 1 molecule of CPE (carbopropylene) -methylester was converted to 1 molecule of Equol. Only the introduced fraction was collected.

  8N sodium hydroxide is added to the collected equol-CPE-methylester bound to each molecule and heated at 50 ° C. for 20 minutes. After heating, the mixture was acidified with hydrochloric acid, extracted with ethyl acetate, and evaporated to dryness to obtain Equol-CPE-acid.

  To 2 mg of the obtained Equol-CPE-acid, 1.5 mg of N-hydroxysuccinimide (NHS) and 1.5 mg of water-soluble carbodiimide (WSC) are added and dissolved in 20 μL of DMSO and reacted at 25 ° C. for 1 hour. , Equol-CPE-NHS was obtained.

  To 250 μL of 20 mg / mL HRP solution (20 mg of horseradish peroxidase dissolved in 1 mL of 50 mM carbonate buffer (pH 9.7)), 10 μL of Equol-CPE-NHS (10 mg / mL DMF solution) was added and reacted at 25 ° C. for 30 minutes. Let After the reaction was completed, the unreacted product was removed by gel filtration to obtain a stock solution of horseradish peroxidase-labeled equol solution.

3. A specimen diluent and a standard diluent 100% human free serum (SERACON II: CD INTERGEN) were used.

4). Composition of test buffer 150 mM sodium chloride, 0.5% bovine serum albumin, 0.01% tween 20, 0.1% proclin 300, 50 μg / mL lomefloxacin hydrochloride, 0.1 μM phosphate buffer containing 5 μg / mL lactoperoxidase (PH 7.5) was used as a test buffer.
Further, 100 mM sodium chloride, 0.025% tween 20-containing 0.3 mM phosphate buffer (pH 7.5) was used as a washing solution.

5. Substrate 0.05% hydrogen peroxide solution, 2.2 mg / mL OPD (o-phenylenediamine dihydrochloride) -containing 50 mM citrate buffer (pH 5.5) was used as a substrate.

6). Reaction stop solution 3N sulfuric acid was used as the reaction stop solution.

7). As an enzyme solution for pretreatment of an enzyme solution specimen, sulfatase (SIGMA) dissolved in 2 mL of 0.1 M acetate buffer (pH 5.0), 2.48 μL of β-glucuronidase (ROCHE) and 100 mg / mL with purified water. ) Prepared by adding 26.4 μL.

Measurement of equol using ELISA kit (direct method)
A sample dilution solution of 200 μL is added to 20 μL of a urine sample to prepare an 11-fold diluted urine sample. To the well of the anti-rabbit goat antibody-immobilized plate, 20 μL of 11-fold diluted urine specimen, 50 μL of horseradish peroxidase-labeled equol solution and 50 μL of anti-equol rabbit antiserum solution are added, and left at 25 ° C. for 1 hour after stirring.

  After removing the reaction solution from the well of the anti-rabbit goat antibody-immobilized plate, the well is washed three times with a washing solution. After washing, 100 μL of the substrate is added to the well of the anti-rabbit goat antibody-immobilized plate, and the plate is allowed to stand at 25 ° C. for 30 minutes. Thereafter, 100 μL of the reaction stop solution is added to the well of the anti-rabbit goat antibody-immobilized plate, the absorbance at a wavelength of 490 nm is measured, and the equol concentration in the sample is calculated from the calibration curve similarly measured.

Measurement of equol using ELISA kit (enzyme treatment method)
A sample dilution solution of 200 μL is added to 20 μL of a urine sample to prepare an 11-fold diluted urine sample. Add 50 μL of the enzyme solution to 50 μL of the 11-fold diluted urine sample, and stir overnight at 37 ° C. after stirring. Thereafter, 50 μL of a neutralizing solution (0.5 M phosphoric acid 2Na (pH 8.8)) is added to obtain an enzyme-treated sample.
20 μL of the enzyme-treated specimen, 50 μL of horseradish peroxidase-labeled equol solution and 50 μL of anti-equol rabbit antiserum solution are added to the wells of the anti-rabbit goat antibody-immobilized plate, and the mixture is allowed to stand at 25 ° C. for 1 hour after stirring.

  After removing the reaction solution from the well of the anti-rabbit goat antibody-immobilized plate, the well is washed three times with a washing solution. After washing, 100 μL of the substrate is added to the well of the anti-rabbit goat antibody-immobilized plate, and the plate is allowed to stand at 25 ° C. for 30 minutes. Thereafter, 100 μL of the reaction stop solution is added to the well of the anti-rabbit goat antibody-immobilized plate, the absorbance at a wavelength of 490 nm is measured, and the equol concentration in the sample is calculated from the calibration curve similarly measured.

Comparison of equol ELISA measurement values with and without enzyme treatment For nine urine samples, equol measurement with an ELISA kit was performed using the direct method and the enzyme treatment method. A comparison of the measured values obtained is shown in FIG. 1. In the direct method, the measured value was as low as about 1/3 compared with the enzyme treatment method, but the correlation coefficient r = 0.993 was high. Showed sex.

ELISA measurement by the equol direct method was carried out on 12 urine samples from which the measurement results by the HPLC method correlated with the HPLC method were obtained. FIG. 2 shows a comparison between the obtained measured value and the measured value by the HPLC method. The measured value of the EISA method showed a low value of about 1/4 compared with the HPLC method. The number r = 0.799 showed a high correlation.

The dilution series of cross-reactive equol and the antigen dilution series for confirmation of cross-reactivity are measured, and the concentration (50% inhibitory concentration: IC50) showing 50% absorbance of standard 0 is compared, and the IC50 of the equol dilution series is compared. The ratio was defined as the cross reaction rate.

  The results are shown in Table 2. Crossability with other substances having a similar structure including daidzein which is a precursor of equol was 1% or less.

  In anticipating antiestrogenic and estrogenic effects, when taking processed soybean products, the equol concentration in urine and blood is measured to determine whether it is effective. It takes time and is not suitable for daily measurement.

  However, according to the present invention, equol in urine and blood can be measured in a short time, and the effect of intake of processed soybean products, including the judgment of the effect of administration of a safe lactic acid bacteria preparation that produces equol, can be estimated. It becomes possible to do.

Claims (8)

  A method for measuring equol in a sample by immunization, wherein the equol is measured without deconjugation from the conjugate.   The method for immunoassay of equol in a sample according to claim 1, wherein the conjugate is a sulfate conjugate or a glucuronic acid conjugate.   The immunoassay method for equol in a sample according to claim 1 or 2, wherein the immunoassay method is an ELISA method.   The immunoassay method for equol in a sample according to claim 1 or 2, wherein the immunoassay method is an RIA method.   The immunoassay method for equol in a sample according to claim 1 or 2, wherein the immunoassay method is an immunochromatography method.   The immunoassay method for equol in a sample according to claim 1 or 2, wherein the immunoassay method is a latex agglutination method.   The immunoassay method according to any one of claims 1 to 6, wherein the antibody used in the method for measuring equol in a sample by an immunoassay is a polyclonal antibody and / or a monoclonal antibody.   The immunoassay method according to any one of claims 1 to 7, wherein the sample is a blood sample or a urine sample.

Images