WO2015064701A1

WO2015064701A1 - Glycoalbumin measurement kit and measurement method

Info

Publication number: WO2015064701A1
Application number: PCT/JP2014/078925
Authority: WO
Inventors: 淳子田中; 悠石毛; 釜堀　政男; 麦前川; 理子岩田; 中村　英博; 健澤崎
Original assignee: 日立化成株式会社
Priority date: 2013-10-31
Filing date: 2014-10-30
Publication date: 2015-05-07
Also published as: JP2015087293A

Abstract

To measure a glycoalbumin (GA)/albumin ratio easily and precisely, the total amount of albumin (103) and GA (104) trapped using anti-albumin antibodies (102) immobilized on a solid phase (101) is normalized and the amount of GA (104) among albumin (103) and GA (104) trapped using anti-GA antibodies (105) is measured.

Description

Glycoalbumin measurement kit and measurement method

Diabetes is a disease in which the blood sugar level increases due to insufficient insulin action, and it can cause stroke and heart disease, so early diagnosis and treatment are necessary. Conventionally, measurement of blood glucose level has been used for diagnosis of diabetes, but there has been a problem that blood glucose level fluctuates due to meals and drinking. On the other hand, the amount of glycated protein such as hemoglobin A1c (HbA1c) and glycoalbumin (GA) is a stable index reflecting blood glucose level over a longer period (several weeks to several months). Therefore, it is useful for determining the success or failure of blood glucose control.

Since the glycated protein is a glycated specific protein such as hemoglobin or albumin according to the blood glucose level, the abundance ratio of the glycated protein per specific protein reflects the long-term blood glucose level. For example, in the case of HbA1c, the abundance ratio of HbA1c to hemoglobin (HbA1c / hemoglobin ratio), and in the case of GA, the abundance ratio of GA to albumin (GA / albumin ratio) is an indicator of long-term blood glucose level.

Since the period in which the abundance ratio of glycated protein is reflected is determined by the half-life of the glycated protein in the body, it is a value that reflects the change in blood glucose level over the past 1 to 2 months for HbA1c and for the past 2 weeks for GA. Become. For this reason, GA is attracting attention as a marker that shows a glycemic control state in a shorter period of time than HbA1c.

HbA1c was standardized by the International Clinical Chemistry Association (IFCC) and defined as a glycated product of the valine residue at the N-terminal of the β chain of hemoglobin. On the other hand, GA is albumin having a lysine residue to which glucose is bound according to the project of the Japanese Association of Clinical Chemistry, Diabetes Related Indicators Special Committee “Establishment of Standard Method for Glycoalbumin Measurement”. Defined as the molar ratio of groups to albumin. That is, the number of sites for judging saccharification is one in HbA1c, whereas in GA, at least four sites known as main saccharification sites are targeted. Thus, HbA1c and GA are different in definition even though they are glycated proteins.

As a method for measuring GA, the HPLC method (Diabetologia, 31, pp.627-631, 1988) was first developed, and then the enzyme method (JP-A-5-192193) and the immunization method (JP-A-2010-261196). As a result of the development, the reaction can be measured easily and in a shorter time than the previous HPLC method. However, in these methods, the amount of albumin and the amount of GA need to be measured separately and converted into a GA / albumin ratio, which makes the measurement operation complicated. Therefore, albumin can be obtained by adsorbing a certain amount of albumin to a solid phase (Japanese Patent Laid-Open No. 2004-242522) or binding an anti-albumin antibody to a fixed solid phase (Japanese Patent Laid-Open No. 5-87809). A method has been developed that can be used to measure the GA / albumin ratio by standardizing the total amount of the protein without separately measuring it.

JP-A-5-192193 JP 2010-261196 A JP 2004-242522 A Japanese Patent Laid-Open No. 5-87809

As a result of intensive studies on a simple and highly accurate method for measuring the GA / albumin ratio to albumin, it is difficult to control the amount of adsorption by a method of solid-phase adsorption of albumin as disclosed in JP-A-2004-242522. It was difficult to standardize with good reproducibility. In addition, in the method of detecting GA using a molecule that recognizes and detects only a sugar such as a phenylboronic acid derivative as disclosed in JP-A-5-87809, all sugars existing in the reaction system other than GA are also detected. Since it reacts, there is a possibility that an error may occur in GA measurement depending on the specimen.

Therefore, in order to measure the abundance ratio of GA to albumin simply and accurately, the object of the present invention is to 1) normalize the total amount of albumin without separately measuring 2) normalization of the total amount of albumin and GA The purpose is to facilitate control of the measurement system in detection and to reduce factors that lower measurement accuracy.

In order to solve the above problems, the total amount of albumin captured using the anti-albumin antibody is normalized, and the GA amount of the albumin captured using the anti-GA antibody is measured. By reacting the specimen with the anti-albumin antibody immobilized on the solid phase, only albumin in the specimen is captured on the solid phase. At this time, the amount of the anti-albumin antibody immobilized on the solid phase is made smaller than the amount of albumin in the specimen. Of the captured albumin, only GA is detected and quantified with an anti-GA antibody. Furthermore, the amount of captured albumin is measured using an anti-albumin antibody that recognizes an epitope different from the immobilized albumin antibody.

As another means, albumin captured using an anti-albumin antibody is measured by physically adsorbing albumin in a sample solution to a solid phase, first measuring the GA amount of the albumin captured using an anti-GA antibody. Measure the amount.

In order to easily dilute the specimen, a filter for introducing the specimen is installed on the flow path, and the specimen that has been dropped onto the filter and diffused into the solution in the flow path is used as the specimen. Since the present invention is a method for directly determining the abundance ratio of GA to albumin, the measurement value is not greatly affected by the dilution factor, and such a simple dilution method can be measured with sufficient accuracy.

The glycoalbumin measurement kit of the present invention comprises a solid phase provided with an immobilized anti-albumin antibody and an electrode, and an enzyme-labeled anti-glycoalbumin antibody. The electrode is preferably used for potential measurement. The solid phase can have a sample introduction port and a sample holding unit, and the sample holding unit can use any of filter paper, non-woven fabric, and porous fiber. It is preferable that the anti-albumin antibody and the electrode are disposed in the flow path, and the anti-albumin antibody is disposed at least upstream and downstream of the electrode. The solid phase can have a sample inlet connected to the flow path.

The method for measuring glycoalbumin of the present invention uses an anti-albumin antibody and an enzyme-labeled anti-glycoalbumin antibody to quantify the ratio of glycoalbumin to albumin in a sample by potential measurement. In this measurement method, the anti-albumin antibody is an immobilized antibody immobilized on a solid phase, the step of supplying a specimen to the solid phase, the step of binding albumin and glycoalbumin in the specimen to the immobilized antibody, Supplying an enzyme-labeled anti-glycoalbumin antibody to the phase, and binding the enzyme-labeled antibody to glycoalbumin bound to the solid phase to form an immobilized anti-albumin antibody-glycoalbumin-enzyme-labeled anti-glycoalbumin antibody complex A step of supplying a substrate to the solid phase, a step of reacting the substrate with a complex enzyme to generate a reaction product, a step of measuring the concentration of the reaction product by potential measurement, and a concentration of the reaction product in the sample. And a step of converting to the glycoalbumin concentration. Here, it is preferable to use a sample diluted with a diluent as the sample. It is preferable that the dilution rate of dilution is 10 times or more and 1000 times or less. Furthermore, the solid phase has a sample holding unit, and has a step of supplying a diluent to the solid phase after the step of supplying the sample to the solid phase, and the sample held in the sample holding unit is brought into contact with the diluent. In this way, the specimen can be diluted.

In the method of the present invention, albumin is captured with an anti-albumin antibody, and then the amount of albumin captured using an anti-GA antibody is measured. The abundance ratio of GA to albumin can be accurately measured. By using an anti-albumin antibody for capturing albumin, variation in the amount of albumin captured can be suppressed. Since the amount of albumin to be captured can be predicted, the amount of anti-GA antibody used can be minimized. Since it is sufficient that the amount of albumin in the sample is larger than the amount of anti-albumin antibody immobilized on the solid phase, the necessary dilution rate can be predicted. By measuring the amount of albumin captured using an anti-albumin antibody that recognizes an epitope different from that of the immobilized albumin antibody, the influence of the immobilized antibody activity can be suppressed even if it fluctuates. It can be measured stably.

When physically adsorbing albumin in a sample to a solid phase, first measure the GA amount of albumin captured using an anti-GA antibody, and then measure the amount of albumin captured using an anti-albumin antibody. Thereby, the influence of the fluctuation | variation of a physical adsorption amount can be suppressed and the abundance ratio of GA with respect to albumin can be measured stably.

The figure which showed the outline of the measurement of GA / albumin ratio. The figure which showed the outline of the procedure which measures GA / albumin ratio. The figure which showed that a fixed amount of human albumin could be capture | acquired by the anti-human albumin antibody fixed solid phase. The figure which showed that the GA / albumin ratio could be detected with a small dependence on the total albumin concentration. The figure which calculated theoretically the relationship between GA / albumin ratio and the density | concentration of GA and an anti- GA antibody complex. The figure which showed the method of correct | amending the amount of human albumins using an anti-human albumin antibody as a detection antibody. The figure which shows an example of the cartridge for GA / albumin ratio measurement. The figure which shows an example of the apparatus for measuring with the cartridge of FIG. The figure which shows the outline | summary of a glycoalbumin measuring kit. The figure which shows the external appearance of the apparatus for measuring with the cartridge of FIG. The figure which shows an example of the measuring method using the cartridge of FIG. 7, and the apparatus of FIG. The figure which shows the introduction method of the sample to a cartridge flow path. The figure which shows the electric potential measurement method using an electrochemical sensor. The figure which shows the example of the positional relationship of the electrode for electric potential measurement, and an antibody fixed part.

FIG. 1 schematically shows a method for measuring a GA / albumin ratio using the present invention. An anti-human albumin antibody 102 is immobilized on the solid phase 101, and the human albumin 103 and GA104 in the sample solution are captured by the anti-human albumin antibody 102. Alkaline phosphatase (AP) -labeled anti-GA antibody (hereinafter referred to as enzyme-labeled anti-GA antibody) 105 recognizes and binds to the saccharification site of GA104 (shown schematically in FIG. 1). When the substrate 106 is added, it is converted into the product 107 by the labeling enzyme of the enzyme-labeled anti-GA antibody 105 (in this case, alkaline phosphatase). The cases where the GA / albumin ratio in the sample solution is high and low are shown in FIGS. 1A and 1B, respectively. Since the amount of GA104 captured by the anti-human albumin antibody 102 differs depending on the GA / albumin ratio, and the amount of the enzyme-labeled anti-GA antibody 105 that binds changes accordingly, comparing the amount of the product 107 with the GA / albumin The ratio can be determined. Note that beads or the like may be used instead of the solid phase 101 as long as the anti-human albumin antibody 102 can be immobilized on the solid phase.

FIG. 2 shows an outline of the procedure for measuring the GA / albumin ratio.

Step 1: Sample solution is added, and human albumin 103 and GA104 in the sample solution are captured by anti-human albumin antibody 102 immobilized on solid phase 101. Step 2: Human in sample solution not bound to antibody by washing Step 3: Add enzyme-labeled anti-GA antibody 105 and bind to GA104 captured by anti-human albumin antibody 102 Step 4: Wash away excess unlabeled enzyme-labeled anti-GA antibody 105 by washing Exclude Step 5: Add substrate 106 for alkaline phosphatase and measure enzyme activity from the amount of product 107. At this time, in a general antigen-antibody reaction, anti-human which is an excess amount of capture antibody than human albumin in the sample solution Albumin antibody is used, but in the present invention, human albumin in the sample solution is used. Characterized by fixing the small anti-human albumin antibody to the solid phase. Under the condition that the immobilization density of the anti-human albumin antibody is the same, if the human albumin concentration in the sample solution is higher than the concentration at which human albumin can bind to all of the anti-human albumin antibody, it is captured on the solid phase. The total amount of human albumin that is equal to the amount of anti-human albumin antibody is constant, and a certain amount of human albumin can be fractionated for each measurement. The concentration of the complex in the antigen-antibody reaction can be expressed by equation (1).

Formula 1

For example, Ab = 10 ⁻⁸ (M), k _on = 10 ⁵ (M ⁻¹ s ⁻¹ ), k _off = 10 ⁻⁴ (s ⁻¹ ), t _b = 10 (min), t _w = 1 ( min), X _b = 9.93 × 10 ⁻⁹ (M) when Ag = 10 ⁻⁶ (M), and X _b = 9.94 × 10 ⁻ when Ag = 10 ⁻⁵ (M). ⁹ (M).

Here, the antibody concentration Ab can be expressed by the equation (2) using the antibody fixing density D, the reaction field volume V, and the antibody fixing area S in the reaction field.

Formula 2

From the equation (2), the conditions under which the anti-human albumin antibody was fixed at a fixed density of 6.5 × 10 ⁻⁹ mol / m ² in a reaction field having a volume of 100 μl and an antibody fixing area of 154 mm ² were as follows: antibody concentration 10 ⁻⁸ M It corresponds to. Since the human albumin concentration in blood is 5.6 to 7.4 × 10 ⁻⁴ M (37 to 49 mg / ml), the anti-human albumin antibody is immobilized at a fixed density of 6.5 × 10 ⁻⁹ mol / m ^2. For the reaction field (reaction field volume 100 μl, antibody immobilization area 154 mm ² ), the serum sample has an albumin concentration sufficient for binding of human albumin to all of the anti-human albumin antibodies on the solid phase.

The immobilization density of the anti-human albumin antibody is preferably 1.7 × 10 ⁻¹⁴ to 3.6 × 10 ⁻⁴ mol / m ² . If the immobilization density of the anti-human albumin antibody is lower than 1.7 × 10 ⁻¹⁴ , three significant digits of the measured value cannot be secured. Conversely, in the reaction field in which the anti-human albumin antibody is immobilized at a fixed density higher than 3.6 × 10 ⁻⁴ mol / m ² , depending on the performance of the antibody, all human albumin contained in the serum sample is anti-human albumin. Since it binds to the antibody, the total amount of human albumin captured on the solid phase varies depending on the concentration of human albumin in the serum sample, and a certain amount of human albumin cannot be captured for each measurement.

FIG. 3 shows that by using the solid phase 101 on which the anti-human albumin antibody 102 is immobilized, a certain amount of human albumin can be captured by the antibody if the concentration of human albumin in the sample solution is a certain level or more. Detailed experimental procedures are shown below.

Biotinylated monoclonal anti-human albumin antibody (capture antibody) was added to a streptavidin-coated microplate and immobilized. After washing with 0.1% Tween20-containing Tris Buffered Saline (hereinafter referred to as TBST), 2% bovine serum albumin (BSA) -containing TBST was added for blocking. The plate was washed with TBST to prepare an anti-human albumin antibody fixed plate.

A dilution series in which human albumin (containing GA) and TBST were mixed was prepared and used as a sample solution. The sample solution was introduced into the anti-human albumin antibody fixed plate, human albumin and GA in the sample solution were reacted with the capture antibody, and excess human albumin was washed with TBST. An alkaline phosphatase-modified monoclonal anti-human albumin antibody (detection antibody) was added to the plate and reacted, and then the excess antibody was washed with TBST.

Bromochloroindolyl phosphate (BCIP), which is a chromogenic substrate for alkaline phosphatase, and a color former, nitro blue tetrazolium (NTB), were added and reacted, and then the reaction was stopped by adding ethylenediaminetetraacetic acid (EDTA) to 595 nm. The absorbance was measured. FIG. 3 shows the relationship between the human albumin concentration in the sample solution and the absorbance. The absorbance was almost constant at a human albumin concentration of 0.05 mg / ml or more, indicating that a certain amount of human albumin could be captured by the capture antibody.

FIG. 4 is a diagram showing that the GA / albumin ratio can be measured with a small dependence on the total human albumin concentration by detecting GA contained in human albumin captured by the anti-human albumin antibody with the anti-GA antibody. It is. Detailed experimental procedures are shown below.

An anti-human albumin antibody fixed plate was prepared in the same manner as in the measurement in FIG. A serum specimen with a known GA / albumin ratio and TBST were mixed to dilute the serum specimen 100-fold and 1000-fold to obtain a sample solution. At this time, the human albumin concentration in the sample solution is desirably equal to or higher than the concentration at which human albumin can bind to all of the immobilized anti-human albumin antibodies. That is, when the anti-human albumin antibody is immobilized at a fixed density of 6.5 × 10 ⁻⁹ mol / m ² in a reaction field having a volume of 100 μl and an antibody immobilization area of 154 mm ² , dilution can be performed up to 5000 times.

The sample solution was introduced into the anti-human albumin antibody fixed plate, human albumin and GA in the sample solution were reacted with the anti-human albumin antibody, and excess specimen was washed with TBST. Monoclonal anti-GA antibody (antibody for detection) modified with alkaline phosphatase was added, reacted with GA captured by the anti-human albumin antibody, and then washed with TBST. In a general antigen-antibody reaction, the concentration of the detection antibody is about one-tenth of that of the capture antibody. However, in the present invention, human albumin is captured by all of the anti-human albumin antibodies that are capture antibodies. The concentration of a certain anti-GA antibody is preferably higher than the concentration of the immobilized anti-human albumin antibody depending on the antibody performance. For example, when the immobilization density of the anti-human albumin antibody is 2.1 × 10 ⁻⁸ mol / m ² , the reaction field volume is 100 μl, and the immobilization area is 154 mm ² , the concentration of the anti-GA antibody is preferably 33 nM or more. . The anti-GA antibody may be a mixture of a plurality of types of monoclonal antibodies or may be a polyclonal antibody. Since there are multiple glycation sites of human albumin, sensitivity and reproducibility can be improved by mixing multiple types of monoclonal anti-GA antibodies having different epitopes.

It should be noted that the relationship between the density of the capture antibody and the concentration of the detection antibody can be generalized using the equations (1) and (2). As described above, if the human albumin concentration in the sample solution is equal to or higher than the concentration capable of binding to all of the anti-human albumin antibodies, the total amount of human albumin captured on the solid phase can be approximated by the number of molecules of the anti-human albumin antibodies. Therefore, by substituting the anti-human albumin antibody concentration Ab of the formula (2) into the human albumin concentration Ag captured by the antibody in the formula (1), the relationship between the density of the captured antibody and the concentration of the detection antibody can be expressed by one formula ( It can be expressed by 3).

Formula 3

After BCIP and NTB were added and reacted, EDTA was added to stop the reaction, and the absorbance at 595 nm was measured. FIG. 4 shows a graph summarizing the relationship between the GA / albumin ratio and the absorbance in the sample solution. Although some background signal was added, absorbance corresponding to the GA / albumin ratio was obtained. According to the equation (3), the GA / albumin ratio and the GA / anti-GA antibody complex concentration theoretically have a linear relationship as shown in FIG. Also in FIG. 4, the relationship between the GA / albumin ratio and the absorbance can be expressed linearly, and the GA / albumin ratio could be measured according to the theory. Furthermore, no significant difference was observed in the absorbance between the 100-fold dilution and the 1000-fold dilution, but the GA / albumin ratio obtained from the calibration curve varied by 1.1 to 1.4 times despite the 10-fold albumin concentration being different. I was able to suppress it. Since human albumin was captured using the immobilized anti-human albumin antibody, the effect was that almost constant human albumin was captured regardless of the 10-fold difference in human albumin concentration. Therefore, even if there is a change in the dilution factor when the specimen is diluted, the GA / albumin ratio can be measured while suppressing the influence of the change.

In the example shown, the sample was diluted 100 times and 1000 times, but the GA / albumin ratio in the sample solution can be obtained in the same manner even at undiluted and low dilution ratios. However, in the case of undiluted or low dilution ratio, the measured value is likely to fluctuate due to non-specific adsorption of human albumin.

As shown in FIG. 6, after detection with an anti-GA antibody as shown in FIG. 6A, an enzyme-labeled anti-human albumin antibody 108 that recognizes an epitope different from the capture antibody is added, and the anti-antibody immobilized as shown in FIG. 6B is immobilized. By measuring the amount of human albumin captured by the human albumin antibody, even if the activity of the immobilized antibody varies, the measured value with the anti-GA antibody can be corrected to further suppress the variation.

After measuring the GA concentration of human albumin bound on a solid phase with an anti-GA antibody, and determining the total amount of human albumin with the anti-human albumin antibody and correcting the GA / albumin ratio, electrostatic interaction or hydrophobic interaction It can also be applied to normalization by adsorbing a certain amount of human albumin on a solid phase by physical adsorption such as action. Since there is a step of correcting the fluctuation of the total amount of human albumin due to physical adsorption, it can be measured with higher accuracy than the conventional method in which the total amount of human albumin is standardized by adsorption to a solid phase as in JP-A-2004-242522. .

FIG. 7 is a view showing an example of a cartridge for GA ratio measurement. 7A is a schematic plan view, and FIGS. 7B to 7D are schematic cross-sectional views. The cartridge includes a solid phase 701, a flow path section 702, and a liquid holding section 703. The solid phase 701 includes solution

introduction detection electrodes

713 and 714, a potential measurement electrode 715, an antibody fixing site 716, and terminals 724 to 726. The flow path unit 702 has a sample introduction port 712,

reagent supply ports

717, 718, 727, 728, and a waste liquid reservoir connection port 720. The liquid holding unit 703 includes a sample holding unit 711, a waste liquid reservoir 719, and an air hole. 721 and

boundary portions

722 and 723. Each positional relationship is explained by following the flow path from the reagent supply port 717. The solution introduction detection electrode 713, the antibody immobilization site 716, the potential measurement electrode 715, the antibody immobilization site 716, and the solution introduction detection electrode 714 flow in this order. It is provided inside the road. A sample introduction port 712 is provided in the upper part of the flow path between the antibody fixing site 716 and the sample introduction detection electrode 714. The approximate dimensions of the cartridge were 20 mm x 10 mm, and the flow path was 1 mm wide x 13 mm long x 0.25 mm high.

For the solid phase 701, a semiconductor substrate such as silicon, a circuit substrate such as glass epoxy, or a film substrate such as polyethylene terephthalate (PET), polypropylene (PP), polycarbonate (PC), or polyimide (PI) is used. Can do. In the channel portion 702, a film in which polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polycarbonate (PC), polyimide (PI) or the like is laminated, ethylene vinyl acetate copolymer (EVA), or the like is used. A thermoplastic resin, an epoxy resin, or a silicone resin such as polydimethylsiloxane (PDMS) can be used. For the liquid holding portion 703, aramid fiber, glass fiber, cellulose fiber, nylon fiber, vinylon fiber, polyester fiber, polyolefin fiber, rayon fiber filter paper, nonwoven fabric or porous fiber may be used. it can. The specimen holder 711 may be outside the specimen inlet 712 as shown in FIG. 7B, or may be between the specimen inlet 712 and the flow path as shown in FIG. 7C, and the flow as shown in FIG. 7D. May be on the road. In FIG. 7B and FIG. 7C, since the specimen holding part 711 separates the flow path from the outside, the pressure loss is large and it is not necessary to close the specimen inlet 712 during measurement. For the

boundary portions

722 and 723, low density polyethylene (LDPE) resin, ethylene vinyl acetate (EVA) resin, polyamide resin, or polyethylene terephthalate (PET) resin can be used. An antibody such as an anti-human albumin antibody is immobilized on the antibody immobilization site 716. The immobilization method may be physical adsorption or chemical bonding. First, after avidin is immobilized, a biotinylated antibody is immobilized. It may be immobilized by an avidin-biotin bond, or an antibody may be immobilized via avidin after immobilizing a biotin-modified molecule.

FIG. 8 is a diagram showing an example of an apparatus for performing measurement with the cartridge of FIG. FIG. 8A shows a top view of the positional relationship between the cartridge 810 and the apparatus 802, and FIG. 8B shows a side view of the connection relationship between the cartridge 810 and the reagent supply port and the electrodes / terminals of the apparatus 802. By setting the cartridge 801 in the apparatus 802, the

reagent supply ports

717, 718, 727, and 728 of the cartridge 801 are fluidly connected to the

reagent supply ports

811, 812, 816, and 817 of the apparatus 802, and the terminal 724 of the cartridge 801 is connected. To 726 and terminals 813 to 815 of the device 802 are electrically connected. Antibody reagent solution 826 is supplied to reagent supply port 811 by pump 825, sample diluent 824 is supplied to reagent supply port 812 by pump 823, and cleaning solution 828 is supplied to reagent supply port 816 by pump 827. Then, the substrate supply liquid 822 is supplied to the reagent supply port 817 by the pump 821. The sample diluent 824 and the cleaning solution 828 may be solutions having the same composition. The reagent supply ports may be provided at one to three locations, and a plurality of reagents may be supplied by branching a flow path connected to the reagent supply port. An AC power source 833 and an AC ammeter 834 are connected to the

terminals

814 and 815. A voltmeter 832 is connected to the terminal 813, and the other terminal of the voltmeter 832 is connected to a reference electrode 831 disposed in a flow path connected to the reagent supply port 817. Control of the

pumps

821, 823, 825, and 827 is performed by the control unit 803, and control and measurement of the AC power source 833, the AC ammeter 834, and the voltmeter 832 are performed by the measurement unit 804. The apparatus 802 includes a display unit 805 for displaying measurement results and messages, and an input unit 806 for a user to input an operation. The

pumps

821, 823, 825, and 827 may be peristaltic pumps, syringe pumps, or diaphragm pumps. The reference electrode 831 may be an internal liquid-type silver-silver chloride reference electrode, a bare silver-silver chloride electrode, or an ion-selective electrode as long as it exhibits a constant potential.

FIG. 9 is a diagram showing an example of a glycoalbumin measurement kit. The glycoalbumin measurement kit 901 accommodated the solid phase 701 provided with the antibody immobilization site 716 on which the anti-albumin antibody is immobilized and the potential measurement electrode 715 shown in FIG. 7, and the antibody reagent solution 826 shown in FIG. It consists of a container.

FIG. 10 is a schematic diagram showing an example of a state in which the cartridge is mounted on the measuring apparatus. The cartridge 801 is set in the measuring device 802 and the lid 1001 is closed.

FIG. 11 is a diagram showing an example of a measurement method using the cartridge of FIG. 7 and the apparatus of FIG. A sample is added to the sample introduction port 712 provided on the flow path (S201). The specimen may be a body fluid collected from a biological sample itself or a pretreatment such as centrifugation, filtration, or dilution as necessary. The sample holding unit 711 is made of an absorbent material such as filter paper. Further, since the periphery of the sample holding unit 711 is surrounded by a boundary portion 722, the added sample is held by the sample holding unit 711. The cartridge is set in the apparatus, and the sample diluent 824 is introduced from the reagent supply port 812 of the apparatus into the reagent supply port 718 of the cartridge using the pump 823 (S202). As the sample diluent, a buffer solution such as Tris （Buffered Saline (hereinafter referred to as TBS) or Phosphate Buffered Saline (hereinafter referred to as PBS) is used. In order to introduce the sample diluent 824 to the sample introduction port 712, solution

introduction detection electrodes

713 and 714 are used. For example, when an AC voltage is applied between the solution

introduction detection electrodes

713 and 714 using the AC power source 833, the current that was substantially zero before the sample diluent 824 was introduced, and the sample diluent 824 was introduced into the solution. When reaching the detection electrode 714, the solution

introduction detection electrodes

713 and 714 are electrically connected to increase. By monitoring this increase in current by the measuring unit 804, it can be detected that the sample diluent 824 has reached the solution introduction detection electrode 714. When the solution introduction detection electrode 714 detects the arrival of the sample diluent 824 (S203), the introduction of the sample diluent 824 from the reagent supply port 718 is stopped (S204). When the sample holder 711 and the sample diluent 824 are kept in contact with each other for a certain time (S205), the components in the sample held in the sample holder 711 are diffused into the sample diluent 824. Since the amount of components in the specimen that diffuses into the specimen diluent 824 depends on the retention time, the dilution ratio of the specimen can be controlled by the retention time.

When a slight amount of the sample diluent 824 is aspirated from the reagent supply port 718 and the sample components diffused in the sample diluent 824 are transported to the antibody fixing site 716 (S206), human albumin in the sample is transferred to the antibody fixing site 716. Captured by immobilized anti-human albumin antibody. After holding for a certain period of time, the sample diluent is further introduced from the reagent supply port 718, and the sample diluent 824 containing the sample is discarded in the waste solution reservoir 719 through the waste fluid reservoir connection port 720 (S207). The flow path is washed with the washing liquid 828 introduced from the reagent supply port 727 (S208), and human albumin not captured by the anti-human albumin antibody immobilized on the antibody immobilization site 716 is removed. The cleaning liquid 828 used for cleaning is also discarded in the waste liquid reservoir 719. The antibody reagent solution 826 is introduced from the reagent supply port 811 of the apparatus into the reagent supply port 717 of the cartridge using the pump 825 (S209). As the antibody reagent solution 826, a solution containing an alkaline phosphatase-labeled anti-GA antibody (labeled anti-GA antibody) or the like is used. Then, the labeled anti-GA antibody in the antibody reagent solution 826 binds to GA captured by the anti-human albumin antibody. After holding for a certain period of time, a washing liquid 828 is introduced from the reagent supply port 727, and the flow path is washed (S210), thereby removing unbound labeled anti-GA antibody. The antibody reagent solution 826 and the cleaning solution 828 are discarded in the waste solution reservoir 719.

The substrate solution 822 is introduced from the reagent supply port 817 of the apparatus into the reagent supply port 728 of the cartridge using the pump 821 (S211). As the substrate solution, a solution containing ascorbic acid phosphate, which is a substrate of alkaline phosphatase, and potassium ferricyanide, which is a mediator, is used. The ascorbic acid phosphate in the substrate solution 822 is hydrolyzed by alkaline phosphatase of the anti-human albumin antibody-GA-labeled anti-GA antibody complex present at the antibody fixing site 716, and the generated ascorbic acid reacts with potassium ferricyanide. Potassium ferrocyanide is generated, and the potential of the potential measuring electrode 715 changes. Therefore, by measuring the potential difference between the potential measuring electrode 715 and the reference electrode 831 with the voltmeter 832 (S212), the amount of captured GA, that is, the GA / albumin ratio in the sample is obtained. In order to obtain the GA / albumin ratio with higher accuracy, a calibration curve representing the relationship between the GA / albumin ratio and the measurement potential is prepared using a sample with a known GA / albumin ratio in a separately prepared cartridge. An unknown sample value may be calculated.

FIG. 12 shows the details of the embodiment of the method for introducing the sample into the cartridge flow path in S201 to S206. FIG. 12 is an explanatory diagram showing details of a method for introducing the sample into the cartridge flow path. In step 1, a sample is dropped onto a sample holding unit 711 made of a filter or the like provided in a part of the flow path. In step 2, a buffer solution is introduced into the flow path and is brought into contact with the specimen holding unit 711 and held for a certain period of time. In step 3, the sample components held in the sample holding unit 711 diffuse into the buffer solution. By this operation, a diluted specimen solution equivalent to the case where the specimen is diluted with a buffer solution is obtained. The dilution rate of the diluted specimen solution can be controlled by the buffer retention time. Finally, in step 4, the diluted specimen solution is transferred to the antibody fixing site 716, and an antigen-antibody reaction is performed. Since the antibody fixing site 716 is upstream of the sample holding unit 711 with the buffer introduction side being upstream, the sample components diffused into the solution from the sample holding unit 711 in the subsequent introduction of the solution in S207 to S212 are antibody Passing through the fixed portion 716 can be suppressed.

In FIG. 12, the side where the buffer solution is introduced may be upstream, and the antibody immobilization site 716 may be downstream from the specimen holding unit 711. The diluted sample solution can be transported to the antibody fixing site 716 in the same procedure as in FIG. Since the antibody fixing part 716 is downstream of the specimen holding part 711, the specimen component diffused from the specimen holding part 711 is supplied to the antibody fixing part 716 even if the diluent is kept flowing. Therefore, instead of holding the diluent as in Steps 2 to 3, the diluent may be continuously fed. In this case, the dilution rate of the diluted specimen solution supplied is controlled by the feeding speed.

Since the introduction of the sample uses diffusion from a filter or the like, it is possible to suppress adsorption of non-specific sample components in the flow channel, which is a concern when introducing the sample into the flow channel, or in the flow channel. . In addition, it is not necessary to measure a trace amount of sample that can be a problem when realizing a high dilution factor of about 100 times. In addition, since the sample is held in the sample holding unit 711 by capillary force, scattering of the sample when handling the cartridge can be suppressed.

The specimen holder 711 can be made of aramid fiber, glass fiber, cellulose fiber, nylon fiber, vinylon fiber, polyester fiber, polyolefin fiber, rayon fiber filter paper, nonwoven fabric, or porous fiber. . Alternatively, one or more pores can be used instead. By using these materials, the sample holding portion 711 can remove or reduce substances in the sample that may cause clogging of the cartridge flow path. In addition, by mixing activated carbon or hydrophobic resin with these materials, lipids or the like in the specimen that can be a factor of inhibition of the immune reaction system can be removed or reduced. At this time, these materials are desirably substances with low reactivity to proteins and carbohydrates in order to prevent an extreme decrease in the abundance of human albumin and GA and fluctuations in the GA / albumin ratio.

FIG. 13 shows the details of the embodiment of the potential measurement method using the electrochemical sensor in S212 below. In the detection step of the antigen-antibody reaction, the antigen-antibody reaction can be detected as an electrochemical signal by adding ascorbic acid phosphate, which is a substrate of the detection antibody-modifying enzyme alkaline phosphatase, and mediator, potassium ferricyanide. Ascorbic acid phosphate is hydrolyzed by alkaline phosphatase into ascorbic acid. Thereafter, dehydroascorbic acid is produced from ascorbic acid, and potassium ferricyanide is reduced to produce potassium ferrocyanide. As a result, the concentration ratio of the oxidizing substance and the reducing substance in the reaction solution changes and the potential of the reaction solution varies. FIG. 13A shows the measurement result of the potential that fluctuated with time due to the action of alkaline phosphatase present in the flow path. However, the potential does not fluctuate even if BSA which is a negative control is present instead of alkaline phosphatase. FIG. 13B is a graph in which the potential is converted to the concentration of potassium ferrocyanide generated using Nernst equation (4).

Formula 4

A measurement result obtained by using an albumin / GA mixed sample having a predetermined GA / albumin abundance ratio is used for calibration, and a GA / albumin abundance ratio in the specimen is obtained from the measurement result of the specimen. As a measurement result to be used, an inflection point as shown in FIG. 13A can be used in addition to the produced potassium ferrocyanide concentration after a certain time and the amount of produced potassium ferrocyanide per unit time. This inflection point is an equivalent amount of potassium ferricyanide and potassium ferrocyanide according to equation (4). While the standard electrode potential is required to obtain the potassium ferrocyanide concentration from the potential at an arbitrary time, it is always the standard electrode potential at the inflection point. Therefore, the measurement using the inflection point is not affected by the fluctuation of the standard electrode potential that occurs every measurement.

Since the measurement sensitivity of the potential measurement method does not depend on the measurement volume, the measurement sample can be made in a small amount without affecting the measurement sensitivity as compared with the oxidation-reduction current method or the absorptiometry. In addition, since the potential measurement method obtains a signal proportional to the logarithm of the concentration of the enzyme-labeled antibody, a wider dynamic range is obtained compared to the oxidation-reduction current method and the spectrophotometric method, which can obtain a signal proportional to the concentration of the enzyme-labeled antibody. It is done.

The combination of antibody modifying enzyme for detection, its substrate and mediator is not limited to the combination of hydrolyzing enzyme and substrate such as alkaline phosphatase, ascorbic acid phosphate and potassium ferricyanide, but also oxidoreductase such as glucose oxidase and glucose and potassium ferricyanide. A combination of redox substances may also be used. The concentration of the mediator is preferably lower than the substrate concentration, and the measurement sensitivity can be adjusted by the concentration ratio of the substrate to the mediator.

As the electrode 715 for potential measurement, an electrode capable of electrochemical measurement is used, such as a noble metal such as gold or platinum, a carbon material such as graphite or carbon black, or a mixture of a carbon material and a noble metal. Unlike current measurement, where the electrode area affects the measurement current, it is not necessary to precisely adjust the size of the electrode in potential measurement.

The relationship between the potential measurement electrode 715 and the antibody fixing site 716 will be described with reference to FIG. 14A shows the case where the antibody is immobilized only on the electrode, FIG. 14B shows the case where the antibody is immobilized in a wider range than the electrode including the electrode, and FIG. 14C shows the flow path centered on the electrode without fixing the antibody to the electrode. Shows the case where the antibody is immobilized at a position sandwiched between the upstream and downstream directions. Each graph shows the concentration of the reaction product when the substrate is added in the state where the immobilized antibody-antigen-enzyme labeled antibody complex is formed. The added substrate is decomposed by the enzyme of the immobilized antibody-antigen-enzyme labeled antibody complex to produce a reaction product, which is spread by diffusion. When the antibody is immobilized only on the electrode, the distribution of the reaction product has a concentration difference on the electrode as shown in FIG. 14A. In the example of this figure, the end of the electrode is about half the center of the electrode. On the other hand, when the antibody is immobilized in a wider range than the electrode including the electrode, the concentration difference on the electrode is suppressed as shown in FIG. In addition, as shown in FIG. 14C, when the antibody is not fixed to the electrode and the antibody is fixed at a position sandwiched from the upstream and downstream directions of the flow path centering on the electrode, a slight concentration difference occurs on the electrode. In this example, the difference is 7%, which is smaller than when the antibody is immobilized only on the electrode as shown in FIG. 14A. In the potential measurement, since the potential fluctuates according to the concentration of the reaction product, the antibody immobilization site 716 is wider than the electrode as shown in FIG. 14B or FIG. 14C so that the concentration of the reaction product is constant on the electrode. It is desirable to arrange the electrodes so as to surround the electrodes. In addition, the antibody fixing site 716 may be disposed so as to surround the electrode from four sides as shown in FIGS. 14D and 14E, and the antibody may be fixed on the electrode. It is desirable that the antibody immobilization site 716 is adjacent to the electrode both when the electrode is sandwiched between the antibody immobilization sites and when the electrode is surrounded.

In addition, this invention is not limited to the above-mentioned Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

101 Solid phase 102 Anti-human albumin antibody 103 Human albumin 104 GA
105 Enzyme-modified anti-GA antibody 106 Substrate 107 Product 108 Enzyme-modified anti-albumin antibody 701 Solid phase 702 Channel portion 703 Liquid holding portion 711 Sample holding portion 712

Sample introduction port

713, 714 Solution introduction detection electrode 715 Potential measurement electrode 716 Antibody Fixed

portion

717, 718, 727, 728 Reagent supply port 719 Waste liquid reservoir 720 Waste liquid reservoir connection port 721

Air holes

722, 723 Boundary portion 724 to 726 Terminal 801 Cartridge 802 Device 803 Control unit 804 Measurement unit 805 Display unit 806 Input unit 811 812, 816, 817 Reagent supply ports 813 to 815

Terminals

821, 823 Pump 822 Substrate liquid 824

Specimen diluent

825, 827 Pump 826 Antibody reagent liquid 828 Washing solution 831 Reference electrode 832 Voltmeter 833 AC power supply 834 AC ammeter 901 Min measurement kit 1001 Cover 1201 Sample

Claims

A solid phase provided with an immobilized anti-albumin antibody and an electrode;
A glycoalbumin measurement kit comprising an enzyme-labeled anti-glycoalbumin antibody.
In the glycoalbumin measurement kit of claim 1,
A glycoalbumin measurement kit, wherein the electrode is for potential measurement.
In the glycoalbumin measurement kit of claim 1,
The glycoalbumin measurement kit, wherein the solid phase has a specimen holding part.
In the glycoalbumin measurement kit according to claim 3,
The specimen holding unit uses a filter albumin, a non-woven fabric, or a porous fiber.
In the glycoalbumin measurement kit of claim 1,
The anti-albumin antibody and the electrode are arranged in a flow path;
The glycoalbumin measurement kit, wherein the anti-albumin antibody is disposed at least upstream and downstream of the electrode.
6. The glycoalbumin measurement kit according to claim 5, wherein the solid phase has a sample introduction port connected to the flow path.
A method for measuring glycoalbumin, characterized by quantifying the ratio of glycoalbumin to albumin in a specimen by potential measurement using an anti-albumin antibody and an enzyme-labeled anti-glycoalbumin antibody.
The measurement method according to claim 7, wherein the anti-albumin antibody is an immobilized antibody immobilized on a solid phase,
Supplying a specimen to the solid phase;
Binding the albumin and glycoalbumin in the specimen to the immobilized antibody;
Supplying an enzyme-labeled anti-glycoalbumin antibody to the solid phase;
Binding the enzyme-labeled antibody to glycoalbumin bound to the solid phase to form an immobilized anti-albumin antibody-glycoalbumin-enzyme-labeled anti-glycoalbumin antibody complex;
Supplying a substrate to the solid phase;
Reacting the substrate with an enzyme of the complex to produce a reaction product;
Measuring the reaction product concentration by potential measurement;
Converting the concentration of the reaction product into a concentration of glycoalbumin in the sample;
In order.
The method for measuring glycoalbumin according to claim 8,
A method for measuring glycoalbumin, comprising using a specimen diluted with a diluent.
The method for measuring glycoalbumin according to claim 9,
The method for measuring glycoalbumin, wherein the dilution ratio of the dilution is 10 times or more and 1000 times or less.
The glycoalbumin measurement method according to claim 8, wherein the solid phase has a specimen holding part,
After the step of supplying the specimen to the solid phase, the step of supplying a diluent to the solid phase,
A method for measuring glycoalbumin, comprising diluting a specimen by bringing the specimen held in the specimen holding part into contact with the diluent.