JP2012215420A - Measuring apparatus and measurement program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately measure a coloration state even when dust or the like exists between a reagent and an imaging device.SOLUTION: A measuring apparatus focuses on an attachment surface to which dust or the like is attached (step S100), obtains a second image by photographing the attachment surface (step 102), performs Fourier transform on the second image (step S104), focuses on a reagent strip (step S106), obtains a third image by photographing the reagent strip (step 108), and computes a removal image in which influence of dust or the like has been removed, based on an image obtained by Fourier transform of a first image photographed by attaching a virtual attached matter to the attachment surface in advance and focusing on the reagent strip, an image obtained by Fourier transform of the second image, and the third image.

Description

  The present invention relates to a measurement apparatus, a measurement program, and a measurement method, and in particular, a chromatographic measurement apparatus for measuring an assay device using a chromatographic method, in particular, a specific binding substance for a test substance is fixed. The present invention relates to a measuring apparatus, a measuring program, and a measuring method for measuring a test line and a coloration state of a control line for determining the end of measurement to inspect a test substance.

  In recent years, there has been an assay device that sends a sample (analyte solution) that may contain a test substance to a carrier and uses an assay method such as immunological measurement to test the test substance simply and quickly. Many devices have been developed, and various devices for in-vitro diagnostics, toxic substances and the like are commercially available. As an example of such a device, one utilizing an immunochromatographic method as shown in Patent Document 1 can be cited. When a device using an immunochromatography method is used, it is easy to operate without requiring heavy equipment / equipment for the determination / measurement. A measurement result is obtained only by leaving still for 5 to 10 minutes. Therefore, measurement methods using assay methods such as immunological measurement are widely used in many situations, such as clinical tests in hospitals or laboratory tests in laboratories, as a simple and rapid measurement method with high specificity. Yes.

  On the other hand, in clinical practice such as clinics, clinics, home medical care, etc., immunochromatographic measurement devices (immunochromatography readers) are used as measuring devices for POCT (Point of Care Testing) treatments that can be easily performed regardless of clinical laboratory specialists. ) Is used. This immunochromatographic measuring apparatus measures the coloration state of the reagent of the loaded device with high sensitivity, and enables high-sensitivity and highly reliable inspection even in a low coloration state where visual judgment is difficult. .

  When measuring the coloration state of this reagent, there may be a case where an erroneous determination is made due to the influence of dust and dust that has entered the measuring apparatus, and reflection from dust or dust on the reagent case containing the reagent. In particular, when dust, dust, or the like is present on the reagent reaction line and the coloration state is a threshold, the sample may be false positive or false negative.

  In Patent Document 2, even if dust other than the measurement component or remaining flow of the labeling substance remains in the immobilized reagent part provided on the chromatographic test piece, the concentration of the measurement component is determined by obtaining an accurate coloration degree. An immunochromatographic measurement apparatus capable of determining the above is disclosed.

JP 2008-139297 A JP 2009-98080 A

  When the reagent case containing the reagent is set in the measuring device, the reagent is photographed, and the coloration state of the reagent is measured based on the photographed image, the reagent case of the measuring device is set as a place where dust or dust is attached. The glass surface or the bottom surface of the reagent case may be considered. Since the adhesion surface to which dust adheres exists between the reagent and the image sensor, if the dust adheres to these adhesion surfaces, the photographed image is blurred and the coloration state of the reagent cannot be measured accurately. There is a case.

  However, the technique described in Patent Document 2 is not assumed for the case where dust or the like is present between the reagent and the imaging element as described above, and it is difficult to accurately measure the coloration state. There was a problem that there was.

  The present invention has been made to solve the above problems, and a measuring apparatus and a measuring program capable of accurately measuring a colored state even when dust or the like is present between a reagent and an image sensor. And it aims at providing a measuring method.

  In order to solve the above-mentioned problem, the measuring device according to claim 1 is provided with an imaging element and a reagent for determining whether or not a test substance is contained in a sample by an immunochromatographic method, and A reagent paper in which a specific binding substance that is colored when the sample contains the test substance is formed in a predetermined test line region, and between the reagent case containing the reagent paper and the imaging element A focusing means for focusing on any of the attached surfaces to which the attached matter can be attached; and a virtual attached matter corresponding to the attached matter that can be attached to the attached surface; A storage unit that stores a first image that is focused and captured by the image sensor, and a second image that is captured by the image sensor while being focused on the attachment surface after the sample is supplied to the reagent paper. Image and the test Control means for controlling the focusing means and the imaging element so as to obtain a third image focused by paper and picked up by the imaging element; the first image; the second image; And calculating means for calculating a removal image obtained by removing the attached image corresponding to the attached matter attached to the attached surface based on the third image.

  According to the present invention, the first image picked up by focusing the reagent paper by attaching the virtual sticking matter to the sticking surface on which dust or the like can adhere, and the second image picked up by focusing on the sticking surface. And the third image picked up in focus on the reagent paper, and the removal image obtained by removing the attached image corresponding to the attached matter attached to the attached surface is calculated. It becomes possible to measure with high accuracy.

  According to a second aspect of the present invention, the computing unit multiplies the first transformed image obtained by Fourier transforming the first image and the second transformed image obtained by Fourier transforming the second image. The removed image may be calculated by performing inverse Fourier transform on the obtained image and subtracting the inverse Fourier transformed image from the third image.

  According to a third aspect of the present invention, when there are a plurality of the attachment surfaces, a plurality of captured images obtained by focusing each of the attachment surfaces and performing Fourier transform, and the plurality of Fourier transform images obtained by performing the Fourier transform. The control means further includes a specifying means for specifying the attached surface to which the attached matter is attached, and the control means obtains the second image by focusing on the attached surface specified by the specifying means. As described above, the focusing unit and the image sensor may be controlled.

  A measurement program according to a fourth aspect of the invention is a measurement program for causing a computer to function as each means constituting the measurement apparatus according to any one of the first to third aspects.

  According to the measurement method of the invention described in claim 5, the imaging element and a reagent for determining whether or not the test substance is contained in the sample are formed by an immunochromatography method, and the test substance is present in the sample. A specific binding substance that is colored when included is a reagent paper that is formed in a predetermined test line region, and a deposit that can be adhered between the reagent case containing the reagent paper and the imaging element. A focusing unit that focuses on one of the surfaces, and a virtual deposit corresponding to a deposit that can be deposited on the deposition surface, and focuses on the reagent paper to focus on the reagent paper. A storage unit that stores a first image captured by the measurement method, wherein the sample is supplied to the reagent paper and then focused on the attachment surface. Take an image with the image sensor Controlling the focusing means and the imaging device to acquire a second image and a third image captured by the imaging device while focusing on the reagent paper, and the first image, Based on the second image and the third image, a removal image obtained by removing the adhesion image corresponding to the adhesion material adhering to the adhesion surface is calculated.

  According to the present invention, even when dust or the like is present between the reagent and the imaging device, the coloration state can be accurately measured.

It is a schematic perspective view which shows a chromatograph measuring apparatus. It is the schematic which shows the inside of the chromatograph measuring apparatus in the II line | wire of FIG. (A) is a schematic top view showing an assay device loaded in a chromatographic measurement apparatus, (B) is a schematic bottom view showing an assay device loaded in a chromatographic measurement apparatus, and (C) is a schematic diagram of (A). It is a schematic sectional drawing which shows the cross section of the device for an assay in the II-II line. It is a figure which shows the device for immunochromatography set on the 1st measurement part. It is a figure which shows a mode that a virtual adhering matter is made to adhere to an adhesion surface, it focuses on a reagent paper, and is image | photographed. It is a flowchart of the process performed by a control part. It is a figure which shows a mode that a focused surface is image | photographed. It is a figure which shows a mode that a reagent paper is focused and image | photographed. It is a block diagram of a computer.

  Hereinafter, although an embodiment of the present invention is described using a drawing, the present invention is not limited to this. In addition, for easy visual recognition, the scale of each component in the drawings is appropriately changed from the actual one.

(Chromatograph measuring device)

  First, the configuration of the chromatographic measurement apparatus 11 according to this embodiment will be described. FIG. 1 is a schematic perspective view showing the appearance of the chromatograph measuring apparatus 11 according to the present embodiment, and FIG. 2 is a schematic view showing the inside when the immunochromatographic device 120 is loaded and cut along line II in FIG. FIG. 3A and 3B are a schematic top view and a schematic bottom view showing the appearance of the immunochromatographic device 120, respectively. FIG. 3C is a schematic cross-sectional view taken along the line II-II in FIG. is there.

  As shown in FIGS. 1 and 2, the chromatograph measurement device 11 operates the casing 110, the screen display unit 111 disposed on the surface of the casing 110, and the menu displayed on the screen display unit 111. The menu operation unit 112 and the power switch 113, the device loading unit 114 for loading the immunochromatographic device 120 into the apparatus, and the first device for acquiring information from the immunochromatographic device 120 constituting the interior of the apparatus 1 A measurement unit 140, a second measurement unit 150, a determination unit 132 that determines the validity of the test result of the test substance and the necessity of amplification based on the information acquired by the first measurement unit 140, a predetermined unit An operation mode selection unit 134 that enables selection of an operation mode for causing the apparatus to perform operation processing, and a storage unit 1 that stores processing programs and various data. 6, an amplification solution supply unit 160 that develops the amplification solution on the insoluble carrier 121 in the immunochromatography device 120, the first measurement unit 140, the second measurement unit 150, the determination unit 132, the operation mode selection unit 134, And a control unit 130 for controlling the amplification solution supply unit 160.

  3A to 3C, the immunochromatographic device 120 includes an insoluble carrier 121 (reagent) having two test lines (A and B) and a control line C, and an insoluble carrier 121. Device housing 122 for housing, solution injection port 123 for injecting a reagent solution into the insoluble carrier 121, and observation for observing the inspection site 121a of the insoluble carrier 121 accommodated in the device housing 122 And an information display unit 125 is provided on the surface of the device casing 122. Note that the observation window 124 is made of a transparent member such as glass or plastic.

  Hereinafter, the measurement apparatus 11 will be described.

(Judgment part)

  The determination unit 132 of the chromatograph measurement device 11 determines the validity of the test result of the test substance and the necessity of amplification based on the coloration state of the test site measured by the first measurement unit 140. is there. Based on the validity determination, the progress of the test (solution development state, capture state of the test substance and labeling substance in the test line, capture state of the labeling substance in the control line, etc.) is normal, and the results obtained thereafter are reliable. Or whether the test is completed successfully and the result is reliable. In these cases, if the reliability is sufficient, the validity determination is made, and if the reliability is not enough, the error determination is made. On the other hand, whether or not it is necessary to amplify the coloration state of the examination site is determined based on the necessity determination of amplification. If necessary, an amplification necessity determination is made, and if not necessary, an amplification unnecessary decision is made. .

  Here, the test line A is a test line for inspecting the detected substance A, the test line B is a test line for inspecting the detected substance B different from the detected substance A, and the control line C is the end of the inspection ( This is a control line indicating that the labeling substance is being delivered to the point. The test line A and the test line B are colored when the test substances A and B are inspected, respectively, and the C line is colored when the test is completed regardless of the presence or absence of the test substance.

(Effectiveness judgment)

  When the determination unit 132 issues an error determination in the progress stage of the inspection, for example, the progress state may not be normal, so even if some inspection result is obtained, it is determined that the reliability of the result is low. And the like. Examples of the case where the progress of the inspection is not normal include a case where the control line is colored despite the initial stage of the inspection because the immunochromatographic device 120 is reused. When the determination unit 132 determines that the test is valid at the end of the test, the determination unit 132 also determines whether the test result is positive or negative. An example of a case where an error determination is issued by the determination unit 132 at the end stage of the inspection is a case where a normal inspection result cannot be obtained because the progress state is not normal.

(Amplification necessity judgment)

  The determination unit 132 determines whether amplification is necessary, for example, because the concentration of the test substance in the sample solution is originally low, or because of the nature of the reagent for the test line (test line and control line). Since the reaction completion time itself is long, there is a case where the progress of the test is predicted to be in a state where the test result cannot be obtained unless a considerable time is required even if the test is allowed to proceed as it is. . Here, it is possible to arbitrarily set at which point in the progress state the amplification necessity determination is performed. Prediction of whether or not a considerable amount of time is required can be predicted based on a change in the color state measured over time, for example, based on a change rate of optical density. When the determination unit 132 determines that amplification is not necessary, for example, because the concentration of the test substance in the sample solution is high or because the reaction completion time of the test itself is short, the progress state has already been confirmed. Or a case where it is predicted that a test result can be obtained without taking a considerable amount of time.

  There is no particular limitation on the timing at which the validity determination and amplification necessity determination are performed in the course of the test, but the most basic inspection process is the necessity determination of amplification and the validity determination after the elapse of a predetermined time from the sample solution injection. In this order, the final inspection result is obtained. The predetermined time is appropriately set by a user such as a doctor in accordance with the purpose of the examination and shortening the time for obtaining the examination result. For example, if the predetermined time is set short, the necessity of amplification can be determined at an early stage and the amplification process can be performed as necessary, so that the time until obtaining the inspection result can be shortened. Further, as will be described later, in order to detect an abnormality in the progress state in the initial stage of inspection, for example, an effectiveness determination can be added to the basic inspection process before the necessity of amplification is determined.

(Operation mode selection part)

  The operation mode selection unit 134 stores an operation mode for causing the apparatus to perform predetermined operation processing set in advance. The stored operation mode is displayed as a menu on the screen display unit 111 via the control unit 130, and the user of the measuring apparatus 11 operates the menu operation unit 112 to select an operation mode from the menu. be able to. As a result, the user can perform measurement suitable for the inspection purpose.

  For example, as an operation mode, the operation mode selection unit 134 causes the measurement unit to measure the optical density and chromaticity of the test line and the control line when a predetermined time elapses after the sample solution is spotted on the insoluble carrier. When the optical density and chromaticity for all lines reach the predetermined values, the determination means determines that amplification is not necessary, and determines the validity of the test result of the test substance without performing amplification. In other cases, the determination means determines that the amplification necessity determination is necessary, the amplification means amplifies the coloration state, and after amplification, the determination means validates the test result of the test substance. An operation mode (first operation mode) for performing sex determination is stored. According to such an operation mode, when there are a plurality of test lines in one immunochromatographic device 120, it is possible to perform a highly reliable test for all the test lines.

  Further, when there are two or more test lines in the immunochromatographic device 120, when the measuring apparatus 11 includes a designation unit that can designate a part of the two or more test lines as an inspection target, For example, as another operation mode, the operation mode selection unit 134 uses the optical density and chromaticity of the test line and the control line as measurement means when a predetermined time elapses after the sample solution is spotted on the insoluble carrier. When the optical density and chromaticity of the test line and control line specified by the specifying means reach a predetermined value, the determination means determines that the necessity of amplification is not necessary and does not perform amplification. In other cases, the test result of the test substance is determined. Otherwise, the determination means determines that the amplification necessity determination is necessary, and the amplification means determines the coloration state. It is the width, after amplification, stores the operation mode to perform the determination of validity of the test result of the test substance to the determining means (second operation mode). According to such an operation mode, it is possible to reduce the useless inspection time and to efficiently inspect the designated test line.

(Measurement part)

  The 1st measurement part 140 measures the coloration state of the test site | part 121a through the observation window 124 of the device 120 for immunochromatography, and acquires the optical information in the test site | part 121a. As shown in FIG. 2, the first measurement unit 140 includes an image sensor 142, a light source 144, and an optical lens (not shown in FIG. 2), and the immunochromatographic device 120 is loaded in the measurement apparatus 11. In this case, they are arranged below the immunochromatographic device 120 so as to face the observation window 124. Then, based on the optical information in the examination site 121a acquired by the first measurement unit 140, that is, the photographed image, the optical density and chromaticity are calculated as the coloration state of the examination site 121a. Although details will be described later, in the present embodiment, a removed image from which the influence of dust and the like is removed is obtained from a photographed image photographed by the image sensor 142, and the coloration of the examination region 121a is obtained based on the obtained removed image. The optical density and chromaticity are calculated as the state.

  The image sensor 142 has, for example, a configuration in which a plurality of photodiodes are arranged in a line or a configuration having an optical sensor such as an area sensor, and generates an output corresponding to the luminance of received light. The light receiving range of the image sensor 142 is a band extending in the longitudinal direction of the immunochromatographic device 120. The light source 144 is a module in which, for example, an LED is built, and is configured to emit white light. The light source 144 may emit, for example, monochromatic light as long as the chromaticity before and after amplification can be distinguished. In the case where the light source 144 includes a plurality of modules, a plurality of modules that emit monochromatic light having different wavelengths may be used. The light emitted from the light source 144 can illuminate the longitudinal direction of the immunochromatographic device 120.

  The second measurement unit 150 irradiates the information display unit 125 of the immunochromatographic device 120 with illumination light, and acquires information displayed on the information display unit 125. The information display unit 125 displays information related to the inspection by handwriting or sticker attachment. Information related to testing includes, for example, information related to the patient from whom the test substance was collected (name, age, sex, etc.) and information related to the sample / reagent used in the test (test substance to be tested, cleaning solution, amplification solution, etc.) Name). The method for acquiring the information is not particularly limited, and the information display unit 125 can be directly imaged or read from the barcode information. As shown in FIG. 2, the second measurement unit 150 includes an image sensor 152 and a light source 154. When the immunochromatographic device 120 is loaded in the measuring apparatus 11, these are arranged above the immunochromatographic device 120. The information display unit 125 is configured to face the information display unit 125. Then, the information related to the inspection acquired by the second measuring unit 150 and the inspection result are linked and managed. The image sensor 152 and the light source 154 are the same as the image sensor 142 and the light source 144, respectively.

(Optical density)

  The optical density is determined by the following equation (1), where I is the intensity of incident light incident on the test site of the assay device and Ir is the intensity of reflected light from the test site.

Optical density = −log 10 (Ir / I) (1)

(Chromaticity)

  The chromaticity is a numerical display of hue and saturation, and is calculated from the RGB luminance signal read by the image sensor. As a chromaticity color system, a general CIE color system can be used.

(Amplification solution supply unit)

  The amplification solution supply unit 160 includes an amplification solution storage pot 162 that stores an amplification solution containing a silver ion-containing compound, an amplification solution injection nozzle 162a that injects the amplification solution from the amplification solution storage pot 162, and silver contained in the amplification solution. A reducing agent storage pot 164 for storing a reducing agent for reducing ions; and a reducing agent injection nozzle 164a for injecting the reducing agent from the reducing agent storage pot 164. Each of the amplification solution storage pot 162 and the reducing agent storage pot 164 includes The stored liquid can be injected into the solution injection port 123 of the immunochromatography device 120 loaded through the respective injection nozzles 162a and 164a. The liquid may be injected by, for example, driving the amplification solution supply unit 160 by a driving device and sequentially or alternately injecting each of the injection nozzles from each other, or by combining two nozzles at the tip portion. You may make it inject | pour simultaneously the liquid supplied from the storage pot.

  The amplification solution supply unit 160 is not limited to the above configuration. For example, a combination of solutions stored in the respective storage pots may be a combination of an amplification solution and a washing solution, or a single pot for storing the amplification solution. Furthermore, the storage pot for storing the solution is not necessarily provided inside the measuring device 11. For example, a storage pot may be provided outside the measuring apparatus (housing 110), and only the injection nozzle may be operated to inject the solution into the solution injection port 123.

(Chromatograph measurement method)

  Next, the chromatographic measurement method of the present invention will be described.

  The measurement method according to this embodiment includes an insoluble carrier 121 having a test line on which a specific binding substance for a test substance is fixed, a control line for determining the end of measurement, and an immobilized labeling substance. An immunochromatographic device 120 is prepared, a sample solution containing a test substance to be tested is injected into the solution injection port 123 of the immunochromatographic device 120, and the sample solution is spotted on the insoluble carrier 121. After a predetermined time has passed, the optical density and chromaticity are measured as the coloration state at the examination site 121a, and amplification necessity determination (first determination) is performed based on the optical density and chromaticity, and the test lines (A and B) ) And at least one of the optical density and chromaticity of the control line C did not reach predetermined values. In such a case, the test line (A and B) and the control line C are cleaned by developing a reducing agent (here, the reducing agent also functions as a cleaning solution) on the insoluble carrier 121 (cleaning process), By developing the amplification solution on the insoluble carrier 121, silver particles formed by reduction of silver ions are deposited on the labeling substance to amplify the color state (amplification process), and the optical density of the amplified color state and The chromaticity is measured, and the validity determination (second determination) is performed based on the optical density and chromaticity. Otherwise, the amplification process (including the cleaning process and the amplification process) is not performed. The effectiveness determination (second determination) is performed based on the optical density and chromaticity at the time of amplification necessity determination (first determination).

  In the chromatographic measurement method according to the present invention, the predetermined time is preferably 5 minutes to 20 minutes. Moreover, it is preferable to perform 2nd determination within 1 minute-20 minutes after 1st determination. That is, by setting the time in this way, it is possible to determine whether or not amplification is necessary at an early stage and to obtain a test result more quickly.

  The angle formed between the developing direction of the sample solution and the developing direction of the cleaning liquid is preferably 45 to 170 degrees, and the insoluble carrier is preferably a porous carrier.

(Sample solution)

  The sample solution that can be analyzed using the assay device of the present invention is a sample solution that may contain a test substance (naturally-occurring substances, toxins, hormones, physiologically active substances such as agricultural chemicals, environmental pollutants, etc.). As long as it is not particularly limited, for example, biological samples, particularly body fluids of animals (particularly humans) (for example, blood, serum, plasma, spinal fluid, tears, sweat, urine, pus, runny nose, Or sputum) or excrement (for example, feces), organs, tissues, mucous membranes and skin, swollen specimens (swabs), gargles, or animals and plants themselves or their dried bodies, which are considered to contain them. And the like diluted with 1.

  The sample solution is used as it is or in the form of an extract obtained by extracting the sample solution with an appropriate extraction solvent, and further, a diluted solution obtained by diluting the extract with an appropriate diluent. Or an extract concentrated by an appropriate method.

(Labeling substance)

  As a labeling substance that can be used in the present invention, metal fine particles (or metal colloids), colored latex particles, enzymes, etc., which are used in general immunochromatography, can be visually confirmed or can be inspected by reaction. It can be used without any particular limitation as long as it is a labeled substance, but when a signal is amplified by metal deposition on the labeled substance by a metal ion reduction reaction using the labeled substance as a catalyst, From the viewpoint of catalytic activity, metal fine particles are preferred.

  As a material for the metal fine particles, a metal simple substance, metal sulfide, other metal alloy, or polymer particle label containing metal can be used. The average particle diameter of the particles (or colloid) is preferably in the range of 1 nm to 10 μm. Here, the average particle diameter is an average value of the diameters of the plurality of particles (the longest diameter of the particles) measured by a transmission electron microscope (TEM). More specifically, a gold colloid, a silver colloid, a platinum colloid, an iron colloid, an aluminum hydroxide colloid, a composite colloid thereof, and the like are preferable, and preferably a gold colloid, a silver colloid, a platinum colloid, and a composite colloid thereof. It is desirable to be. In particular, gold colloid and silver colloid are suitable in terms of appropriate particle diameters, and gold colloid is red and silver colloid is yellow, which is more preferable in view of high visibility. By using a gold colloid, the chromaticity of the label changes before and after amplification by performing an amplification process using a silver ion-containing compound (the gold colloid is colored red, and after amplification, the reduced silver ions become gold As described later, this change can be used for inspection error determination. The average particle size of the metal colloid is preferably about 1 to 500 nm, more preferably 1 to 100 nm.

(Specific binding substance)

  The specific binding substance is not particularly limited as long as it has an affinity for the test substance. For example, when the test substance is an antigen, an antibody against the antigen, the test substance is a protein, Aptamers for metal ions or low molecular weight organic compounds, and nucleic acids such as DNA and RNA having sequences complementary to these when the test substance is a nucleic acid such as DNA and RNA. Examples include biotin when the substance is avidin, and a complex that specifically binds to the substance when the test substance is a specific peptide. In the above example, the relationship between the specific binding substance and the test substance can be exchanged. For example, when the test substance is an antibody, an antigen against the antibody can be used as the specific binding substance. Furthermore, a compound or the like partially containing a substance having affinity for the test substance as described above can also be used as the specific binding substance.

  Specific examples of the antibody include an antiserum prepared from the serum of an animal immunized with the test substance, an immunoglobulin fraction purified from the antiserum, and a spleen cell of the animal immunized with the test substance. Monoclonal antibodies obtained by cell fusion, or fragments thereof [eg, F (ab ′) 2, Fab, Fab ′, or Fv] can be used. These antibodies can be prepared by a conventional method.

(Signal signal amplification)

  In the case of an assay using a metal colloid label, metal sulfide label, other metal alloy label, or polymer particle label containing a metal as the labeling substance, the signal of the metal-based label can be amplified. Specifically, after the complex of the test substance and the labeling substance is formed, a silver ion supplied from a compound containing silver such as an inorganic silver salt or an organic silver salt and a reducing agent are brought into contact with each other, and the silver ion is reduced by the reducing agent. When silver particles are produced by reduction, the silver particles are deposited on the metal label using the metal label as a nucleus, so that the metal label is amplified and the analysis of the test substance can be performed with high sensitivity. .

(Insoluble carrier)

  The insoluble carrier 121 includes a sample addition pad that is a portion where the specimen solution is dropped, a labeling substance holding pad to which a labeling substance that can bind to the test substance is fixed, and a test site to which a specific binding substance for the test substance is fixed. And an absorption pad that absorbs the sample solution and the like. The test site is provided with two test lines to which different specific binding substances are fixed, but the number of test lines is not limited to this, and may be one or three or more. By using a plurality of test lines, when a plurality of test substances are included in the sample solution, a plurality of test substances can be efficiently inspected at a time. The material of the insoluble carrier 121 is preferably porous, for example, a nitrocellulose membrane, a cellulose membrane, an acetylcellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, glass fiber, a nonwoven fabric, a cloth, or a thread. Preferably mentioned.

  On the chromatographic carrier, a specific binding substance for the test substance is immobilized, and a test line or a control site is produced if desired. The specific binding substance may be directly immobilized on a part of the chromatographic carrier by physical or chemical binding, or the specific binding substance may be physically or chemically attached to fine particles such as latex particles. The fine particles may be trapped on a part of the chromatographic carrier and immobilized.

  The labeling substance holding pad is prepared by preparing a suspension containing the labeling substance described above, applying the suspension to an appropriate pad (for example, a glass fiber pad), and then drying it. . Thereby, when the sample solution is injected into the insoluble carrier, the labeling substance is combined with the test substance while being developed together with the test substance, and is flowed to the examination site.

  The sample addition pad is a part for spotting a sample (specimen solution or the like), and also a part for filtering insoluble matter particles or the like in the sample. In addition, in order to prevent the analyte in the sample solution from adsorbing non-specifically on the material of the sample addition part and reducing the accuracy of the analysis during the analysis, the sample addition pad is pretreated with non-specific adsorption. Sometimes used.

  The absorption pad is a part where the added sample is physically absorbed by the chromatographic movement and absorbs and removes the unreacted labeling substance that is not insolubilized in the inspection line of the chromatographic carrier. The chromatographic speed after the chromatographic tip of the added sample reaches the absorber varies depending on the material, size, etc. of the absorbent, so it is possible to set a speed that suits the measurement of the test substance. it can.

(Amplification solution)

  The amplification solution is a solution capable of causing a signal amplification by generating a colored or luminescent compound or the like by the contained drug reacting with the catalytic action of the labeling substance or the test substance. For example, a silver ion solution that causes metal silver to be deposited by physical development on a metal label. Details include general books in the field of photographic chemistry (for example, “Basics of Revised Photo Engineering-Silver Salt Photo Edition” (Japan Photographic Society, Corona), “Photochemistry” (Akira Sakurai, Photo Industry Publishing) ), “Latest Prescription Handbook” (Shinichi Kikuchi et al., Amico Publishing Co., Ltd.)), so-called developers can be used. For example, when a physical developer containing a silver ion-containing compound is used as the amplification solution, the silver ions in the solution can be reduced with a silver ion reducing agent centering on a metal colloid or the like that serves as a development nucleus.

  As another example, an enzyme reaction is used. For example, a solution of a phenylenediamine compound and a naphthol compound, which becomes a dye by the action of a peroxidase label and hydrogen peroxide, can be used. Further, it may be a chromogenic substrate for detecting horseradish peroxidase as described in the non-patent document "Staining using clinical test Vol. 41 no. 9 p. 1020 H2O2-POD system". Further, the chromogenic substrate described in JP-A-2009-156612 can be particularly preferably used. A system using a metal catalyst such as platinum fine particles instead of the enzyme may be used.

  As an example using another enzyme, there is a system in which alkaline phosphatase is used as a label and color is developed using 5-bromo-4chloro-3-indolyl-phosphate disodium salt (BCIP) as a substrate. As described above, the reaction that gives a color is given as a representative, but any combination of enzyme and substrate, such as those generally used in enzyme immunoassays, may be used, and even a chemiluminescent substrate is a substrate that emits fluorescence. May be.

(Silver ion-containing compound)

  As the silver ion-containing compound, an organic silver salt, an inorganic silver salt, or a silver complex can be used. Preferably, it is a silver ion-containing compound having high solubility in a solvent such as water, and examples thereof include silver nitrate, silver acetate, silver lactate, silver butyrate, and silver thiosulfate. Particularly preferred is silver nitrate. The silver complex is preferably a silver complex coordinated to a ligand having a water-soluble group such as a hydroxyl group or a sulfone group, and examples thereof include hydroxythioether silver. The inorganic silver salt or silver complex is generally contained in an amount of 0.001 mol / m 2 to 0.2 mol / m 2, preferably 0.01 mol / m 2 to 0.05 mol / m 2 as silver.

(Silver ion reducing agent)

  As the silver ion reducing agent, any inorganic or organic material or a mixture thereof can be used as long as it can reduce silver ions to silver.

  Preferred examples of the inorganic reducing agent include reducible metal salts and reducible metal complex salts whose valence can be changed by metal ions such as Fe2 +, V2 +, and Ti3 +. When using an inorganic reducing agent, it is necessary to complex or reduce the oxidized ions to remove or render them harmless. For example, in a system using Fe2 + as a reducing agent, a complex of Fe3 + that is an oxide can be formed using citric acid or EDTA and rendered harmless. In this system, it is preferable to use such an inorganic reducing agent, and more preferable is a metal salt of Fe2 +.

  Note that detailed description of specific examples such as amplification necessity determination that can be analyzed using the assay device according to the present embodiment, detailed determination before and after amplification based on optical density and chromaticity, and the like will be omitted. However, for example, the method and method described in Japanese Patent Application No. 2009-22610 filed by the present applicant can be used.

(Acquisition of removed images with dust removed)

  Next, a process for obtaining a removed image obtained by removing the influence of dust and the like from a photographed image photographed by the image sensor 142 will be described.

  FIG. 4 shows a state where the immunochromatographic device 120 is set on the first measurement unit 140. Although not shown in FIGS. 1 to 3, the image sensor 142 and the optical lens 143 of the first measurement unit 140 are formed above the glass surface 145, and the immunochromatographic device 120 is set on the glass surface 145. The

  The optical lens 143 can be moved in the arrow Y direction in FIG. 4 under the control of the control unit 130, and can be focused on the reagent paper S (insoluble carrier 121), the observation window 124, and the glass surface 145. It is configured as follows.

  In the case of such a configuration, a deposit G such as fine dust or dust adheres to the observation window 124 formed of a transparent member such as glass or plastic of the glass surface 45 of the first measurement unit 140 or the device for immunochromatography 120. There is a case. When the reagent paper S is photographed in a state where dust or dust adheres to the glass surface 145 or the observation window 124, the photographed image is blurred due to the dust or dust, and the test lines A and B of the reagent paper S are based on the photographed image. When determining the coloration state of the control line C, there is a risk of erroneous determination.

  For this reason, in this embodiment, a removed image obtained by removing the influence of dust and dust from the image photographed by the image sensor 142 is acquired, and the coloration state of the test line and the control line is determined based on the removed image.

  Although the details will be described later, this removed image is obtained as follows. First, as shown in FIG. 5, a virtual deposit K assumed as dust or the like adhering to the adhesion surface F (observation window 124 or glass surface 145) of dust or the like is adhered to the adhesion surface F so that the focus is on the reagent paper. The optical lens 143 is moved so as to match S, and the reagent paper S is photographed by the image sensor 142. This captured image is used as the first image h1, and the first image is subjected to Fourier transform, and this is stored in advance in the storage unit 136 as the first converted image H1. In FIG. 5, the shape of the immunochromatographic device 120 is simplified and described, and the observation window 124 and the glass surface 145 are represented as an adhesion surface F to which dust or the like adheres. The same applies to FIGS.

  The virtual deposit K can be, for example, PS (polystyrene) particles, and the size thereof can be, for example, a sphere having a diameter of about 20 μm. The reason why the size of the PS particles is set in this way is that, in the present embodiment, the minimum resolution of the focus surface is, for example, about 20 μm, and the most likely dust size is about 10-20 μm. is there. Note that the type and size of the virtual deposit K is not limited to this, and may be appropriately determined according to the minimum resolution of the focus surface and the size of assumed dust.

  Next, processing executed by the control unit 130 will be described with reference to a flowchart shown in FIG.

  In the process shown in FIG. 6, the immunochromatographic device 120 is set in the device loading unit 114, the menu operation unit 112 is operated, the operation mode is selected, the measurement is instructed, and the sample solution (sample) is converted into the insoluble carrier. This is executed after a predetermined time has elapsed since the spotting, that is, when the coloration state of the test line or control line can be appropriately measured.

  First, in step S100, the optical lens 143 is moved to focus on the adhesion surface F as shown in FIG.

  In step S102, the adhesion surface F is photographed by the image sensor 142, and the photographed image is acquired as the second image h2.

  In step S104, the acquired second image is subjected to Fourier transform, and this is defined as a second transformed image H2.

  In step S106, as shown in FIG. 8, the optical lens 143 is moved so that the reagent paper S is in focus.

  In step S108, the reagent paper S is photographed by the image sensor 142, and the photographed image is set as a third image h3.

  In step S110, based on the first converted image H1, the second converted image H2, and the third image h3, a removed image h4 from which the influence of dust or the like has been removed is obtained by the following equation.

h4 = h3-F [H0] (1)

H0 = H1 × H2 (2)

Here, F [] represents an inverse Fourier transform function. The converted image H0 obtained by the above equation (2) is a first converted image H1 which is an image obtained by Fourier transforming an image obtained by attaching the virtual deposit K to the adhesion surface F and focusing on the reagent paper S. The image obtained by focusing on the adhesion surface F is multiplied by the second converted image H2, which is an image obtained by Fourier transform. For this reason, it can be said that H0 is an image obtained by Fourier transforming an image of the deposit G such as dust actually adhered to the adhesion surface F at the position of the reagent paper S.

  Therefore, H0, which is an image obtained by Fourier transforming an image of dust or the like at the position of the reagent paper S from the image h3 photographed in focus on the reagent paper S according to the above equation (1), is obtained by the inverse Fourier transform function F []. By subtracting the inverse Fourier transformed image F [H0], a removed image h4 from which the influence of dust or the like is removed is obtained. By measuring the coloration state of the test line and the control line using the removed image h4 by the method described above, the coloration state of the test line and the control line can be accurately measured.

  In order to obtain a removed image from which dust and the like adhering to both the observation window 124 and the glass surface 145 are removed, the following may be performed.

  First, the processes of steps S100 to S104 are performed on both the observation window 124 and the glass surface 145, and a second converted image H2A, which is an image obtained by Fourier transforming an image photographed in focus on the observation window 124, the glass surface 45. H2B, which is an image obtained by performing Fourier transform on an image taken with the focus on the image, is acquired.

  In step S110, a removed image h4 is obtained by the following equations (3) to (5).

h4 = h3-F [H0A] -F [H0B] (3)

H0A = H1 × H2A (4)

H0B = H1 × H2B (5)

  Further, the above-described processing may be executed after specifying which of the observation window 124 and the glass surface 145 has dust or the like attached thereto. In this case, prior to the processing of FIG. 6, first, a second transformed image H2A, which is an image obtained by performing Fourier transform on an image photographed with the observation window 124 in focus, and an image photographed with focus on the glass surface 145 are Fourier transformed. H2B which is the converted image is acquired. When dust is not attached, the Fourier transformed image should be flat in all frequency regions, but when dust is attached, it peaks at a frequency corresponding to dust. There should be. Therefore, it is possible to specify the attached surface to which dust or the like is attached depending on whether or not there is a peak in the second converted image H2A and the second converted image H2B. Then, after the attached surface is specified, the processes after step S100 may be executed on the specified attached surface.

  As described above, in the present embodiment, the image F [H0 obtained by performing the inverse Fourier transform on H0, which is an image obtained by Fourier transforming an image such as dust at the position of the reagent paper S from the image h3 photographed with the reagent paper S in focus. ] To obtain a removed image h4 from which the influence of dust or the like is removed, and the coloration state of the test line or the control line is measured based on this image. Thereby, the coloration state of a test line or a control line can be accurately measured.

  The control unit 130 can be configured by a computer 130 as shown in FIG. The computer 130 includes a CPU (Central Processing Unit) 130A, a ROM (Read Only Memory) 130B, a RAM (Random Access Memory) 130C, a non-volatile memory 130D, and an input / output interface (I / O) 130E via a bus 130F. It is a connected configuration. In this case, the processing program shown in FIG. 6 is written in the storage unit 136 or the non-volatile memory 130D, and is read and executed by the CPU 130A, whereby the computer 130 functions as the control unit 130.

  In the said embodiment, although the immunochromatography method was demonstrated as an assay method, the assay method of this invention is not limited to this. A system that does not use a so-called immune reaction may be used, for example, a system that captures a test substance with a nucleic acid such as DNA or RNA without using an antibody, or another small molecule having affinity for the test substance or A system in which a test substance is captured by a peptide, protein, complex-forming substance, or the like may be used.

11 Chromatographic Measurement Device 114 Device Loading Unit 120 Immunochromatographic Device 121 Insoluble Carrier 122 Device Case 124 Observation Window 130 Control Unit 132 Determination Unit 134 Operation Mode Selection Unit 136 Storage Unit 140 Measurement Unit 142 Image Sensor 143 Optical Lens 144 Light Source 145 Glass surface

Claims (5)

An image sensor;
A specific binding substance that is colored when a reagent for determining whether or not the sample contains a test substance is formed by the immunochromatography method and the sample contains the test substance is predetermined. Focusing means for focusing on any of the reagent paper formed in the test line region, the reagent case in which the reagent paper is accommodated, and the adhesion surface on which the deposit can adhere between the imaging element,
Storage means for storing a first image captured by the imaging element by attaching a virtual deposit corresponding to an deposit that can be adhered to the adhesion surface on the adhesion surface and focusing on the reagent paper;
After the sample is supplied to the reagent paper, a second image that is imaged by the imaging element while focusing on the adhesion surface, and a second image that is imaged by the imaging element while focusing on the reagent paper Control means for controlling the focusing means and the image sensor so as to obtain the image of
An arithmetic means for calculating a removal image obtained by removing the adhesion image corresponding to the adhesion material adhered to the adhesion surface based on the first image, the second image, and the third image;
Measuring device. The computing means performs inverse Fourier transform on an image obtained by multiplying the first transformed image obtained by Fourier transforming the first image and the second transformed image obtained by Fourier transforming the second image, and the inverse Fourier transform is performed. The measuring apparatus according to claim 1, wherein the removed image is calculated by subtracting the converted image from the third image. When there are a plurality of the attachment surfaces, each of the plurality of picked-up images picked up by focusing on each attachment surface is subjected to Fourier transform, and the attached matter adheres based on the Fourier transform images obtained by the Fourier transform. Further comprising a identifying means for identifying the attached surface,
The said control means controls the said focusing means and the said image pick-up element so that it may focus on the said adhesion surface specified by the said specification means, and may acquire the said 2nd image. measuring device.   The measurement program for functioning a computer as each means which comprises the measuring apparatus of any one of Claims 1-3. An image sensor;
A specific binding substance that is colored when a reagent for determining whether or not the sample contains a test substance is formed by the immunochromatography method and the sample contains the test substance is predetermined. Focusing means for focusing on any of the reagent paper formed in the test line region, the reagent case in which the reagent paper is accommodated, and the adhesion surface on which the deposit can adhere between the imaging element, Storage means for storing a first image picked up by the imaging device by attaching a virtual attachment corresponding to an attachment that can adhere to the attachment surface onto the attachment surface and focusing on the reagent paper; A measurement method executed by a measurement device provided,
After the sample is supplied to the reagent paper, a second image that is imaged by the imaging element while focusing on the adhesion surface, and a second image that is imaged by the imaging element while focusing on the reagent paper Controlling the focusing means and the image sensor so as to obtain an image of 3;
A measurement method for calculating a removed image from which an attached image corresponding to an attached matter attached to the attached surface is removed based on the first image, the second image, and the third image.

Images