Method for quantification and optical measurement device

Abstract

This method for quantification of a substance to be detected in a specimen solution comprises using an inspection instrument including a flow path through which the specimen solution flows in one direction, the instrument being provided with, in order from the upstream side to the downstream side of the flow path, a first detection region in which a capture substance that specifically binds to the substance to be detected is immobilized and a second detection region in which the capture substance is immobilized, to determine the quantity of the substance to be detected contained in the specimen solution on the basis of the sum of a first signal intensity detected in accordance with the amount of the substance to be detected that is captured in the first detection region and a second signal intensity detected in accordance with the amount of the substance to be detected that is captured in the second detection region after the specimen solution is caused to flow from the upstream side to the downstream side of the flow path.

Description

Quantitative method and optical measurement device

[0001] The present invention relates to a method for quantifying a substance to be detected in a sample and an optical measurement device. This application claims priority based on Japanese Patent Application No. 2025-011093 filed in Japan on January 27, 2025, the content of which is incorporated herein by reference.

[0002] Conventionally, immunochromatography has been carried out in the medical field using a test device in which an antibody that captures a substance to be detected is immobilized on a nitrocellulose membrane, for example, to qualitatively or quantitatively measure the presence of a substance to be detected in a sample solution such as urine or blood (for example, Patent Document 1).

[0003] Japanese Patent No. 7226878

[0004] In immunochromatography, a predetermined region of a flow path composed of a nitrocellulose membrane or the like is defined as a detection region. A capture substance such as an antibody that specifically binds to the substance to be detected is immobilized linearly (in a line) in this detection region and functions as a test line. That is, when the sample solution that develops in the flow path passes through the test line, the substance to be detected is captured by the immobilized antibody. When the captured substance to be detected accumulates on the test line, a signal that increases or decreases according to the amount of the captured substance to be detected can be measured on the test line using a labeling substance that binds to the substance to be detected.

[0005] However, in the conventional method, when detecting the signal of the test line with a device, it may not be possible to ensure a wide measurement range corresponding to a wide range of concentrations of the substance to be detected contained in the sample solution. Specifically, by increasing the amount of the labeling substance (for example, gold colloid-labeled antibody) or the solid-phase substance (for example, antibody), the sensitivity on the low-concentration side of the substance to be detected can be improved. However, when the measurement range on the low-concentration side is thus expanded, problems may occur in the measurement on the high-concentration side. Such problems include: 1) the problem that the signal emitted from the test line is too strong and exceeds the upper limit of the measurement performance of the measuring device; 2) since there is a limit to the amount of antibody that can be included in one test line, when the antigen concentration is high, the upper limit of the measurement performance by one test line is exceeded, and the measurement range on the high-concentration side is reduced, etc.

[0006] The present invention provides a quantitative method that can quantify a substance in a sample at high concentrations without compromising measurement accuracy at low concentrations.

[0007] The present invention includes the following embodiments: [1] A method for quantifying a substance to be detected in a sample solution, wherein a testing instrument is provided with a channel through which the sample solution flows in one direction, and a first detection region in which a capture substance that specifically binds to the substance to be detected is solidified and a second detection region in which the capture substance is solidified are provided in order from the upstream side to the downstream side of the channel, and after the sample solution is circulated from the upstream side to the downstream side of the channel, the amount of the substance to be detected contained in the sample solution is determined based on the sum of a first signal intensity detected according to the amount of the substance to be detected captured in the first detection region and a second signal intensity detected according to the amount of the substance to be detected captured in the second detection region. [2] The method for quantifying according to [1], wherein the amount of the capture substance solidified in the first detection region in the channel is less than the amount of the capture substance solidified in the second detection region. [3] A quantitative method according to [1] or [2], wherein, when determining the amount of the substance to be detected contained in the sample solution, a calibration curve created using a standard sample of known concentration of the substance to be detected and the testing instrument is used. [A] A quantitative method according to any one of [1] to [3], wherein the flow path is further provided with a labeling substance holding section containing a labeling substance that specifically binds to the substance to be detected, located upstream of the first detection region. [B] A quantitative method according to any one of [1] to [3] and [A], wherein the flow path is further provided with a sample supply section, which is a part to which the sample solution is supplied, located upstream of the first detection region or upstream of the labeling substance holding section. [C] A quantitative method according to any one of [1] to [3] and [A] to [B], wherein the flow path is further provided with one or more additional detection regions downstream of the second detection region in which the capture substance is solidified, and the amount of the substance to be detected contained in the sample solution is determined based on the sum of the signal intensities detected in each detection region according to the amount of the substance to be detected captured in each detection region.[4] An optical measuring device comprising: an irradiation unit that irradiates a first detection area and a second detection area of ​​a testing instrument for quantifying a substance to be detected in a sample solution with measurement light; a measurement unit that measures the signal intensity emitted from the first detection area and the second detection area; and a determination unit that determines the amount of the substance to be detected in the sample solution based on the sum of the signal intensities emitted from the first detection area and the second detection area. [5] The optical measuring device according to [4], wherein the testing instrument is mounted within the irradiation range of the measurement light of the irradiation unit, the first detection area and the second detection area are provided sequentially from upstream to downstream in the flow path of the testing instrument, and a capture substance that specifically binds to the substance to be detected is immobilized in the first detection area and the second detection area, respectively. [6] The optical measuring device according to [5], wherein the amount of the capture substance immobilized in the first detection area in the flow path is less than the amount of the capture substance immobilized in the second detection area. [7] The optical measuring device according to any one of items [4] to [6], wherein the determination unit determines the amount of the substance to be detected contained in the sample solution, and uses a standard sample with a known concentration of the substance to be detected and a calibration curve created using the testing instrument.

[0008] According to the quantitative analysis method of the present invention, two or more test lines with a captured substance solidified on the test phase are provided, and the concentration of the substance to be detected in the sample is calculated by summing the signals obtained from the two or more test lines. Therefore, quantitative analysis can be performed even in the high concentration range without reducing the measurement accuracy in the low concentration range of the substance to be detected.

[0009] Figure 1 is a schematic top view of an example of an inspection instrument usable in the present invention. Figure 2 is a schematic side view of an example of an inspection instrument usable in the present invention. Figure 3 is an exploded view of the housing of the inspection instrument used in the embodiment. Figure 4 is a block diagram showing an example of the optical measuring device of the present invention.

[0010] ≪Quantitative Method≫ The first aspect of the present invention is a method for quantifying a substance to be detected in a sample solution, using a testing instrument equipped with a channel through which the sample solution flows in one direction. Hereinafter, an example of an embodiment of the testing instrument will be described, with reference to the drawings, as a testing instrument for immunochromatography. The testing instrument used in this aspect is not limited to a testing instrument for immunochromatography, and any instrument equipped with a channel through which the sample solution can flow can be used.

[0011] Figure 1 shows a schematic top view of inspection instrument 1, an example of an inspection instrument usable in this embodiment. Figure 2 is a schematic side view of inspection instrument 1. The arrows in Figures 1 and 2 indicate the direction in which the sample solution 20 flows.

[0012] The testing device 1 includes an absorbent member that forms a channel 13 through which the sample solution 20 moves (unfolds). Specifically, the channel 13 includes a sample supply unit 11, a detection unit 12, a blood cell separation membrane 14, a labeling substance holding unit 15, and an absorbent pad 16. The channel 13 is supported by a base material 17. The detection unit 12 is provided with a first detection region 12a and a second detection region 12b, and a control line 18 is provided downstream of the second detection region 12b. Although not shown, one or more additional detection regions may be provided between the second detection region 12b and the control line 18.

[0013] The sample supply unit 11 is the part to which the sample solution 20 containing the substance to be detected is supplied. Examples of materials for the sample supply unit 11 include nonwoven fabric, cellulose filter paper, paper, glass fiber, fiberglass, acrylic fiber, nylon fiber, and various textiles. The sample supply unit 11 may also contain optional additives such as buffers, sugars, and polyethylene glycol (PEG) for mixing with the sample solution.

[0014] The sample solution 20 is not particularly limited as long as it contains the substance to be detected, and examples include biological samples derived from humans or animals. Specifically, it may be, for example, blood, culture supernatant, urine, feces, cerebrospinal fluid, saliva, sweat, ascites, or cell or tissue extracts. The blood may be whole blood, plasma, serum, or blood that has been pre-treated in any way. The sample solution 20 may contain additives such as preservatives and surfactants.

[0015] The substances to be detected in the sample solution 20 are the substances detected in the detection unit 12. The substances to be detected are not particularly limited, but examples include cancer markers, hormones, infectious diseases, autoimmune substances, plasma proteins, TDM, coagulation / fibrinolysis, amino acids, peptides, proteins, genes, and cells. Specifically, these include CEA, AFP, ferritillin, β2 micro, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1 and 2, fecal occult blood, urinary β2 micro, PIVKA-2, urinary BTA, insulin, E3, hCG, HPL, LH, HCV antigen, HBs antigen, HBs antibody, HBc antibody, HBe antigen, HBe antibody, HTLV-1 antibody, HIV antibody, toxoplasma antibody, syphilis, ASO, and type A insulin. Influenza antigen, influenza A antibody, influenza B antigen, influenza B antibody, coronaviruses such as MERS-CoV, SARS-CoV, SARS-CoV-2, coronavirus 229E, coronavirus OC43, and coronavirus NL63, rota antigen, adenovirus antigen, rota-adenovirus antigen, group A streptococcus, group B streptococcus, Candida antigen, C. difficile, Cryptococcus antigen, Lera bacteria, meningococcal antigen, granulobacter elastase, Helicobacter pylori antibody, O157 antibody, O157 antigen, leptospirosis antibody, Aspergillus antigen, MRSA, RF, total IgE, LE test, CRP, IgG, A, M, IgD, transferrin, urinary albumin, urinary transferrin, myoglobin, C3 / C4, SAA, LP(a), α1-AC, α1-M, haptoglobin, microtransferrin, AP Examples include R score, FDP, D-dimer, plasminogen, AT3, α2PI, PIC, PAI-1, protein C, coagulation factor X3, type IV collagen, hyaluronic acid, GHbA1c, NT-proBNP, BNP, PCT, troponin, and various other antigens, antibodies, viruses, bacteria, amino acids, peptides, proteins, DNA, cells, allergens, pesticide residues, and harmful substances.

[0016] The detection unit 12 is installed on a water-absorbing material placed within the flow path 13. Examples of water-absorbing materials include porous membranes, specifically nitrocellulose membranes, cellulose mixed esters, and the like.

[0017] The detection unit 12 includes a line-shaped first detection region on which a first antibody, which is a capture substance that specifically binds to the substance to be detected, is immobilized, and a line-shaped second detection region on which a second antibody, which is a capture substance that specifically binds to the substance to be detected, is immobilized. Here, the substance to be detected to which the first antibody binds and the substance to which the second antibody binds are the same substance. The site (epitope) to which the first antibody binds and the site (epitope) to which the second antibody binds may be the same or different, but from the viewpoint of improving measurement accuracy, it is preferable that the first antibody and the second antibody are the same antibody and that they recognize the same epitope.

[0018] When the detection unit 12 is used to quantify the substance to be detected by providing one or more additional line-shaped detection regions downstream of the second detection region, it is preferable that the antibody immobilized in the third detection region and subsequent detection regions is the same antibody as the first antibody and recognizes the same epitope. When the detection unit 12 is used to detect a substance different from the substance to be detected by providing one or more additional line-shaped detection regions upstream or downstream of the second detection region, it is preferable that the antibody immobilized in the third detection region and subsequent detection regions is a different antibody from the first antibody.

[0019] In this embodiment, if the capture substance used is an antibody, the antibody may be a full-length antibody or a fragmented antibody. Examples of fragmented antibodies include F(ab) and F(ab'). ２ Examples include the above. The type, subtype, and animal from which the antibody used in this embodiment originates are not particularly limited. Any antibody capable of capturing the substance to be detected may be used.

[0020] The capture substance used in this embodiment may be any substance that can specifically bind to the substance to be detected, and may not be an antibody. Examples include DNA aptamers, peptides, proteins other than antibodies, biotin, and the like.

[0021] The testing device 1 may optionally include a labeling substance holding section 15. The labeling substance holding section 15 is located between the sample supply section 11 and the detection section 12. The labeling substance holding section 15 contains a labeling substance that specifically binds to the substance to be detected. An example of a labeling substance is a labeling section and a third antibody that binds to the labeling section and specifically binds to the substance to be detected. Here, it is preferable that the site (epitope) of the substance to which the third antibody binds is different from that of the first and second antibodies. By using a third antibody with a different epitope, the third antibody can further bind to the substance to be detected that has been bound to the first and second antibodies.

[0022] The labeled portion constituting the labeled substance may be particles insoluble in water and buffer solutions. The particles are not particularly limited, but examples include metal colloid particles such as gold colloid, platinum colloid, silver colloid, and selenium colloid. The particles may also be colored latex particles obtained by coloring styrene-based latex such as polystyrene latex, colored silica particles obtained by coloring silica consisting of a three-dimensional structure of silicon and oxygen atoms, colored cellulose particles obtained by coloring cellulose particles, or carbon black.

[0023] The labeling portion constituting the labeling substance may be fluorescent particles. Examples of fluorescent particles include particles containing europium complexes, rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic hydrocarbon-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, pyromecene-based dye molecules, diketopyrrolopyrrole-based dye molecules, phthalocyanine-based dye molecules, as well as Alexa Fluor® (registered trademark, manufactured by Invitrogen), BODIPY® (registered trademark, manufactured by Invitrogen), Cy® (registered trademark, manufactured by GE Healthcare), DY® (registered trademark, manufactured by DYOMICS), HiLyte® (registered trademark, manufactured by Anaspec), DyLight® (registered trademark, manufactured by ThermoScientific), ATTO® (registered trademark, manufactured by ATTO-TEC), MFP® (registered trademark, manufactured by Mobitec), and fluorescent proteins.

[0024] The labeling portion of the labeling substance may be an enzyme. Examples of enzymes include horseradish peroxidase and alkaline phosphatase.

[0025] The method of binding the label portion constituting the labeling substance to the third antibody is not particularly limited and includes, for example, binding by physical adsorption, binding by covalent bonding, and binding by a combination thereof.

[0026] In the testing device 1, the labeling substance may exist in a state impregnated in a dedicated pad (conjugate pad), or a part of the sample supply unit 11 may function as a labeling substance holding unit. Alternatively, the labeling substance may be added to the sample solution as a labeling substance-containing reagent, separately from the testing device, and mixed with the sample solution.

[0027] The testing device 1 may optionally be equipped with a blood cell separation membrane 14. The blood cell separation membrane 14 separates blood cells, such as red blood cells, contained in the sample solution 20 from the sample. An example of a blood cell separation membrane 14 is a porous membrane having pores of a diameter sufficient to separate blood cells. As an example, the dimensions of blood cells are: red blood cells have a diameter of 7 to 8 μm, a thickness of approximately 2 μm, white blood cells have a diameter of 6 to 30 μm, and platelets have a diameter of 2 to 3 μm. As an example, the pore diameter of the porous membrane of the blood cell separation membrane 14 is 2 to 100 μm, preferably 2 to 30 μm, and more preferably 2 to 5 μm. The blood cell separation membrane may also be an asymmetric porous membrane in which the pore diameter decreases in the thickness direction, in which case the pore diameter of the part with the smallest pore diameter should be within the above range.

[0028] In the example of the testing device 1, the sample supply unit 11, the labeling substance holding unit 15, the blood cell separation membrane 14, and the detection unit 12 are arranged in that order from upstream to downstream in the flow direction (arrow direction) of the sample solution 20, with each layer overlapping at least partially with the upper and lower layers to form a flow path 13. The flow path 13, in particular the detection unit 12, is supported by a substrate 17. An absorbent pad 16 is positioned downstream of the control line 18 on the membrane containing the detection unit 12.

[0029] The absorption pad 16 absorbs any excess sample that has passed through the detection unit 12. Examples of materials that can be used for the absorption pad 16 include, but are not limited to, cellulose filter paper, paper, glass fiber, fiberglass, acrylic fiber, nylon fiber, and various textiles.

[0030] The base material 17 may be a plastic base material, or a plastic adhesive sheet having an adhesive layer on a plastic base material. Examples of materials for the plastic base material and the plastic adhesive sheet include, but are not limited to, polyester, polystyrene, polypropylene, and polyvinyl chloride.

[0031] The testing device 1 may include a control line 18 for confirming whether the test was performed correctly. For example, the control line 18 can be equipped with a function to emit a signal when the sample solution 20 reaches the control line 18. The mechanism of the control line 18 can be the same as that of conventional immunochromatography.

[0032] The testing instrument 1 may be housed in a housing (see Figure 3) having a sample addition hole and a detection window. The testing instrument 1 is housed in a housing such that the sample supply unit 11 and the detection unit 12 are positioned in the housing, corresponding to the sample addition hole and detection window, respectively.

[0033] In the inspection device 1 described above, the detection unit 12 is provided with a first detection region 12a in which a capture substance that specifically binds to the substance to be detected is solidified, and a second detection region 12b in which the capture substance is solidified, in order from the upstream side to the downstream side of the flow path 13.

[0034] In the flow path 13, it is preferable that the amount of captured substance solidified in the first detection region is less than the amount of captured substance solidified in the second detection region 12b. With this configuration, the slope of the calibration curve becomes steeper, and the influence of measurement errors on the measurement results can be reduced, so that more accurate measurements are possible whether the concentration of the substance to be detected is high or low.

[0035] In the quantitative analysis method of this embodiment, after the sample solution 20 is circulated (expanded) from the upstream side to the downstream side of the flow path 13, the amount of the substance to be detected contained in the sample solution 20 is determined based on the sum of a first signal intensity detected according to the amount of the substance to be detected captured in the first detection area 12a and a second signal intensity detected according to the amount of the substance to be detected captured in the second detection area 12b. Reliability can be increased by basing the determination on the sum of two signals compared to basing it on a single signal. When based on a single signal, reliability decreases due to the signal being too low or too high depending on the concentration of the substance to be detected, but in the quantitative analysis method of this embodiment, since it is based on the sum of two signals, such a decrease in reliability can be prevented. Furthermore, if any number of other detection areas are provided downstream of the second detection area, reliability can be further increased by determining the amount of the substance to be detected in the sample solution 20 based on the sum of the signals obtained from each detection area.

[0036] In the quantitative method of this embodiment, it is preferable to use a calibration curve when determining the amount of the substance to be detected contained in the sample solution 20. A calibration curve can be created by using a standard sample with a known concentration of the substance to be detected as the sample solution 20, performing the quantitative method of this embodiment using the testing instrument 1, calculating the sum of the detected first signal intensity and the second signal intensity, and measuring the sum of signal intensities corresponding to the known concentrations. A calibration curve can also be created in the same manner when any number of other detection regions are further provided downstream of the second detection region. That is, by using the standard sample as the sample solution 20 and performing the quantitative method of this embodiment, a calibration curve can be created based on the correspondence between the sum of signals obtained from each detection region and the known concentration of the substance to be detected contained in the standard sample.

[0037] <<Optical Measuring Device>> A second aspect of the present invention is an optical measuring device comprising at least an irradiation unit, a measuring unit, and a judgment unit. The optical measuring device of this aspect can be used in the quantitative method of the first aspect. The optical measuring device of this aspect may or may not have the inspection instruments described in the first aspect attached to it.

[0038] Figure 3 shows a block diagram of an optical measuring device 40 as an example of an embodiment of the optical measuring device according to this embodiment. The optical measuring device 40 comprises an operation unit 41, a sensor unit 42, a notification unit 43, a storage unit 44, and a control unit 45. The control unit 45 integrally controls the operation of the operation unit 41, the sensor unit 42, the notification unit 43, and the storage unit 44.

[0039] The operation unit 41 receives instructions from the user of the optical measuring device 40. The operation unit 41 may be composed of hardware keys such as switches, or of software keys such as a liquid crystal touch panel. The instructions received by the operation unit 41 are input to the control unit 45.

[0040] The sensor unit 42 optically detects optical signals (e.g., color change or light emission) emitted from each detection area, including the first and second detection areas of the inspection instrument (e.g., the inspection instrument 10 described above), and from the control line. The sensor unit 42 comprises an installation section on which the inspection instrument is installed, an illumination section 51, and a measurement section 52. The installation section is within the illumination range of the measurement light from the illumination section 51. The illumination section 51 includes a light source (e.g., an LED) that irradiates light onto each detection area. The measurement section 52 includes a light-receiving element (e.g., a photodiode) or an image sensor (e.g., a CCD or CMOS) that receives optical signals from each detection area and the control line. Each detection area and the control line of the inspection instrument installed in the installation section are illuminated by the illumination section 51, and the optical signals from the inspection instrument are measured by the measurement section 52. The acquired optical signals are input to the control unit 45.

[0041] The notification unit 43 notifies the user of the optical measuring device 40 of various information (for example, the amount of the substance to be detected contained in the sample solution determined by the judgment unit 54). The notification unit 43 can display information in the form of images or text on a display panel such as a liquid crystal display.

[0042] The storage unit 44 stores control programs and control data executed by the control unit 45, and also stores various types of information such as analysis results. The storage unit 44 is composed of an information storage medium such as flash memory, hard disk, ROM, or RAM.

[0043] The control unit 45 operates according to a control program and controls the operations of the operation unit 41, the sensor unit 42, the notification unit 43, and the storage unit 44. Also, the control unit 45 operates according to a control program and functions as an image processing unit 53 and a determination unit 54.

[0044] When acquiring the optical signals in each detection area and the control line by imaging, the optical measurement device of this embodiment includes an image processing unit 53. Based on the image acquired by the sensor unit 42, the image processing unit 53 generates luminance information quantified by the luminance of the image from the intensities of the optical signals in each detection area and the control line of the inspection instrument. For example, the signal intensity of the first detection area is used as the first luminance information, the signal intensity of the second detection area is used as the second luminance information, and the signal intensity of the control line is used as the reference luminance information. Similarly, when there is another detection area downstream of the second detection area, the signal intensities of each detection area are generated as individual luminance information.

[0045] The determination unit 54 quantitatively analyzes the detected substance in the specimen solution based on the signal intensity acquired by the measurement unit 52 or the respective luminance information generated by the image processing unit 53. Specifically, the amount of the detected substance in the specimen solution is determined based on the sum of the signal intensities of each detection area in the inspection instrument. At this time, the determination unit 54 can refer to a relationship value (for example, a calibration curve) indicating the relationship between the concentration of the detected substance in the specimen solution and the sum of the signal intensities of each detection area. The relationship value is stored in the storage unit 44, and the control unit 45 can read out and refer to the relationship value from the storage unit 44 as needed for its processing.

[0046] Also, the determination unit 54 detects that the specimen solution has flowed appropriately through the flow path of the inspection instrument based on the signal intensity emitted from the control line. The determination unit 54 generates the results of the above analysis.

[0047] In the above configuration, the control unit 45 operates according to a control program, and when an analysis start instruction is input from the operation unit 41, it executes a predetermined analysis process. The predetermined analysis process includes the steps of timing the elapsed time of a predetermined reaction time from the start of analysis, having the sensor unit 42 measure the testing instrument after the predetermined reaction time has elapsed, quantifying the signal intensity of each detection area from the acquired testing instrument, and generating analysis results based on the obtained quantified information. The reaction time is set appropriately as the time required for the sample solution to flow sufficiently through the flow path of the testing instrument to the control line.

[0048] The control unit 45 notifies the notification unit 43 of the analysis results generated through the predetermined analysis process described above. The control unit 45 stores the analysis results in the storage unit 44.

[0049] The hardware of the control unit 45 includes electrical circuits having semiconductor elements and circuit elements, including various known processors (CPU, FPGA, PLD, ASIC, etc.). Each processing unit of the control unit 45 (image processing unit 53, decision unit 54) may be composed of a single processor, or of a combination of two or more processors of the same or different types. Alternatively, multiple processing units may be composed of a single processor.

[0050] Examples of configuring multiple processing units with a single processor include, firstly, a configuration in which one or more CPUs and software combine to form a single processor, as is common in computers such as clients and servers, and this processor functions as multiple processing units. Secondly, a configuration in which a processor is used that realizes the functions of the entire system, including multiple processing units, on a single IC chip, as is common in systems on a chip.

[0051] In the following, unless otherwise specified, units such as "%" and "parts" refer to "mass percent" and "parts by mass" based on mass.

[0052] <Preparation of Immunochromatography Test Device> A) Sample Pad Preparation 62.99 μL / cm² of 20 mM MOPS (pH 7.2) containing 0.5% trehalose is added to a fiberglass pad (Lydall). ２ The sample pad was soaked in the liquid at the specified ratio, dried, and used as the sample pad.

[0053] B) Preparation of anti-NT-proBNP antibody The anti-NT-proBNP antibody used in the following tests was obtained by immunizing mice with recombinant NT-proBNP protein (human brain natriuretic peptide precursor N-terminal fragment) as the antigen, using a method commonly used by those skilled in the art to produce monoclonal antibodies.

[0054] C) Preparation of Gold Colloid Solution To 100 mL of boiling purified water, 1 mL of 1% (w / v) triammonium citrate aqueous solution was added and stirred. Subsequently, 1 mL of 1% (w / v) tetrachlorogold(III) aqueous solution was added and stirred for 10 minutes, after which the reaction mixture was cooled to obtain a gold colloid solution. This gold colloid solution was diluted with purified water so that the absorbance (unit: OD / mL) at the maximum absorption wavelength of the gold colloid was 1.

[0055] D) Preparation of gold colloid-labeled anti-NT-proBNP antibody solution To the above 1 OD / mL gold colloid solution (pH 7.0), NT-proBNP monoclonal antibody diluted to 46.2 μg / mL with 2 mM Tris-HCl buffer (pH 7.0) was added and stirred at room temperature for 10 minutes. To this mixture of gold colloid and antibody, purified water containing 10% (w / v) BSA was added and stirred at room temperature for 5 minutes. Then, the mixture was centrifuged at 11900 × g for 45 minutes at 10°C. After removing the supernatant, 1 mL of Conjugate Dilution Buffer (SCRIPS Laboratories) was added to the obtained precipitate to suspend the conjugate and obtain an anti-NT-proBNP antibody conjugate.

[0056] E) Preparation of gold colloid-labeled KLH (KLH conjugate) for the control line To 20 mL of the gold colloid solution with an absorbance of 1 (unit: OD / mL) described above, 1 mL of KLH (keyhole limpet hemocyanin; Sigma-A) dissolved in 2 mmol / L phosphate buffer to a concentration of 620 μg / mL was added and the mixture was stirred at room temperature for 10 minutes. To the mixture of gold colloid and KLH, 1 mL of 10% bovine serum albumin (BSA) aqueous solution was added and the mixture was stirred at room temperature for 5 minutes. Then, the mixture was centrifuged at 10°C for 45 minutes, the supernatant was removed, and 1 mL of conjugate diluent was added to the resulting precipitate to suspend the conjugate and obtain the KLH conjugate.

[0057] F) Conjugate Pad Preparation: A detection reagent solution was prepared by mixing 3 OD / mL of anti-NT-proBNP antibody conjugate, 0.75 OD / mL of KLH conjugate, 2.4% trehalose, 1.5% Neo Protein Saver (TOYOBO), and 20 mM MOPS (pH 7.2). The detection reagent solution was then dispensed at a rate of 66.93 μL / cm³ onto a Glass Fiber Diagnostic Pad (Merck Millipore). ２ The mixture was soaked in at the specified liquid-to-liquid ratio, dried, and used to create a conjugate pad.

[0058] G) For the antibody-immobilized membrane fabrication test line, a solution was prepared by adding sucrose and anti-NT-proBNP monoclonal antibody to 10 mM PBS pH 7.2 to final concentrations of 2.5% and 2 mg / mL, respectively. For the control line, rabbit anti-KLH polyclonal antibody (Bethyl) was diluted and prepared in the same manner as above. The anti-NT-proBNP monoclonal antibody solution was applied in a line to the upstream side of a strip-shaped nitrocellulose membrane (UniSart CN150 white backing, Sartorius) in the sample deployment direction to form one (comparative example) or two (example) test lines. The anti-KLH polyclonal antibody solution was applied in the same manner downstream from the formed test line in the sample deployment direction at a certain interval to form one control line. The membrane coated with the antibody solution was dried to obtain an antibody-immobilized membrane. Each antibody solution was applied using an immunochromatography dispenser "XYZ3050" (BIO DOT), set to a dispensing rate of 1 μL / cm. The prepared antibody-immobilized membranes were incorporated into test strips (described later) and then cut to a size of 25 mm in the sample development direction and 6 mm in the width perpendicular to the sample development direction. In the two antibody-immobilized membranes prepared, the comparative example with one test line had a distance of 8 mm between the test line and the control line. In the example with two test lines, the distance between the two test lines and between the test line and the control line was set to 4 mm each.

[0059] H) Test Strip Preparation [Materials] - Sample pad prepared as described above - Conjugate pad prepared as described above - Blood cell separation membrane Vivid PSGF-SM (Lot. 9152PLQ1, Nippon Pall Co., Ltd.) - Antibody-immobilized membrane prepared as described above - End pad 470 (Lot. 1757772522, GE Healthcare) - Backing sheet (Cat. LC-59474, Lot. 077065-02, KENOSHYA Co., Ltd.) - Clear Mylar ARcare 7815 (Lot. 072921A, Adhesive Research Co., Ltd.)

[0060] [Manufacturing Method] The components were bonded together as shown in Figures 1 and 2. First, the antibody-immobilized membrane was attached to the backing sheet (plastic adhesive sheet), which is the base material 17. At this time, one or two test lines (anti-NT-proBNP antibody coated area) were placed on the upstream side of the unfolding as the detection area 12, and one control line (anti-KLH antibody coated area) was placed on the downstream side of the unfolding. Next, the blood cell separation membrane 14, the conjugate pad which is the labeling substance holding area 15, the sample pad which is the sample supply area 11, and the end pad which is the absorption pad 16 were attached as shown in the figure. In addition, a transparent protective film called Clear Mylar (not shown) was placed on top so as to cover the exposed area of ​​the antibody-immobilized membrane and the absorption pad 16. The laminate of these components was cut to a width of 6 mm when viewed from above to form the test strip (immunochromatographic test piece), which is the testing instrument 1. The size of the prepared test strip was 25 mm in length in the direction of sample unfolding and 6 mm in width perpendicular to the direction of sample unfolding. This test strip was housed inside a plastic housing and used as a test device (inspection instrument) for the following tests.

[0061] Figure 3 shows an exploded view of the housing described above. The container portion 31 is provided with multiple fixing pins 35 that can hold and secure the sides of the test strip. With the test strip 1, which is fixed to the multiple fixing pins, housed inside, the lid portion 32 covers the container portion 31 and is secured by a fitting mechanism (not shown), forming an integrated housing (storage case). The lid portion 32 is provided with a first opening 33 (through hole for sample dropping) located above the sample pad, and a second opening 34 (through hole for observing each line) located above the test line and control line.

[0062] [Test Method] A) Preparation of NT-proBNP-containing samples Low-concentration NT-proBNP-containing plasma and high-concentration NT-proBNP-containing plasma were mixed to prepare sample groups with different NT-proBNP concentrations. The NT-proBNP concentration in each sample was measured using Elecsys Reagent NT-proBNP II (Roche), Elecsys NT-proBNP II Calibrator v2 (Roche), and cobas e801 (Roche).

[0063] B) Sample measurement using an immunochromatographic test device for NT-proBNP measurement. 120 μL of the sample was dropped onto the sample pad of the test device prepared as described above. After standing for 10 minutes, the reflectance absorbance of the test line was measured using RapidPia (Sekisui Medical Co., Ltd.).

[0064] [Evaluation of the effect of increasing the number of test lines on reflectance absorbance] Samples with NT-proBNP concentrations of 42, 141, 290, 538, 1035, 2027, 3516, and 4509 pg / mL were dropped onto immunochromatography test devices equipped with one or two test lines at a solid-phase antibody concentration of 1.0 mg / mL (Comparative Example 1 and Example 1, respectively), and the reflectance absorbance at the test lines was measured. For Example 1, the sum of the reflectance absorbances measured at each of the two test lines was calculated. The results are shown in Tables 1 and 2.

[0065] The reflectance absorbance of Comparative Example 1 (referred to as sensitivity in the table) was similar to that of the test line upstream in the sample development direction (hereinafter referred to as TL1) in Example 1. Increasing the number of test lines to two did not affect the reflectance absorbance of TL1. In Example 1, color development was observed in the test line downstream in the sample development direction (hereinafter referred to as TL2) when measuring samples of all concentrations. The sum of the reflectance absorbances of TL1 and TL2 at each antigen concentration increased with increasing antigen concentration in the sample. The sum of the reflectance absorbances of TL1 and TL2 measured in Example 1 at each antigen concentration was greater than the reflectance absorbance measured in Comparative Example 1 for all samples. In particular, the reflectance absorbance of Example 1 when measuring samples with an NT-proBNP concentration of 141 pg / mL or less was more than 40% higher than that of Comparative Example 1, indicating that the lower limit of detection was improved in Example 1 compared to Comparative Example 1. Furthermore, across the entire concentration range, the sensitivity increase per unit antigen concentration in Example 1 was greater than that of Comparative Example 1. This result means that by using the test device of Example 1, it is possible to draw a steeper calibration curve than in Comparative Example 1. By using the test device of Example 1, the influence of sensitivity fluctuations due to measurement errors on the measurement results can be suppressed, and higher measurement accuracy than in Comparative Example 1 can be ensured.

[0066] In Table 1, the "increase in sensitivity per unit antigen concentration" is calculated as (68.0 - 29.1) / (141 - 42) = 0.393 for the antigen concentration range of 42 pg / mL to 141 pg / mL in the sample. The same applies to other intervals. In Table 2, the "increase in sensitivity per unit antigen concentration" is calculated as (98.6 - 47.7) / (141 - 42) = 0.514 for the antigen concentration range of 42 pg / mL to 141 pg / mL in the sample. The same applies to other intervals.

[0067]

[0068]

[0069] [Evaluation of the effect of changes in TL2 solid-phase antibody concentration on reflectance absorbance] An immunochromatography test device (Examples 2 and 3, respectively) was equipped with two test lines: one with a solid-phase antibody concentration of 0.5 mg / mL on the upstream side of the sample development direction (hereinafter referred to as TL1), and two test lines with solid-phase antibody concentrations of 0.5 mg / mL or 2.0 mg / mL on the downstream side of the sample development direction (hereinafter referred to as TL2). Samples prepared to have NT-proBNP concentrations of 42, 141, 290, 538, 1035, 2027, and 3516 pg / mL were dropped onto the test lines, and the reflectance absorbance in each test line was measured. The sum of the reflectance absorbances measured in each of the two test lines is shown in Tables 3 and 4.

[0070] In both Examples 2 and 3, the sum of the reflectance absorbances of TL1 and TL2 increased with increasing antigen concentration in the sample. When measuring samples with an NT-proBNP concentration of 141 pg / mL or higher, the sum of the reflectance absorbances of TL1 and TL2 was greater in Example 3, where the solid-phase antibody concentration of TL2 was higher than in Example 2. Furthermore, the increase in sensitivity per unit antigen concentration was also greater in Example 3 than in Example 2. This result means that by increasing the solid-phase antibody concentration of TL2 above that of TL1, it is possible to draw an even steeper calibration curve, thereby suppressing the influence of measurement errors on the measurement results.

[0071]

[0072]

[0073] [Evaluation of the impact of increasing the number of test lines on the measurement range] Immunochromatography test devices equipped with one test line at solid-phase antibody concentrations of 0.2, 0.5, or 2.0 mg / mL (Comparative Examples 3-1, 3-2, and 3-3, respectively) and immunochromatography test devices equipped with two test lines: one with a solid-phase antibody concentration of 0.2 mg / mL or 0.5 mg / mL upstream in the sample development direction (hereinafter referred to as TL1) and one with a solid-phase antibody concentration of 2.0 mg / mL downstream in the sample development direction (hereinafter referred to as TL2) (Examples 3-1 and 3-2, respectively) were used. Samples prepared to have NT-proBNP concentrations of 0, 40, 523, 4519, 8016, and 12012 pg / mL were dropped onto each device, and the reflectance absorbance at the test lines was measured. For Examples 3-1 and 3-2, the sum of the reflectance absorbances measured at each of the two test lines was calculated. The results are shown in Tables 5, 6, 7, 8, and 9.

[0074] When the NT-proBNP concentration in the sample was low, the increase in sensitivity per unit antigen concentration was greater in the order of Comparative Example 3-1, Comparative Example 3-2, and Comparative Example 3-3, suggesting that the sensitivity improved with increasing solid-phase antibody concentration. However, in Comparative Example 3-1, the sensitivity was similar when measuring samples with NT-proBNP concentrations of 0 pg / mL and 40 pg / mL, indicating poor measurement performance at low values. In Comparative Examples 3-2 and 3-3, the increase in sensitivity per unit antigen concentration was small when measuring samples containing high concentrations of NT-proBNP. On the other hand, Examples 3-1 and 3-2 showed a good increase in sensitivity per unit antigen concentration for all concentration ranges of samples used for measurement. These results mean that by calculating the concentration of the substance to be detected in the sample by summing the signals obtained from the two test lines in each example, it was possible to quantify the substance at high concentrations without reducing the measurement accuracy at low concentrations.

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] [Simultaneous reproducibility when measuring samples with low concentrations of NT-proBNP] Using the immunochromatography test device of Example 3-2, a sample with an NT-proBNP concentration of 40 pg / mL was measured five times. Table 10 shows the results of calculating the mean, standard deviation, and coefficient of variation for TL1 sensitivity, TL2 sensitivity, and the sum of TL1 sensitivity and TL2 sensitivity. The coefficient of variation for the sum of TL1 sensitivity and TL2 sensitivity was smaller than the coefficient of variation for the sensitivity of TL1 and TL2 individually. It is thought that the measurement error was reduced because antigens that did not bind to the solid phase antibody in the upstream TL1 could bind to the solid phase antibody in the downstream TL2.

[0081]

[0082] 1... Testing device, 11... Sample supply unit, 12... Detection unit, 12a... First detection area, 12b... Second detection area, 13... Flow channel, 14... Blood cell separation membrane, 15... Labeling substance holding unit, 16... Absorption pad, 17... Substrate, 18... Control line, 20... Sample solution, 31... Container unit, 32... Lid unit, 33... First opening, 34... Second opening, 35... Fixing pin

Claims

1. A method for quantifying a substance to be detected in a sample solution, comprising: using a testing instrument equipped with a channel through which the sample solution flows in one direction, wherein the channel is provided with, in order from the upstream side to the downstream side, a first detection region in which a capture substance that specifically binds to the substance to be detected is solidified, and a second detection region in which the capture substance is solidified, and after the sample solution has been circulated from the upstream side to the downstream side of the channel, the amount of the substance to be detected contained in the sample solution is determined based on the sum of a first signal intensity detected according to the amount of the substance to be detected captured in the first detection region and a second signal intensity detected according to the amount of the substance to be detected captured in the second detection region.

2. The quantitative method according to claim 1, wherein, in the flow path, the amount of the captured substance solidified in the first detection region is less than the amount of the captured substance solidified in the second detection region.

3. The quantitative method according to claim 1 or 2, wherein, when determining the amount of the substance to be detected contained in the sample solution, a standard sample with a known concentration of the substance to be detected and a calibration curve created using the testing instrument are used.

4. An optical measuring device comprising: an irradiation unit that irradiates a first detection area and a second detection area of ​​a testing instrument for quantifying a substance to be detected in a sample solution with measurement light; a measurement unit that measures the signal intensity emitted from the first detection area and the second detection area; and a determination unit that determines the amount of the substance to be detected in the sample solution based on the sum of the signal intensities emitted from the first detection area and the second detection area.

5. The optical measuring device according to claim 4, wherein the inspection instrument is mounted within the irradiation range of the measurement light of the irradiation unit, the first detection region and the second detection region are provided sequentially from the upstream side to the downstream side in the flow path of the inspection instrument, and a capture substance that specifically binds to the substance to be detected is immobilized in the first detection region and the second detection region, respectively.

6. The optical measuring device according to claim 5, wherein, in the flow path, the amount of the captured substance solidified in the first detection region is less than the amount of the captured substance solidified in the second detection region.

7. The optical measuring apparatus according to any one of claims 4 to 6, wherein the determination unit uses a standard sample with a known concentration of the substance to be detected and a calibration curve created using the testing instrument when determining the amount of the substance to be detected contained in the sample solution.

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