Method for detection, reagent kit, and method for manufacturing reagent

Abstract

To perform deconjugation of a detection target more efficiently.SOLUTION: The method for detection according to the present disclosure includes: a first step (S1) of preparing a sample including a conjugate as a detection target; and a second step (S3) of preparing a first solution by adding an enzyme derived from coliform bacteria which deconjugate the conjugate to the sample.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a detection method, a reagent kit, and a method for producing a reagent for measuring the concentration of a detection target. [Background technology]

[0002] Patent Document 1 discloses a technique for detecting a target substance that has formed a conjugate with another substance, in which the target substance is detected after deconjugating the conjugate. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2007-256044 A Summary of the Invention [Problem to be solved by the invention]

[0004] It is desired to perform deconjugation of a detection target that has formed a conjugate more efficiently than ever before. [Means for solving the problem]

[0005] A detection method according to one embodiment of the present disclosure includes a first step of preparing a sample containing a conjugate to be detected, and a second step of adding an enzyme derived from Escherichia coli that deconjugates the conjugate to the sample to prepare a first solution.

[0006] A reagent kit according to one embodiment of the present disclosure includes an enzyme derived from Escherichia coli that deconjugates a conjugate to be detected, and an antibody against the conjugate to be detected.

[0007] A method for producing a reagent according to one embodiment of the present disclosure includes a first preparation step of preparing a first enzyme solution, which is a solution containing an enzyme derived from Escherichia coli that deconjugates a conjugate to be detected, and a second preparation step of adding an antibody to be detected to the first enzyme solution to prepare a second enzyme solution. Effect of the Invention

[0008] According to one aspect of the present disclosure, deconjugation of the detection target can be performed more efficiently than ever before. [Brief description of the drawings]

[0009] [Figure 1] 1 is a flowchart illustrating an example of the flow of a detection method according to the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram showing an overview of a detection method according to the present disclosure. [Diagram 3] FIG. 2 is a functional block diagram showing a configuration of a sensor device. [Figure 4] FIG. [Diagram 5] FIG. [Figure 6] FIG. 1 is a schematic diagram showing an overview of a conventional detection method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] [Embodiment] Hereinafter, one embodiment of the present disclosure will be described.

[0011] <Detection method overview> First, the detection method according to the present disclosure will be outlined with reference to Fig. 1. Fig. 1 is a flow chart showing an example of the flow of the detection method according to the present disclosure.

[0012] The detection method according to the present disclosure is a method for measuring the concentration of the target substance by detecting the target substance contained in a specimen. The detection method according to the present disclosure includes step S1 (first step) of preparing a specimen containing a conjugate of the target substance, and step S3 (second step) of adding an enzyme derived from E. coli that deconjugates the conjugate to the specimen to prepare a first solution. The detection method may also include step S2 (fourth step) of preparing a second solution containing the enzyme derived from E. coli to be added to the specimen after step S1. The detection method may also include step S4 of holding the first solution at a first temperature for a first time after step S3 (second step). The detection method may also include step S5 (third step) of detecting the target substance after step S4. When the detection method according to the present disclosure includes step S2, in step S3, the first solution is prepared by adding a second solution containing the enzyme derived from E. coli to the specimen.

[0013] Here, "detection" means determining whether or not the target substance is present in a sample. "Measurement" means measuring the concentration or amount of the target substance contained in a sample. "Measurement" may be considered as a subordinate concept of "detection."

[0014] The detection target may be any of steroid hormones, flavonoids, or metabolites of steroid hormones or flavonoids. The detection target may be any of isoflavones or metabolites of isoflavones. The detection target may be any of daidzein, genistein, glycitein, or equol. The specimen may be urine, saliva, blood, or the like, which may contain the above-mentioned detection target. As an example, the specimen may be urine, and the detection target may be equol contained in the urine. The enzyme may be added to the specimen obtained from the subject without pretreatment.

[0015] Furthermore, the conjugate may be a substance that contains at least one of the glucuronide conjugate to be detected and the sulfate conjugate to be detected, as described above.

[0016] When urine is used as a sample, equol exists in the urine as a glucuronide or sulfate conjugate. When the equol concentration is measured, these conjugates are deconjugated.

[0017] Conventionally, in order to measure a target substance that has formed a conjugate using an antibody, an enzyme having both β-glucuronidase and sulfatase activity has been used for the purpose of deconjugating both glucuronide conjugates and sulfate conjugates.

[0018] In contrast, the inventors of the present disclosure have found that, as long as the enzyme used has β-glucuronidase activity, the use of an enzyme with low sulfatase activity does not have a significant effect on the detection results of the target substance when an enzyme with high activity in both β-glucuronidase and sulfatase is used. In other words, they have found that even an enzyme with low sulfatase activity can be used for deconjugation. This allows more types of enzymes to be used for deconjugation of the target substance that has formed a conjugate. Specifically, they have found that there is no significant difference in the detection results of equol between the use of Helix pomatia, an enzyme that has been used conventionally for deconjugation, and the use of an enzyme derived from Escherichia coli.

[0019] According to the detection method of the present disclosure, the glucuronide conjugate can be deconjugated by adding a first solution containing an enzyme derived from Escherichia coli to deconjugate the conjugate.

[0020] Fig. 2 is a schematic diagram showing an overview of the detection method according to the present disclosure. As shown in Fig. 2, the method according to the present disclosure can measure the concentration of the detection target by utilizing a competitive method.

[0021] First, a specimen P containing a detection target X is prepared. The specimen P may contain a detection target X2 present alone, a glucuronide conjugate X3 of the detection target X2, and a sulfate conjugate X4 of the detection target X2. The detection target X2, the detection target glucuronide conjugate X3, and the detection target sulfate conjugate X4 of the detection target X2 are collectively referred to as "detection target X." A second solution Q containing an enzyme Y and an antibody Z of the detection target X is added to the specimen P to prepare a first solution R. As a result, the detection target glucuronide conjugate X3 contained in the first solution R is deconjugated to become the detection target X2.

[0022] In the first solution R, simultaneously with the deconjugation, an antigen-antibody reaction (hereinafter, antibody reaction) occurs between the target substance X and the antibody Z that reacts with the target substance X. As a result, the first solution R contains the antibody Z2 that reacted with the target substance X and the antibody Z3 that did not react with the target substance X (unreacted antibody). In addition, as shown in FIG. 2, in the solution R, the sulfate conjugate X4 also binds to the antibody Y.

[0023] For example, as shown by reference numeral 201 in Fig. 2, the amount of unreacted antibody Z3 can be measured by introducing a first solution R into a sensor device 1 including a sensor 2 (detection unit 23) having a substance A that reacts with antibody Z3 on its surface. Based on the measurement result, the concentration of target substance X contained in the specimen P can be calculated. For example, substance A may be a substance in which target substance X, which is an antigen of antibody Z, is labeled with BSA (Bovine serum albumin).

[0024] <Sensor device 1> 3 is a functional block diagram showing the configuration of the sensor device 1. The sensor device 1 is a device for detecting a detection target contained in a specimen. The sensor device 1 includes a sensor 2 capable of detecting the detection target, and a control unit 3.

[0025] The sensor 2 may be, for example, a sensor that uses a surface acoustic wave, a QCM (Quartz Crystal Microbalance), a SPR (Surface Plasmon Resonance), or a FET (Field Effect Transistor). That is, the sensor 2 may be capable of converting between an electrical signal and an elastic wave, a QCM, a SPR, a FET, or the like. The sensor 2 according to one embodiment is a sensor that uses an elastic wave. That is, by using the sensor 2, the sensor device 1 can detect a change in an elastic wave based on the presence of an antibody against the detection target as a change in an electrical signal.

[0026] In the sensor device 1, the control unit 3 can detect, for example, the detection target based on the electrical signal input from the sensor 2. The control unit 3 can also calculate, for example, the concentration of the detection target contained in the sample.

[0027] 4 shows a plan view of the sensor 2. The sensor 2 includes a substrate 22, a detection unit 23 located on the substrate 22, and a pair of first IDT (Inter Digital Transducer) electrodes 25a arranged on the substrate 22 so as to sandwich the detection unit 23. The substrate 22 can support the first IDT electrodes 25a and the like.

[0028] (substrate) The substrate 22 is a piezoelectric substrate. For example, the substrate 22 may be a substrate containing a single crystal having piezoelectricity, such as lithium tantalate, quartz, etc. In this embodiment, the substrate 22 is a piezoelectric substrate containing quartz.

[0029] (Detection unit) Substance A that reacts with an antibody against the detection target is immobilized on detection unit 23. Substance A that reacts with an antibody against the detection target may be a substance that contains the same structure as the detection target. For example, when the detection target is equol, substance A may be BSA-labeled equol.

[0030] The detection unit 23 can change the propagation characteristics of the elastic waves of the substrate 22 by reacting an antibody against the detection target with the substance A. Specifically, the detection unit 23 can change the weight on the substrate 22 or the viscosity of the liquid in contact with the surface of the substrate 22, for example, by reacting an antibody against the detection target with the substance A. This changes the characteristics (speed, phase, amplitude, etc.) of the elastic waves propagating through the substrate 22. Therefore, the sensor 2 can detect a change in the surface acoustic wave corresponding to the concentration of the detection target.

[0031] The pair of first IDT electrodes 25a can generate a surface acoustic wave (SAW: Surface Acoustic Wave) between the pair of first IDT electrodes 25a. In the sensor device 1, an electric signal is input to one of the pair of first IDT electrodes 25a. The input electric signal is converted into an elastic wave propagating toward the detection unit 23 and is transmitted from one IDT electrode 25a. The transmitted elastic wave passes through the detection unit 23. The other IDT electrode 25a can receive the elastic wave that has passed through the detection unit 23. The received elastic wave is converted into an electric signal. The pair of first IDT electrodes 25a may be formed of a metal material such as gold, chromium, or titanium. The pair of first IDT electrodes 25a may be a single-layer electrode made of a single material, or a multi-layer electrode made of multiple materials.

[0032] (Reference part) The sensor 2 may have a reference portion 24 located on the substrate 22. The sensor 2 may also have a pair of second IDT electrodes 25b located on the substrate 22 so as to sandwich the reference portion 24. In the sensor device 1, an electrical signal is input to one of the pair of first IDT electrodes 25b. The input electrical signal is converted into an elastic wave propagating toward the reference portion 24 and is transmitted from one IDT electrode 25b. The transmitted elastic wave passes through the reference portion 24. The other IDT electrode 25b can receive the elastic wave that has passed through the reference portion 24. The received elastic wave is converted into an electrical signal.

[0033] The reference section 24 can function as a control for the detection section 23. Specifically, unlike the detection section 23, the substance A is not fixed to the reference section 24. Therefore, the sensor device 1 can detect the detection target based on an electrical signal (detection signal) based on an elastic wave that has passed through the detection section 23, using an electrical signal (reference signal) based on an elastic wave that has passed through the reference section 24 as a reference. Specifically, if the sample contains the detection target, an antibody against the detection target reacts with the substance A, resulting in a difference between the detection signal and the reference signal. Therefore, for example, the sensor device 1 can detect the detection target by taking the difference between the detection signal and the reference signal.

[0034] 5 shows a cross-sectional view of the sensor 2. The sensor 2 has a flow path member 26. The flow path member 26 functions as a passage for the specimen. The flow path member 26 opens on the upper surface of the flow path member 26 and has a supply port 27 for supplying the specimen and a discharge port 28 for discharging the specimen. When the specimen supplied from the supply port 27 reaches the detection unit 23, the sensor 2 detects the detection target in the detection unit 23, and then discharges the specimen from the discharge port 28.

[0035] The sensor device 1 may directly detect the target object 5. For example, an antibody against the object 5 may be located in the detection unit 23 of the sensor 2 as substance A. The antibody against the object 5 may be immobilized as substance A on the detection unit 23 of the sensor 2. That is, for example, the object may be directly reacted with the immobilized substance A. In this case, the amount of the object contained in the specimen can be calculated from the change in the characteristics of the elastic wave generated during the reaction.

[0036] The sensor device 1 may include an output unit 4 which is a display device such as a display capable of displaying information indicating the measured concentration of the target substance. The control unit 3 may cause the output unit 4 to output information indicating the measured concentration of the target substance. This allows a person who has performed a measurement using the sensor device 1 to know the concentration of the target substance contained in the sample.

[0037] Furthermore, the output unit 4 may be a communication module capable of transmitting the information to an external device communicatively connected to the sensor device 1. In this case, the output unit 4 may transmit information indicating the measurement results and the like acquired from the control unit 3 to the external device.

[0038] The sensor device 1 may also include other members (not shown), such as a heating device for heating the first solution and a timer for measuring the heating time.

[0039] <Detection method details> A non-limiting example of the detection method according to the present disclosure will be described in detail below with reference to Fig. 1 again. The following description is based on the premise that steps S2 and S4 are also performed, but steps S2 and S4 are not essential steps.

[0040] (Step S1) First, a specimen containing conjugates to be detected is prepared (step S1: first process). The specimen may be prepared by the subject providing the specimen, or by someone other than the subject. For example, when the specimen is equol contained in urine, in step S1, urine of the subject whose equol concentration is to be measured is sampled as the specimen. The sampled urine may contain equol conjugates. The subject may be, for example, a human, or an animal such as a dog, cat, or cow.

[0041] (Step S2) After step S1, the method may include a step S2 (fourth step) of preparing a second solution containing an enzyme derived from E. coli to be added to the specimen. The pH of the second solution may be 6.0 or more and 7.5 or less. By preparing a second solution with an adjusted pH in advance and mixing it with the specimen, it is possible to easily adjust the pH of the first solution to a pH suitable for the deconjugation reaction. In order to more easily adjust the pH of the first solution to a pH suitable for the deconjugation reaction, the pH of the second solution may be 6.4 or more and 7.0 or less.

[0042] When the enzyme is added as the second solution, the second solution may further contain an antibody against the target of detection. In this flow, the antibody is contained in the second solution together with the enzyme derived from E. coli. By adding the enzyme and the sample to the sample at the same time as the second solution, deconjugation by the enzyme derived from E. coli and binding of the antibody to the target of measurement can be performed simultaneously, and the time required to measure the target of measurement can be shortened.

[0043] The antibody against the detection target may include any of antibodies against steroid hormones, flavonoids, or metabolites of steroid hormones or flavonoids. This allows steroid hormones, flavonoids, or metabolites of steroid hormones or flavonoids to be the detection target. The detection target may include any of isoflavones or metabolites of isoflavones. This allows isoflavones or metabolites of isoflavones to be the detection target. The antibody against the detection target may include any of daidzein antibodies, genistein antibodies, glycitein antibodies, or equol antibodies. This allows daidzein, genistein, glycitein, or equol to be the detection target.

[0044] The second solution may be prepared so as to contain the enzyme and antibody at the desired concentrations. The second solution may be prepared by mixing an enzyme solution obtained by diluting the stock solution to 1% with a 0.2 mg / ml antibody solution in a 1:1 volume ratio. The solvent for the enzyme solution may contain 30 mM phosphate, 450 mM NaCl, and 0.015% Tween (registered trademark) 20, and may have a pH of about 6.4 to 6.6. The solvent for the antibody solution may contain 10 mM phosphate, 150 mM NaCl, and 0.005% Tween (registered trademark) 20, and may have a pH of about 7.0 to 7.7.

[0045] (Step S3) After step S2, a second solution containing an enzyme derived from E. coli that deconjugates the conjugate is added to the prepared specimen to prepare a first solution (step S3: second process). The first solution means a solution containing at least the specimen and the enzyme derived from E. coli.

[0046] The enzyme added to the sample may be an enzyme having β-glucuronidase activity. The sulfatase activity of the enzyme may be lower than that of the β-glucuronidase derived from an organism or an organ. The β-glucuronidase derived from an organism or an organ may be one selected from the β-glucuronidases of Helix pomatia, Helix aspersa, Patella Vulgata, and bovine liver.

[0047] As an enzyme having β-glucuronidase activity but low sulfatase activity, for example, an enzyme derived from Escherichia coli can be used. In the following, an example will be described in which β-glucuronidase derived from Escherichia coli is used as the enzyme.

[0048] The pH of the first solution containing the sample and the E. coli-derived enzyme may be 6.0 or more and 7.5 or less. E. coli-derived enzymes such as β-glucuronidase are highly active in an environment of pH 6.0 or more and 7.5 or less. Therefore, by setting the pH of the first solution after preparation to 6.0 or more and 7.5 or less, the β-glucuronidase activity of the E. coli-derived enzyme contained in the first solution can be increased. In other words, by setting the pH of the first solution to 6.0 or more and 7.5 or less, the time required for deconjugation of the conjugate to be detected can be further shortened.

[0049] The method for adjusting the pH of the first solution is not particularly limited. For example, an enzyme derived from Escherichia coli may be added to a specimen as a second solution containing the enzyme, and then an acidic or basic solution may be added to adjust the pH to 6.0 to 7.5 to prepare the first solution. At this time, a buffer may be added as the acidic or basic solution. The buffer may be, for example, a phosphate buffer, a HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, or a Tris (tris (hydroxymethyl) aminomethane) buffer.

[0050] Alternatively, for example, an enzyme derived from Escherichia coli may be added to a specimen as a second solution that is a buffer containing the enzyme, so that the pH of the first solution is adjusted to 6.0 or more and 7.5 or less. If the second solution is a buffer, the pH of the first solution when the second solution is added to the specimen can be more easily adjusted to a range that enhances β-glucuronidase activity. If the second solution is a buffer, the second solution may be, for example, a phosphate buffer, a HEPES buffer, or a Tris buffer. In this case, the pH of the second solution that is a buffer may be 6.0 or more and 7.5 or less.

[0051] Step S3 (the second step) may be performed using a previously prepared reagent kit. As an example, the reagent kit used in step S3 includes an enzyme derived from Escherichia coli that deconjugates the conjugate to be detected, and an antibody against the conjugate to be measured.

[0052] The reagent kit may contain an enzyme derived from Escherichia coli as a second solution having a pH of 6.0 to 7.5 inclusive. Furthermore, the reagent kit may contain an antibody against the detection target as the second solution.

[0053] FIG. 6 is a schematic diagram showing a conventional method for measuring a target substance. Conventionally, when deconjugating a target substance in a sample, deconjugation of both glucuronide conjugates and sulfate conjugates was performed. For this reason, it was necessary to use an enzyme having both β-glucuronidase and sulfatase activities and high activity at about pH 5. Since the antibody reaction for detecting the target substance is performed at about pH 6.0 to 7.5, the conventional method required a two-step procedure of performing a deconjugation reaction, adjusting the pH, and then performing the antibody reaction.

[0054] Specifically, as shown in Fig. 6, solution S containing enzyme Y2 is added to solution P containing a specimen, and a deconjugation reaction is carried out at about pH 5. After that, solution U containing antibody Z is added to the solution after the deconjugation reaction, and an antibody reaction is carried out at about pH 6.0 to 7.5, thereby obtaining solution V containing antibody Z2 resulting from the reaction between the detection target X and antibody Z, and unreacted antibody Z3.

[0055] In addition, the deconjugation reaction using enzyme Y2, which has both β-glucuronidase and sulfatase activities, took approximately 30 minutes.

[0056] As described above, it has been found that the enzyme used in the deconjugation reaction for detecting the target substance only needs to have β-glucuronidase activity, and does not need to have sulfatase activity.

[0057] In the detection method according to the present disclosure, an enzyme that has β-glucuronidase activity but no sulfatase activity and is highly active at around pH 6.0 to 7.5 can be used. Therefore, in this method, by adding the enzyme and the antibody to the sample, it is possible to carry out the deconjugation reaction and the antibody reaction together in one step.

[0058] In addition, when an enzyme having β-glucuronidase activity but not sulfatase activity is used, the time required for the deconjugation reaction is shorter than that required in the past. For example, when β-glucuronidase derived from Escherichia coli is used as the enzyme, the time required for the deconjugation reaction is about 3 minutes.

[0059] (Step S4) In step S4, prior to step S5 (the third step), the first solution is held at a first temperature for a first time. The first time may be 1 minute or more and 5 minutes or less. The first temperature may be 20° C. or more and 40° C. or less.

[0060] By performing step S4 before step S5, which is the detection step, the deconjugation reaction can be allowed to proceed, and by performing measurements while the deconjugation reaction is allowed to proceed, the reproducibility of the measurements can be improved. In addition, by setting the first time to 1 to 5 minutes, the deconjugation reaction can be allowed to proceed, improving the reproducibility of the measurements, and the tests can be performed quickly.

[0061] (Step S5) Step S5 (third step) may be performed by directly using the first solution prepared in step S3 (second step) and incubated in step S4. Step S5 may be performed by using a sensor device 1 including a sensor 2, and a substance that reacts with an antibody against the target substance may be immobilized on the surface of the detection unit 23 of the sensor 2. The sensor device 1 may be a device including a sensor 2 that uses a surface acoustic wave (SAW), a quartz crystal microbalance (QCM), a surface plasmon resonance (SPR), or a field effect transistor (FET). Alternatively, step S5 may be performed by using a device capable of detecting the target substance by using immunochromatography or an enzyme-linked immunosorbent assay (ELISA).

[0062] By using the sensor device 1 as described above, the target substance contained in the first solution can be detected.

[0063] Specifically, a first solution is introduced onto the detection unit 23 of the sensor device 1. When the first solution is introduced, unreacted antibodies contained in the first solution react with the substance A immobilized on the detection unit 23. The sensor 2 outputs a signal generated by the reaction of the substance A in the detection unit 23 to the control unit 3.

[0064] The control unit 3 obtains a signal value from the sensor 2 according to the amount of the substance that has reacted with the antibody. After the first solution is introduced into the sensor 2, the control unit 3 measures the amount of change in the signal value per unit time, and measures the amount of the antibody that has reacted with the substance based on the amount of change. The time for measuring the amount of change may be, for example, 3 to 5 minutes. The antibody that has reacted with the substance is an antibody that has not reacted with the detection target. Therefore, the control unit 3 can measure the amount of unreacted antibody based on the amount of change in the signal value.

[0065] Since the concentration of the antibody contained in the first solution is known, the control unit 3 can measure the amount of the antibody that has reacted with the analyte by measuring the amount of unreacted antibody. Also, the control unit 3 can measure the concentration of the analyte that has reacted with the antibody, i.e., the analyte contained in the first solution, by measuring the amount of the antibody that has reacted with the analyte.

[0066] In this manner, the concentration of the detection target contained in the sample is measured using the sensor device 1.

[0067] Step S5 (third step) may be performed using a sensor device 1 capable of detecting the target substance. In the sensor device 1, an antibody that reacts with the target substance may be immobilized on the surface of the detection unit 23 of the sensor 2. In this case, the control unit 3 of the sensor device 1 may directly measure the concentration of the target substance.

[0068] Specifically, an equol antibody, which is an antibody against equol to be measured, is immobilized in advance on detection section 23 on the surface of sensor 2 of sensor device 1.

[0069] Next, a solution containing the enzyme at a desired concentration is prepared using the enzyme solution and a buffer solution with a pH of 6.0 to 7.5. A sample containing the target substance is mixed with the prepared solution to cause a deconjugation reaction. That is, when the step 5 is performed using a sensor device 1 capable of detecting the target substance, a second solution not containing an antibody is prepared in step S2.

[0070] For example, one to five minutes after the solution and the specimen are mixed, the mixture is introduced onto the detection section of the sensor 2 of the sensor device 1. Then, the object to be measured binds to the antibody immobilized on the detection section .

[0071] After the mixed liquid has been introduced, for example, 3 to 5 minutes later, controller 3 measures the amount of change in the signal value output from sensor 2 and calculates the concentration of equol, the detection target contained in the sample.

[0072] Even with the above-mentioned method, it is possible to measure the concentration of the detection target.

[0073] <Reagent manufacturing method> Hereinafter, a method for producing the reagent (second solution) used in the above-mentioned detection method will be described.

[0074] A method for producing a reagent according to the present disclosure includes a first preparation step of preparing a first enzyme solution, which is a solution containing an enzyme derived from Escherichia coli that deconjugates a conjugate to be detected, and a second preparation step of adding an antibody to be detected to the first enzyme solution to prepare a second enzyme solution.

[0075] In the first preparation step, a stock solution containing an enzyme may be diluted with a solvent to 1% to prepare a first enzyme solution. The solvent for the first enzyme solution may contain 30 mM phosphate, 450 mM NaCl, and 0.015% Tween (registered trademark) 20, and may have a pH of about 6.4 to 6.6.

[0076] In the second preparation step, a second enzyme solution may be prepared by adding a solution containing an antibody to the first enzyme solution. For example, the second enzyme solution may be prepared by mixing the first enzyme solution with a 0.2 mg / ml antibody solution in a volume ratio of 1:1. The solvent for the antibody solution may contain 10 mM phosphate, 150 mM NaCl, and 0.005% Tween (registered trademark) 20, and the pH may be about 7.0 to 7.7.

[0077] The method may further include a third preparation step of adding a buffer to the second enzyme solution to prepare a second solution having a pH of 6.0 to 7.5. The buffer may be, for example, a phosphate buffer, a HEPES buffer, or a Tris buffer.

[0078] In this manner, the second solution is prepared. The prepared second solution is added to a specimen to prepare the first solution used in measuring the target substance.

[0079] 〔summary〕 As described above, the detection method according to aspect 1 of the present disclosure includes a first step of preparing a sample containing a conjugate to be detected, and a second step of adding an enzyme derived from Escherichia coli that deconjugates the conjugate to the sample to prepare a first solution.

[0080] In the detection method according to aspect 2 of the present disclosure, in the above aspect 1, the conjugate may include at least one of a glucuronide conjugate of the detection target and a sulfate conjugate of the detection target.

[0081] In the detection method according to aspect 3 of the present disclosure, in aspect 1 or 2, the enzyme may have β-glucuronidase activity.

[0082] In the detection method according to aspect 4 of the present disclosure, in any one of aspects 1 to 3, the sulfatase activity of the enzyme may be lower than the sulfatase activity of β-glucuronidase derived from one organism or organ selected from Helix pomatia, Helix aspersa, Patella Vulgata, and bovine liver.

[0083] In the detection method according to aspect 5 of the present disclosure, in any one of aspects 1 to 4, the first solution may have a pH of 6.0 or higher and 7.5 or lower.

[0084] In a detection method according to aspect 6 of the present disclosure, in any one of aspects 1 to 5, the enzyme is added as a second solution that contains the enzyme, and the pH of the second solution may be 6.0 or more and 7.5 or less.

[0085] In the detection method according to aspect 7 of the present disclosure, in the above aspect 6, the second solution may be a buffer solution.

[0086] In a detection method according to aspect 8 of the present disclosure, in any one of aspects 1 to 7, the enzyme is added as a second solution that is a solution containing the enzyme, and the second solution may further contain an antibody against the target substance.

[0087] A detection method according to a ninth aspect of the present disclosure, in the eighth aspect, may further comprise an antibody against a steroid hormone, a flavonoid, or a metabolite of a steroid hormone or a flavonoid.

[0088] The detection method according to aspect 10 of the present disclosure, in any one of aspects 1 to 9, further comprises a third step of detecting the target substance, and the third step may be performed using the first solution as it is.

[0089] The detection method according to embodiment 11 of the present disclosure, in any one of embodiments 1 to 10, includes a third step of detecting the target substance, and the third step may be performed by utilizing immunochromatography, an Enzyme-Linked Immuno Sorbent Assay (ELISA) method, or a surface acoustic wave (SAW), a Quartz Crystal Microbalance (QCM), a Surface Plasmon Resonance (SPR), or a Field Effect Transistor (FET).

[0090] A detection method according to aspect 12 of the present disclosure, in any one of aspects 1 to 11, includes a third step of detecting the target substance, the third step being carried out using a sensor device equipped with a detection unit, and a substance that reacts with an antibody against the target substance may be located on the surface of the detection unit.

[0091] A detection method according to aspect 13 of the present disclosure, in any one of aspects 1 to 12, includes a third step of detecting the target substance, the third step being carried out using a sensor device equipped with a detection unit, and an antibody that reacts with the target substance may be located on the surface of the detection unit.

[0092] The detection method according to aspect 14 of the present disclosure, in accordance with aspect 6, may further include a fourth step of preparing the second solution before the second step.

[0093] In the detection method according to aspect 15 of the present disclosure, in either aspect 12 or 13, the first solution may be held at the first temperature for a first time prior to the third step.

[0094] In the detection method according to aspect 16 of the present disclosure, in aspect 15, the first time period may be 1 minute or more and 5 minutes or less.

[0095] In the detection method according to aspect 16 of the present disclosure, in either aspect 15 or 16, the first temperature may be 20° C. or higher and 40° C. or lower.

[0096] A detection method according to an eighteenth aspect of the present disclosure is the method of any one of the first to seventeenth aspects, wherein the detection target is any one of a steroid hormone, a flavonoid, or a metabolite of a steroid hormone or a flavonoid.

[0097] A detection method according to a nineteenth aspect of the present disclosure is the method according to any one of the first to eighteenth aspects, wherein the sample is urine or blood.

[0098] A reagent kit according to embodiment 20 of the present disclosure includes an enzyme derived from Escherichia coli that deconjugates a conjugate to be detected, and an antibody against the conjugate to be detected.

[0099] The reagent kit according to Aspect 21 of the present disclosure, in Aspect 20, may further comprise the enzyme as a second solution having a pH of 6.0 or higher and 7.5 or lower.

[0100] The reagent kit according to Aspect 22 of the present disclosure is the same as Aspect 21, and may contain the antibody as the second solution.

[0101] A method for producing a reagent according to aspect 23 of the present disclosure includes a first preparation step of preparing a first enzyme solution, which is a solution containing an enzyme derived from Escherichia coli that deconjugates a conjugate to be detected, and a second preparation step of adding an antibody to be detected to the first enzyme solution to prepare a second enzyme solution.

[0102] The method for producing a reagent according to aspect 24 of the present disclosure, in accordance with aspect 23, may further include a third preparation step of adding a buffer solution to the second enzyme solution to prepare a second solution having a pH of 6.0 or more and 7.5 or less. [Explanation of symbols]

[0103] 1 Sensor device 2 Sensors 23 Detection unit P specimen Q 2nd solution R 1st solution S1 1st process S2 Project 4 S3 Project 2 S4 Project 3 X indicates the target Y enzyme Z Antibody

Claims

1. A first step of preparing a sample containing a conjugate to be detected; a second step of adding an enzyme derived from Escherichia coli that deconjugates the conjugate to the sample to prepare a first solution.

2. The detection method according to claim 1 , wherein the conjugate includes at least one of a glucuronide conjugate of the target substance and a sulfate conjugate of the target substance.

3. The method according to claim 1, wherein the enzyme has β-glucuronidase activity.

4. The method according to claim 1, wherein the sulfatase activity of the enzyme is lower than the sulfatase activity of β-glucuronidase derived from one organism or organ selected from Helix pomatia, Helix aspersa, Patella Vulgata, and bovine liver.

5. The detection method according to claim 1 , wherein the pH of the first solution is 6.0 or more and 7.5 or less.

6. The enzyme is added as a second solution, the second solution being a solution containing the enzyme; The detection method according to claim 1 , wherein the pH of the second solution is 6.0 or more and 7.5 or less.

7. The method of claim 6 , wherein the second solution is a buffer solution.

8. The enzyme is added as a second solution, the second solution being a solution containing the enzyme; The detection method according to claim 1 , wherein the second solution further contains an antibody against the target substance.

9. The method of claim 8 , wherein the antibody comprises an antibody against a steroid hormone, a flavonoid, or a metabolite of a steroid hormone or a flavonoid.

10. A third step of detecting the detection target, The detection method according to claim 1 , wherein the third step is carried out using the first solution as is.

11. A third step of detecting the detection target, 2. The detection method according to claim 1, wherein the third step is carried out by utilizing immunochromatography, an Enzyme-Linked Immuno Sorbent Assay (ELISA), or a surface acoustic wave (SAW), a Quartz Crystal Microbalance (QCM), a Surface Plasmon Resonance (SPR), or a Field Effect Transistor (FET).

12. A third step of detecting the detection target, The third step is carried out using a sensor device including a detection unit, The detection method according to claim 1 , wherein a substance that reacts with an antibody against the detection target is located on the surface of the detection section.

13. A third step of detecting the detection target, The third step is carried out using a sensor device including a detection unit, The detection method according to claim 1 , wherein an antibody that reacts with the detection target is located on the surface of the detection section.

14. The detection method according to claim 6 , further comprising a fourth step of preparing the second solution before the second step.

15. 13. The method of claim 12, wherein prior to the third step, the first solution is maintained at the first temperature for a first period of time.

16. The method of claim 15, wherein the first time period is greater than or equal to 1 minute and less than or equal to 5 minutes.

17. The detection method according to claim 15 , wherein the first temperature is equal to or higher than 20° C. and equal to or lower than 40° C.

18. The detection method according to claim 1 , wherein the detection target is any one of a steroid hormone, a flavonoid, and a metabolite of a steroid hormone or a flavonoid.

19. The detection method according to claim 1 , wherein the sample is urine, saliva or blood.

20. an enzyme derived from Escherichia coli that deconjugates the conjugate to be detected; and an antibody against the target substance.

21. The reagent kit according to claim 20 , comprising the enzyme as a second solution having a pH of 6.0 to 7.

5.

22. 22. The reagent kit of claim 21, comprising the antibody as the second solution.

23. a first preparation step of preparing a first enzyme solution which is a solution containing an enzyme derived from Escherichia coli that deconjugates the conjugate to be detected; and a second preparation step of adding the antibody to be detected to the first enzyme solution to prepare a second enzyme solution.

24. The method for producing a reagent according to claim 23, further comprising a third preparation step of adding a buffer solution to the second enzyme solution to prepare a second solution having a pH of 6.0 to 7.5.

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