JP5319651B2 - Analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing physiological active substance to be measured with high accuracy without performing a complex pretreatment on a biological sample. <P>SOLUTION: An analyzing method includes the steps of: forming a removed sample by removing only physiological active substance to be measured from a biological sample; forming an analytical curve (reference curve) by analyzing density of physiological active substance of sample reference solution obtained by adding known density of the physiological active substance to the removed sample in accordance with a predetermined analyzing method; and then analyzing the density of the physiological active substance in the biological sample in accordance with the analyzing method using the analytical curve. <P>COPYRIGHT: (C)2012,JPO&amp;INPIT

Description

  The present invention relates to an analysis method for analyzing the concentration of a physiologically active substance such as cortisol in a specimen.

  For example, cortisol is one of corticosteroids and is contained in blood, urine, saliva and the like. Changes in the concentration of cortisol in these are considered to be related to the stress response and are recognized as typical stress biomarkers.

  A typical quantitative analysis method for such biologically active substances in biological samples such as blood, urine, and saliva includes separation by liquid chromatography (Liquid Chromatography: LC) and mass spectrometry (MS). There is LC / MS to detect in In addition, the components separated by LC (column) are ionized by MS in the first stage, divided by mass, and further, the separated ions collide with an inert gas to be dissociated, and the dissociated ions are in the second stage. There is LC / MS / MS which analyzes with MS. In these LCs, so-called high performance liquid chromatography (HPLC) is used.

  Radioimmunoassay (radioimmunoassay: RIA) and enzyme area assay (ELISA) are also used for the analysis of physiologically active substances such as cortisol.

  In the analysis using the liquid chromatography described above, high measurement accuracy can be obtained even in an extremely low concentration region. However, in order to detect with high sensitivity, there are various types in biological samples other than the physiologically active substance to be analyzed. There is a great deal of interference from these substances (bioactive substances). For example, as is well known, in a column intended for separation of a target substance, the separation performance deteriorates due to the presence of a substance having a large molecular weight different from that of the substance. For this reason, in LC / MS and LC / MS / MS, various pretreatments are usually performed to remove these interference components.

  Here, in this type of analysis (quantification), measurement is performed using a sample standard solution with a known concentration, a calibration curve indicating the correlation between the standard substance and measurement data for the standard substance is created, and the actual sample is obtained. It is used for measurement. Since LC / MS and LC / MS / MS are pretreated as described above, the sample standard solution used as a reference for preparing a calibration curve is an ideal solution that does not contain interfering components. Should be used.

  On the other hand, RIA and ELISA using antigen-antibody reaction are less susceptible to interference by substances other than the target physiologically active substance to be analyzed, and therefore can be analyzed to some degree with high sensitivity without requiring much complicated pretreatment. It is a feature. If biological samples such as blood, urine, and saliva can be quickly analyzed without pretreatment, it is very promising as a real-time analysis or on-site analysis technique.

M. Groschl, "Current Status of Salivary Hormone Analysis", Clinical Chemistry, vol.54, no.11, pp1759-1769, 2008. Shuhei Izawa, Katsuhiko Suzuki, “Comparison of Salivary Cortisol Measurement Kits—Correlation between Salivary and Plasma Cortisol and Comparison between Measurement Methods”, Journal of Complementary and Alternative Medicine, Vol. 4, No. 3, 113- 118, 2007. U. Erdbruegger et al., "Circulating endothelial cells: A novel marker of endothelial damage", Clinica Chimica Acta, vol.373, pp.17-26, 2006. K. Aoki et al., "Quantitative analysis of reversible diffusion-controlled currents of redox soluble species at interdigitated array electrodes under steady-state conditions", J. Electroanal. Chem., Vol.256, pp.269-282, 1988.

  However, even in RIA and ELISA, when a biologically active substance is analyzed (quantified) with high accuracy like LC / MS or LC / MS / MS, substances other than the target physiologically active substance in biological samples (interfering components) ) Cannot be ignored. Here, a calibration curve is also used in analysis methods such as RIA and ELISA. However, if a calibration curve using an ideal sample standard solution that does not contain interfering components is used, there is no influence of interfering ions in the measurement of such sample standard solution. There is a problem that measurement error increases.

  The present invention has been made to solve the above-described problems, and is intended to enable analysis of a target physiologically active substance with high accuracy without performing complicated pretreatment on a biological sample. Objective.

  The analysis method according to the present invention includes a first step of preparing a removed sample from which only a physiologically active substance to be measured is removed from a biological sample, and a physiological activity of a sample standard solution in which a physiologically active substance having a known concentration is added to the removed sample. A second step of preparing a calibration curve by analyzing the concentration of the substance by a predetermined analysis method and a third step of analyzing the concentration of the physiologically active substance in the biological sample by using the calibration curve by the analysis method.

  In the analysis method described above, in the first step, after mixing magnetic beads modified with protein G and antibodies of biologically active substances into a part of the biological sample, the magnetic beads are adsorbed to a magnet, thereby a part of the biological sample. Only the physiologically active substance can be removed. In this case, in the first step, the magnetic beads modified with protein G adsorbed by the antibody are mixed with a part of the biological sample, and then the magnetic beads are adsorbed to the magnet, so that the physiological beads are more physiological. Only the active substance needs to be removed. The physiologically active substance is, for example, cortisol.

  As described above, according to the present invention, since a calibration curve is prepared using a removed sample from which only a physiologically active substance to be measured is removed from a biological sample, complicated preprocessing for the biological sample is performed. Therefore, it is possible to obtain an excellent effect that a physiologically active substance can be analyzed with high accuracy.

FIG. 1 is a flowchart for explaining an analysis method according to an embodiment of the present invention. FIG. 2 is a flowchart for explaining in detail the analysis method according to the embodiment of the present invention. 3 is a characteristic diagram showing a calibration curve prepared from the sample standard solution, the reference standard solution A, and the reference standard solution B in Example 1. FIG. FIG. 4 shows the cortisol concentration analysis results of saliva samples S1, S2, and S3 using the calibration curve shown in FIG. 3, and the cortisol concentration analysis results of saliva samples S1, S2, and S3 using LC / MS / MS. FIG. FIG. 5 is a configuration diagram illustrating a configuration of a measurement apparatus that performs electrochemical measurement. FIG. 6 is a perspective view showing the configuration of the electrode tip 504. FIG. 7 is a characteristic diagram showing a calibration curve prepared from the sample standard solution and the reference standard solution A in Example 2. FIG. 8 shows the cortisol concentration analysis results of saliva samples S1, S2, and S3 using the calibration curve shown in FIG. 7, and the cortisol concentration analysis results of saliva samples S1, S2, and S3 using LC / MS / MS. FIG.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a flowchart for explaining an analysis method according to an embodiment of the present invention. In this analysis method, first, in step S101, a removed sample is prepared by removing only the physiologically active substance to be measured from the biological sample. Next, in step S102, a calibration curve (standard curve) is created by analyzing the concentration of the physiologically active substance in the sample standard solution obtained by adding the above-mentioned physiologically active substance having a known concentration to the removed sample by a predetermined analysis method. Thereafter, in step S103, the concentration of the physiologically active substance in the biological sample is analyzed using the calibration curve by the above analysis method. In step S101, a part of the biological sample is collected and used, and in step S103, actual measurement may be performed using the remaining biological sample.

  In the present embodiment described above, the sample standard solution and the biological sample are in the same state with components other than the physiologically active substance to be measured. For this reason, in the analysis at the time of creating a calibration curve and the analysis of an actual biological sample, the influence from substances other than the physiologically active substance to be measured is equivalent. In other words, if there is a difference between the analysis when creating the calibration curve and the actual analysis of the biological sample, only the concentration of the physiologically active substance to be measured is obtained. Therefore, according to the present embodiment, since the concentration of the target physiologically active substance can be measured in a state in which the influence of the substance other than the target physiologically active substance is excluded, a highly accurate analysis can be performed. In addition, preparation of a sample standard solution used for preparing a calibration curve is easy without requiring complicated processing because it is sufficient to remove a physiologically active substance to be measured, which is a known component.

  In the present embodiment, in the actual analysis of the biological sample, it is not necessary to pre-process the biological sample. For example, as pretreatment, generally, other physiologically active substances that interfere with detection of a physiologically active substance to be measured are removed from a biological sample. However, it is not clear what interfering components are contained in the biological sample, and it is not easy to remove all interfering components from the biological sample. On the other hand, in the present embodiment, analysis can be performed more easily because the analysis can be performed without removing the disturbing component that is an unknown component.

  Next, the analysis method in the present embodiment will be described in more detail using the flowchart of FIG.

  First, in step S201, magnetic beads modified with protein G are prepared. As protein G magnetic beads, commercially available “Dynabeads Protein G” (manufactured by DYNAL) may be used. Further, the target bioactive substance antibody is modified on the magnetic beads modified with protein G. For example, when the target physiologically active substance is cortisol, the cortisol antibody to be used may be “Cortisol antibody [XM210]” (ab1949; manufactured by Abcam).

  For example, 0.2 to 1 μg of cortisol antibody may be mixed with 50 μL of magnetic beads modified with protein G. Mixing may be performed by inversion mixing at room temperature (about 20 ° C.) for about 10 to 30 minutes.

  Next, in step S202, a part of the biological sample is fractionated, and magnetic beads in which an antibody of a physiologically active substance to be measured (for example, cortisol antibody) is modified via protein G are applied to the fractionated sample. Mix. For example, the magnetic beads may be mixed with the sample sample and mixed by inversion for about 10 to 60 minutes at room temperature.

  In the above-described inversion mixing treatment, if the treatment time is too long, the concentration of the antibody used is too high, and the binding of the antibody to protein G modified on the magnetic beads is weak, the target There is a possibility that the antibody bound with the physiologically active substance to be separated from the magnetic beads. In such a case, a magnetic bead modified with protein G may be newly added and, for example, mixed by inverting at room temperature for about 10 to 60 minutes. By this addition, the floating antibody can be collected, so that the target physiologically active substance in the sample can be more reliably removed. For example, in the case of a cortisol antibody, as an example of the mixing ratio of the magnetic beads to which the antibody is not fixed and the biological sample, the condition of mixing 100 to 150 μL of the biological sample per 20 μL of the magnetic beads is preferable.

  Next, in step S203, the magnetic beads are separated from the preparative sample. For example, a magnet is placed on the outside (bottom) of the container containing the sample, and the magnetic beads are drawn to the magnet position to be collected and fractionated. Just take it out. When this method for removing a physiologically active substance is applied to cortisol, the concentration of cortisol remaining in the preparative sample from which cortisol has been removed is analyzed by LC / MS / MS, and is 0.03 ng / mL or less. It was confirmed that the lower limit of quantification of cortisol by LC / MS / MS was 0.03 ng / mL. Thus, it has been confirmed that the above-mentioned cortisol removal using magnetic beads is effective.

  In step S201, magnetic beads modified with protein G are prepared (manufactured), and in step S202, magnetic beads modified with protein G and antibodies are added to the sample to be mixed. Also good. In any case, the target physiologically active substance can be selectively removed from the biological sample by using an antibody against the physiologically active substance. In other words, any physiologically active substance in which an antibody exists can be selectively removed from a biological sample.

  Next, in step S204, a sample standard solution is prepared by adding a physiologically active substance with a known concentration to the removed sample from which the physiologically active substance has been removed. For example, when the physiologically active substance is cortisol, a cortisol buffer solution in which cortisol is dissolved in a phosphate buffer or a carbonate buffer may be added to the removed sample to obtain a sample standard solution. In this case, in order to maintain the characteristics of the biological sample, the amount of the buffer solution with respect to the removed sample is preferably 1/1000 or less. Further, the sample standard solution may be prepared in a total amount of about 100 to 500 μL as a sufficient amount for preparing a calibration curve. Moreover, when a biological sample is saliva, it is good to produce a some sample standard solution so that the cortisol density | concentration in a sample standard solution may be 3-10 types within the range of 0-80 ng / mL.

  Next, in step S205, the concentrations of a plurality of sample standard solutions each having a different concentration of physiologically active substance prepared as described above are analyzed using a predetermined analysis method. The analysis method may be RIA, ELISA, electrochemical detection method, or the like from the point that pretreatment of a biological sample is unnecessary.

  For example, in the analysis of cortisol by ELISA, enzyme-labeled cortisol and a sample standard solution are competitively reacted on a plate on which a cortisol antibody is immobilized, and color development due to the reaction between the adsorbed enzyme-labeled cortisol and the substrate is detected by absorbance measurement. An example of a combination of enzyme and substrate is first horseradish peroxidase (HRP) and 3,3 ', 5,5'-trimethylbenzidine (TMB) (combination A). There is also a combination of alkaline phosphatase (ALP) and p-nitrophenyl phosphate (pNPP) (combination B). In the case of combination A, 450 nm absorption of TMB oxide is used as a detection signal. In the case of the combination B, the absorption at 405 nm of pNP (p-nitrophenol) is used as a detection signal.

  In the analysis of cortisol by electrochemical detection, enzyme-labeled cortisol and sample standard solution are subjected to a competitive reaction on a plate to which a cortisol antibody is immobilized, and a substance produced by the reaction between the adsorbed enzyme-labeled cortisol and a substrate is oxidized. Detect by reduction reaction. As an example of a combination of an enzyme and a substrate, there is a combination of alkaline phosphatase (ALP) and p-aminophenyl phosphate (pAPP) in addition to the above two combinations used in ELISA (Combination C). There is also a combination of acetylcholinesterase (AChE) and acetylthiocholine (ATCh) (combination D).

  In the case of the combination A, the oxidation reaction of unreacted TMB (reduced form of TMB) is measured on the electrode, and the response current value is used as a detection signal. In the case of the combination C, the oxidation reaction of thiocholine as a reaction product is measured on the electrode, and the response current value is used as a detection signal.

  In the case of the combination B, the oxidation reaction of the reaction product pNP and the reaction product is measured on the electrode, and the response current value is used as the detection signal. In the case of the combination D, the oxidation reaction of pAP (p-aminophenol) as a reaction product is measured on the electrode, and the response current value is used as a detection signal.

  Furthermore, the amplification effect by a redox cycle can also be utilized using a comb-shaped electrode etc. (refer nonpatent literature 4). In this case, the reduction reaction of pNP and pAP can be measured on the electrode, and the response current value can be used as a detection signal. When the response current value of the reduction reaction is used as the detection signal, the current value becomes smaller than that of the oxidation reaction due to the efficiency of capturing and reacting the oxidant with the reduction side electrode. However, in this case, since responses from substances other than pNP and pAP hardly contribute, there is a feature that the quantitative accuracy is improved as compared with the case where the response current value of the oxidation reaction is used as a detection signal.

  In any analysis method, in order to further improve the accuracy of the calibration curve, it is preferable to perform two or more sets of measurements to obtain an average value and a measurement error.

  Next, in step S206, a calibration curve is created based on the combination (correlation) of the measured value obtained as a result of the above-described analysis and the known concentration value. The calibration curve is a graph in which the concentration value is shown corresponding to the measured value. A regression analysis is performed on the measured value obtained in the concentration range of a predetermined physiologically active substance, and the result may be used as a calibration curve. For example, in the case of ELISA, a calibration curve can be obtained by regression fitting using a 4-parameter logistic function, exponential decay function, polynomial function, etc. in the range of 0 to 80 ng / mL.

  Next, in step S207, the biological sample is measured by the analysis method used in the calibration curve creation. In this analysis, measurement is performed without pretreatment.

  Finally, in step S208, a concentration value corresponding to the measurement value obtained in the measurement in step S207 is obtained (calculated) using a calibration curve.

  Hereinafter, it demonstrates in detail using an Example. Hereinafter, cortisol will be described as an example of a physiologically active substance to be measured.

[Example 1]
First, Example 1 will be described.

  First, as biological samples, saliva samples within 30 minutes after waking up were individually collected from several adult men and women. 2-3 mL of each person's saliva was directly collected in a test tube, stored frozen immediately after collection, and thawed before measurement. The collection time was about 5 to 10 minutes, although there were individual differences. Separate these samples for calibration curve creation and concentration measurement, and for calibration curve creation, in order to prevent bias due to individual differences, equal amounts of samples taken from several individuals other than those for concentration measurement They were mixed and used.

  Next, preparation of a sample standard solution that is as close as possible to the properties of a biological sample from the above-described saliva sample will be described. A commercially available magnetic bead “Dynabeads Protein G” modified with protein G (manufactured by DYNAL) is prepared. Next, these magnetic beads were mixed at a ratio of 1 μg of a cortisol antibody solution (ab1949, abcam) per 50 μL of the magnetic beads, and mixed by inverting at room temperature for 30 minutes to modify the cortisol antibody on the magnetic beads.

  120 μL of saliva sample was mixed with 25 μL of magnetic beads modified with this cortisol antibody, and mixed by inverting for 30 minutes at room temperature to remove cortisol in saliva. Magnetic beads in the solution were collected at the bottom using a neodymium magnet from the outside of the bottom of the container, and only the biological sample in the supernatant (top of the container) was collected and used as a removed sample. About this removal sample, residual cortisol quantitative analysis was performed using LC / MS / MS, it confirmed that the cortisol density | concentration was 0.03 ng / mL or less, and confirmed that the process of removing cortisol was effective.

  A known amount of cortisol buffer solution is added to the removed sample from which cortisol has been removed to prepare a sample standard solution with a known cortisol concentration. The cortisol buffer solution is a solution obtained by dissolving hydrocortisone (manufactured by SIGMA, H0135) in a phosphate buffer solution (1 × Dulbecco's PBS) to 1 mg / mL. This cortisol buffer solution was diluted stepwise using the above-described removed sample to prepare seven types of sample standard solutions of 0, 2, 5, 10, 20, 40, and 80 ng / mL. In order to keep the properties of the biological sample as much as possible, the amount of the cortisol buffer solution relative to the removed sample is 1/1000 or less. Moreover, the preparation amount of the sample standard solution of each concentration was set to 300 μL as a sufficient amount for preparing a calibration curve.

  Further, as a reference to the sample standard solution described above, a reference standard solution was prepared by a technique used in the conventional method. First, a phosphate buffer solution (1 × Dullbecco'sPBS; pH 7) is used as a buffer solution, and the above-mentioned cortisol buffer solution is serially diluted to obtain seven types of 0, 2, 5, 10, 20, 40, and 80 ng / mL. Reference standard solution A was prepared. In addition, carbonate buffer (pH 9.6) is used as a buffer, and the above-mentioned cortisol buffer solution is serially diluted to prepare seven reference standard solutions B of 0, 2, 5, 10, 20, 40, and 80 ng / mL. Produced.

  A commercially available ELISA kit (Salivary Cortisol ELISA, SLV-2930, DRG) was used for analysis of cortisol concentration.

  First, a calibration curve was prepared as follows. First, among samples collected from several adult men and women on a plate modified with a cortisol antibody, a pre-treated saliva sample (S1, S2, S3) with an arbitrarily selected cortisol concentration and a sample standard 100 μL each of the solution, reference standard solution A, and reference standard solution B were added, and then 200 μL of enzyme-labeled cortisol (HRP-labeled cortisol) was added to each well for competition reaction. These were placed on a shaker and reacted at room temperature for 1 hour, and then washed 3 times with a washing buffer (400 μL / well). Finally, 100 μL of the substrate solution (TMB solution) was added to each well and allowed to stand at room temperature for 30 minutes to obtain color by reaction between the enzyme and the substrate.

  Thereafter, 100 μL of a reaction stop solution (dilute sulfuric acid) was added to each well, and the absorbance was measured twice with a plate reader at a wavelength of 450 nm to obtain the average value of the degree of color development. Here, as the cortisol concentration in the sample standard solution, reference standard solution A, and reference standard solution B, or saliva sample increases, the amount of the enzyme-labeled cortisol adsorbed to the cortisol antibody decreases, so The amount is also reduced. Therefore, quantitative analysis is possible by converting the decrease in absorbance into the cortisol concentration.

  Next, by fitting the absorbance data obtained from the set of the sample standard solution, the reference standard solution A, and the reference standard solution B using the “two phase exponential decay” function as an exponential decay function, each standard sample set A calibration curve was obtained in a concentration range of 0 to 80 ng / mL of cortisol. These calibration curves are shown in FIG. In FIG. 3, a black circle line is a calibration curve based on the sample standard solution, a black square line is a calibration curve based on the reference standard solution A, and a black triangle line is a calibration curve based on the reference standard solution B.

  As shown in FIG. 3, the calibration curve obtained from the sample standard solution in the present embodiment has an absorbance at the same cortisol concentration than the calibration curve obtained from the reference standard solutions A and B used in the conventional method. Was found to be low.

  Next, the cortisol concentration of saliva samples S1, S2, and S3 was determined using each calibration curve described above. Further, LC / MS / MS quantitative analysis was also performed on the saliva samples S1, S2, and S3. In LC / MS / MS quantitative analysis, each saliva sample is centrifuged to remove the high-molecular component mucin, the supernatant is separated, an internal standard sample is added, and organic solvent extraction is performed. Samples that were purified and separated using pretreatment were prepared, and LC / MS / MS quantitative analysis was performed.

  FIG. 4 shows the cortisol concentration analysis results of saliva samples S1, S2, and S3 using the calibration curves described above, and the cortisol concentration analysis results of saliva samples S1, S2, and S3 using LC / MS / MS. In FIG. 4, (a) is an analysis result using a calibration curve with a sample standard solution, (b) is an analysis result using a calibration curve with a reference standard solution A, and (c) is a reference standard. It is the analysis result using the calibration curve by the solution B, (d) is the analysis result by LC / MS / MS.

  As shown in FIG. 4, the cortisol concentration obtained from the calibration curve of the conventional method (reference standard solutions A and B) is calculated as a higher concentration than the quantitative analysis result by LC / MS / MS at a low concentration. First, in the analysis result (b) using the calibration curve with the reference standard solution A, the concentration is about 1.5 times higher than the analysis result by LC / MS / MS. Further, in the analysis result (c) using the calibration curve by the reference standard solution B, the concentration is about twice as high as the analysis result by LC / MS / MS.

  In contrast to these results, the analysis result (a) of the cortisol concentration in S1, S2, and S3 using the calibration curve of the sample standard solution is close to the analysis result based on the analysis result by LC / MS / MS (error ± 5 %)).

  As described above, by creating a calibration curve in ELISA using a sample standard solution that is as close as possible to the properties of a biological sample, a calibration curve that includes all the effects of substances other than cortisol can be obtained. It was shown that quantitative errors due to the use of the solution can be eliminated. From this example, it was shown that according to the present embodiment, a highly accurate determination method of cortisol in an extremely low concentration region is provided.

[Example 2]
Next, Example 2 will be described.

  A saliva sample as a biological sample was collected in the same manner as in Example 1. In addition, a sample standard solution and a reference standard solution A were prepared in the same manner as in Example 1.

  In Example 2, analysis of salivary cortisol concentration was performed by an electrochemical measurement method for a solution obtained by reacting cortisol with an antigen-antibody. Cortisol antigen-antibody reaction was performed using ELISA plates (maxisorp, NUNC). Cortisol antibody (ab1949, abcam) was adjusted to a concentration of 2.5 μg / mL and allowed to stand overnight at 4 ° C. under the condition of 200 μL / well, and the antibody was immobilized on the plate. Next, after washing 3 times with a washing buffer (400 μL / well), blocking agent (2% Block Ace, manufactured by Snow Brand Milk Products) was left at 37 ° C. under a condition of 350 μL / well for 2 hours to perform blocking. .

  To this plate, 100 μL each of the untreated saliva sample (S1, S2, S3) with unknown cortisol concentration, sample standard solution and reference standard solution A was added, and then the enzyme (ALP) label adjusted to 2 μg / mL 200 μL of cortisol (EC152, Michigan Diagnostis LLC) was added to each well for competition reaction. These were placed on a shaker and reacted at room temperature for 30 minutes, and then washed three times with a washing buffer (400 μL / well).

  Finally, 100 μL of a substrate solution (1 mM p-aminophenyl phosphate sodium salt (Funakoshi), diethanolamine buffer solution or carbonate buffer solution) is added to each well and left at room temperature for 10 minutes. The reaction produced pAP. Each solution was placed in a test tube, and the reacted solution was stored at 4 ° C. until immediately before measurement.

  Here, the electrochemical measurement will be described. The electrochemical measurement in Example 2 is performed using the measuring apparatus shown in FIG. This measuring device includes an electrode chip 504 including a working electrode 501, a counter electrode 502, and a reference electrode 503 having two electrode structures facing each other at a comb tooth portion, a potentiostat 505 and a coulometer 506 to which the respective electrodes are connected. And a recorder 507. The potentiostat 505 controls the current flowing through the working electrode 501 and the counter electrode 502 so that the voltage between the working electrode 501 and the reference electrode 503 becomes a set value, and the coulometer 506 flows into the working electrode 501. Measure the current.

  For example, comb electrodes (gold electrodes having a width of 10 μm and intervals of 5 μm) manufactured by BAS Corporation as shown in the perspective view of FIG. In this electrode chip 504, a working electrode 501, a counter electrode 502, and a reference electrode 503 are arranged on a substrate 500 having a plate thickness of 0.5 mm and 12 × 20 (mm). For example, a sample is dropped from the burette 601 onto the two electrode structure portions facing each other at the comb tooth portion of the working electrode 501.

  In addition, an electrode terminal 501 a and an electrode terminal 502 bee are connected to the working electrode 501, an electrode terminal 502 a is connected to the counter electrode 502, and an electrode terminal 503 is connected to the reference electrode 503.

  By using the working electrode 501 having the two electrode structures facing each other at the comb tooth portion described above, the amplification effect by the well-known redox cycle is utilized, and the redox reaction of pAP is traced by the constant potential method. The pAP oxidized at one oxidation side electrode (+0.25 V) of the working electrode 501 is reduced at the other reduction side (−0.2 V) of the working electrode 501, and redox is repeated at two adjacent electrodes. Current amplification occurs. When the response current value of the reduction reaction is used as a detection signal, the current value is smaller than that of the oxidation reaction due to the efficiency of capturing and reacting the oxidant with the reduction side electrode, but the response from substances other than pAP is Since it hardly contributes, there is a feature that the quantitative accuracy is improved as compared with the case where the response current value of the oxidation reaction is used as the detection signal.

  Here, as the cortisol concentration in the sample standard solution or saliva sample increases, the amount of the enzyme-labeled cortisol adsorbed to the cortisol antibody decreases, so that the amount of pAP produced also decreases. Therefore, the amount of decrease in the response current value (∝pAP concentration) from pAP can be converted into cortisol concentration and quantitative analysis can be performed.

  Calibration in the concentration range of 0-80 ng / mL of cortisol, which differs for each standard sample set, by fitting the current value data obtained from each standard sample set using the “two phase exponential decay” function as the exponential decay function Got a line. These calibration curves are shown in FIG. In FIG. 7, a black circle line is a calibration curve using the sample standard solution, and a black square line is a calibration curve using the reference standard solution A.

  From FIG. 7, the calibration curve prepared from the sample standard solution according to the present embodiment obtained from the sample standard solution as close as possible to the properties of the biological sample has the same cortisol concentration as the calibration curve prepared from the reference standard solution A. It was found that the current value of was high.

  Next, the cortisol concentration of saliva samples S1, S2, and S3 was determined using each calibration curve described above. Further, LC / MS / MS quantitative analysis was also performed on the saliva samples S1, S2, and S3. In LC / MS / MS quantitative analysis, each saliva sample is centrifuged to remove the high-molecular component mucin, the supernatant is separated, an internal standard sample is added, and organic solvent extraction is performed. Samples that were purified and separated using pretreatment were prepared, and LC / MS / MS quantitative analysis was performed.

  FIG. 8 shows the cortisol concentration analysis results of saliva samples S1, S2, and S3 using the calibration curves described above, and the cortisol concentration analysis results of saliva samples S1, S2, and S3 using LC / MS / MS. In FIG. 8, (a) is an analysis result using a calibration curve with a sample standard solution, (b) is an analysis result using a calibration curve with a reference standard solution A, and (c) is LC / It is the analysis result by MS / MS.

  As shown in FIG. 8, the cortisol concentration obtained from the calibration curve of the conventional method (reference standard solution A) is calculated as a concentration several times lower than the quantitative analysis result by LC / MS / MS at a low concentration. On the other hand, it was found that the analysis result (a) of the cortisol concentration in S1, S2, and S3 using the calibration curve by the sample standard solution shows a value close to the analysis result by the analysis result by LC / MS / MS. .

  As described above, a calibration curve that includes all the effects of substances other than cortisol can be obtained by creating a calibration curve in an electrochemical measurement method using a sample standard solution that is as close as possible to the properties of a biological sample. It was shown that quantitative errors caused by using an ideal solution can be eliminated. Thus, according to this Embodiment, it was shown that the highly accurate determination method of cortisol in a very low concentration area | region is provided.

  The present invention is not limited to the embodiment described above, and many modifications and combinations can be implemented by those having ordinary knowledge in the art within the technical idea of the present invention. It is obvious. For example, in the above-described embodiment, for preparing a calibration curve, samples collected from several individuals other than those for concentration measurement are mixed and used in equal amounts. However, the present invention is not limited to this. The sample for concentration measurement may be used as it is for preparing a calibration curve.

  Moreover, in the Example mentioned above, although cortisol was demonstrated to the example as a bioactive substance, it does not restrict to this. For example, analysis of other hormones in saliva such as testosterone, estradiol, progesterone, estrogen, dehydroepiandrosterone sulfate (DHEA-S), and epidermal growth factor (EGF) An effect is obtained. Further, the same effect as described above can be obtained even when analyzing stress marker hormones contained in saliva such as amylase, chromogranin A, and immunoglobulin A (IgA). The same effects as described above can be obtained even when analyzing other hormones including adrenocortical hormones such as aldosterone.

Claims (4)

A first step of preparing a removed sample from which only a physiologically active substance to be measured is removed from a biological sample;
A second step of preparing a calibration curve by analyzing the concentration of the physiologically active substance in a sample standard solution obtained by adding the physiologically active substance of known concentration to the removed sample by a predetermined analysis method;
And a third step of analyzing the concentration of the physiologically active substance in the biological sample using the calibration curve by the analysis method. The analysis method according to claim 1,
In the first step, after mixing magnetic beads modified with protein G and the antibody of the physiologically active substance into a part of the biological sample, the magnetic beads are adsorbed to a magnet, whereby a part of the biological sample is obtained. And removing only the physiologically active substance. The analysis method according to claim 2,
In the first step, after mixing magnetic beads modified with protein G adsorbed with the antibody into a part of the biological sample, the magnetic beads are adsorbed to a magnet, thereby a part of the biological sample And removing only the physiologically active substance. The analysis method according to any one of claims 1 to 3,
The analysis method, wherein the physiologically active substance is cortisol.