JP2011214858A - Chromatograph measuring method, and insoluble carrier and measuring device used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable enhancement of a measurement system in a chromatograph measuring method.SOLUTION: The chromatograph measuring method includes the steps of irradiating an inspection region 21a of an insoluble carrier 21 including a detection part T and surroundings of the detection part T with ultraviolet light L to be absorbed by a labeled substance, using the labeled substance having absorption characteristics in a wavelength band of the ultraviolet light L; detecting light Lf to be produced from the inspection region 21a, attributable to irradiation with the ultraviolet light L; and measuring a presence and/or amount of a test object substance, depending on a dark part of the inspection region related to this light, attributable to the absorption characteristics of the labeled substance.

Description

  The present invention relates to a chromatographic measurement method for measuring an assay device using a chromatographic method, and an insoluble carrier and a measurement apparatus used therefor, in particular, a detection unit to which a substance exhibiting specific binding to a test substance is fixed. The present invention relates to a chromatographic measurement method for measuring the coloration state to determine the presence and / or amount of a test substance, and to an insoluble carrier and a measurement apparatus used therefor.

  In recent years, a sample (analyte solution) that may contain a test substance is sent to an insoluble carrier, and the presence and / or amount of the test substance can be conveniently and rapidly measured using an assay such as immunological measurement. A chromatographic measurement method has been developed. Along with this, a large number of devices used for the above-described measurement methods have been developed, and various devices for measuring in vitro diagnostics, poisons, and the like are commercially available. An example of such a device is one utilizing an immunochromatographic method. When using a device that uses the immunochromatography method, it is easy to operate because it does not require heavy equipment and equipment to measure the test substance. Then, a measurement result is obtained only by leaving still for about 5 to 10 minutes. For this reason, measurement methods using assay methods such as immunological measurement are widely used in many situations, for example, clinical tests in hospitals or laboratory tests in laboratories, as a simple, rapid and highly specific measurement method. Yes.

  On the other hand, in clinical practice such as clinics, clinics, home medical care, etc., immunochromatograph measuring devices (immunochromatography readers) are used as POCT (Point of Care Testing) measuring devices for simple measurements regardless of clinical laboratory specialists. ) Is used. This immunochromatographic measuring apparatus measures the coloration state of the reagent in the line-shaped detection unit of the loaded device with high sensitivity, and performs highly sensitive and reliable measurement even in the coloration state where visual judgment is difficult. Make it possible.

  For example, in Patent Document 1, in order to improve a decrease in reproducibility when the color state of a reagent of a device is visually determined, the color state is imaged by an image sensor, and the brightness of each pixel of the image sensor is determined. A method is disclosed in which a corresponding gradation image is acquired, the degree of coloration of the detection unit is calculated based on the gradation image, and the measurement result is determined.

Japanese Patent No. 3541232

  However, in the immunochromatographic measurement as described above, when measuring the light emission characteristics of the label itself on the insoluble carrier, the autofluorescence of the insoluble carrier made of a polymer film such as nitrocellulose becomes noise, resulting in measurement accuracy. There is a problem that decreases.

  The present invention has been made in view of the above problems, and it is an object of the present invention to provide a chromatographic measurement method capable of improving the measurement system in the chromatographic measurement method, and an insoluble carrier and a measuring apparatus used therefor. is there.

In order to solve the above problems, the chromatographic measurement method according to the present invention is:
A sample solution that may contain a test substance is developed on an insoluble carrier having a detection part to which a substance exhibiting specific binding properties to the test substance is fixed, and the sample solution is brought into contact with the detection part, whereby the specific binding is performed. In a chromatographic measurement method for measuring the presence and / or amount of a test substance fixed to a detection unit via a substance exhibiting sex,
A sample solution is brought into contact with the detection unit, and a labeling substance having a light absorption characteristic in the wavelength band of ultraviolet light is brought into contact with the detection unit.
Irradiate ultraviolet light that is absorbed by the labeling substance onto the inspection site of the insoluble carrier including the detection unit and the periphery of the detection unit,
Detect the light generated from the examination site due to the irradiation of this ultraviolet light,
The presence / absence and / or amount of the test substance is measured by the dark part of the test site relating to the light generated from the test site, which is caused by the light absorption characteristics of the labeling substance.

  In the present specification, the “substance exhibiting a specific binding property to a test substance” means a substance that specifically recognizes and binds to the test substance, such as an antibody against an antigen or a cofactor to a protein.

  “Determining the presence or absence of a test substance and / or measuring its amount” means qualitatively determining the presence or absence of a test substance, quantitatively measuring the presence of a test substance, or It means to measure the degree of activity of a substance.

  “Having light absorption characteristics in the wavelength band of ultraviolet light” means having light absorption characteristics in at least a part of the wavelength band of ultraviolet light.

  “With the sample solution brought into contact with the detection unit”, with the labeling substance brought into contact with the detection unit, the labeling substance placed in a dry state between the test site in the insoluble carrier and the part where the sample solution is dropped is placed on the sample solution. When the sample solution is dissolved in the sample solution at the time of development and brought into contact with the sample solution, or after the sample solution is brought into contact with the detection unit, a solution containing a labeling substance is separately supplied to the test site. Including meaning.

  “Contacting the labeling substance with the detection part” means that the labeling substance itself is supplied to the detection part (test site) and brought into contact, or a metal ion-containing compound is supplied to an insoluble carrier and the metal ions are reduced. This means that metal fine particles are generated as the labeling substance, and the metal fine particles are brought into contact with the detection part (test site).

  “Inspection site” is a predetermined portion of an insoluble carrier that includes the detection unit and the surroundings of the detection unit, and is an area that can contrast the coloration state of the detection unit with the surrounding coloration state. Means a selected part. The examination site need not include all of the detection units, and may be selected to include a part of the detection units. Further, it is not necessary that the inspection site is selected so that the detection unit and its surroundings are one continuous region. The first region including at least a part of the detection unit and the first region May be selected to be a non-continuous region and a second region including the periphery of the detection unit.

  The term “light generated from the test site” is meant to include reflected and scattered light of ultraviolet light at the test site, as well as autofluorescence from the test site and fluorescence from the fluorescent material added by the fluorescence treatment of the insoluble carrier. is there.

  Furthermore, in the chromatographic measurement method according to the present invention, the fluorescence generated from the test site when excited by ultraviolet light can be detected as the light generated from the test site.

  Furthermore, in the chromatographic measurement method according to the present invention, it is preferable to use an insoluble carrier that has been subjected to a fluorescence treatment so as to emit fluorescence when excited by ultraviolet light. In this case, the fluorescence treatment can form a fluorescent layer that is excited by the ultraviolet light and emits fluorescence in the portion of the examination site that is shaded by the irradiated ultraviolet light.

  In the present specification, “fluorescence treatment” means that an insoluble carrier is provided with a fluorescence mechanism that emits fluorescence when excited by ultraviolet light.

  “Part of the test site that is shaded by the irradiated ultraviolet light” means a part other than the part irradiated with ultraviolet light without passing through and scattering through the insoluble carrier due to the three-dimensional shape of the insoluble carrier. Means the part of the test site (including the inside of the insoluble carrier). For example, when one surface of a plate-like insoluble carrier is irradiated with ultraviolet light, the other surface can be cited as the shadow part, and ultraviolet light is irradiated from one direction to the side of the cylindrical insoluble carrier. In such a case, the opposite side surface can be cited as the shaded portion.

  Further, in the chromatographic measurement method according to the present invention, it is preferable to detect fluorescence through a filter that transmits fluorescence and blocks ultraviolet light, and it is preferable to use silver fine particles as a labeling substance.

Insoluble carrier for chromatography according to the present invention,
An insoluble carrier having a detection part to which a substance exhibiting specific binding property to a test substance is fixed, and having a fluorescence mechanism that emits fluorescence when excited by ultraviolet light .

  Furthermore, in the insoluble carrier for chromatographs according to the present invention, the fluorescence mechanism is a portion of the inspection site of the insoluble carrier including the detection portion and the periphery of the detection portion, and is shaded by the irradiated ultraviolet light. A fluorescent layer that is formed in a portion and emits fluorescence when excited by ultraviolet light is preferable.

  The insoluble carrier for chromatography according to the present invention is preferably provided with a dropping portion for dropping the sample solution, and a labeling substance disposed in a dry manner between the dropping portion and the test site.

The chromatographic measurement apparatus according to the present invention is
Insoluble in which a fluorescence mechanism that emits fluorescence when excited by ultraviolet light is provided at a test site including the detection unit and the periphery of the detection unit has a detection unit to which a substance exhibiting specific binding to the test substance is fixed A carrier;
A label contact means for contacting a test substance with a labeling substance having an absorption characteristic in the wavelength band of ultraviolet light;
A light source that emits ultraviolet light that is absorbed by the labeling substance, and an irradiation means that irradiates the inspection site with ultraviolet light;
A detection means comprising a photodetector and detecting by the photodetector the fluorescence generated from the examination site when excited by the ultraviolet light;
A measuring means for measuring presence / absence and / or amount of a test substance by a dark part of a test site relating to fluorescence detected by the detection means, which is caused by the light absorption characteristic of the labeling substance. is there.

  In the chromatographic measurement apparatus according to the present invention, the fluorescence mechanism is preferably a fluorescent layer that is formed in a portion of the examination site that is shaded with respect to the irradiated ultraviolet light and emits fluorescence when excited by the ultraviolet light. .

  In the chromatographic measurement method according to the present invention, a labeling substance having a light absorption characteristic in the wavelength band of ultraviolet light is brought into contact with the detection unit, and then the ultraviolet light absorbed by the labeling substance is irradiated to the examination site. Detecting the light generated from the examination site due to irradiation, and measuring the presence and / or amount of the test substance in the dark part of the examination site related to this light caused by the light absorption characteristics It is. As a result, in the chromatographic measurement method, the measurement accuracy can be improved because ultraviolet light is absorbed by the light absorbing label.

  Further, the insoluble carrier for chromatography and the chromatographic measurement apparatus according to the present invention are provided with a fluorescence mechanism that emits fluorescence when excited by ultraviolet light, so that the detection target is made fluorescent in the chromatographic measurement method. In this case, fluorescence can be generated more efficiently, and further improvement in measurement accuracy can be realized.

It is the schematic which shows an example of the measuring apparatus for performing the chromatograph measuring method of 1st Embodiment. It is a schematic top view which shows the external appearance of the chromatographic device used for the chromatographic measuring method. It is a schematic bottom view which shows the external appearance of the chromatographic device used for the chromatographic measurement method. It is a general | schematic cutting part end view in the II line | wire of FIG. 2A. It is a schematic sectional drawing of the insoluble support | carrier used for the chromatographic measurement method of 1st Embodiment. It is a schematic sectional drawing of the insoluble support | carrier used for the chromatographic measurement method of 2nd Embodiment. It is a schematic perspective view which shows the measuring apparatus for performing the chromatograph measuring method of 3rd Embodiment. It is a general | schematic cutting part end elevation which shows the inside of the chromatograph measuring apparatus in the II-II line | wire of FIG. It is a figure which shows the gradation image obtained in the Example. It is a figure which shows the gradation image obtained by the comparative example. The pixel values in the direction perpendicular to the length direction of the insoluble carrier are averaged for the inspection site (region X) in the gradation image obtained in the example, and this average value is one-dimensionally in the length direction of the insoluble carrier. It is a figure which shows the graph of the lightness arranged in. For the inspection site (region Y) in the gradation image obtained in the comparative example, the pixel values in the direction perpendicular to the length direction of the insoluble carrier are averaged, and this average value is one-dimensionally in the length direction of the insoluble carrier. It is a figure which shows the graph of the lightness arranged in.

  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.

<First embodiment of chromatographic measurement method>
A chromatographic measurement method according to the first embodiment will be described. In this embodiment, the presence / absence of the antigen is determined by a sandwich method using a test substance as an antigen, a substance exhibiting specific binding to the test substance as an antibody, and a labeling substance (absorption label) that absorbs ultraviolet light. Alternatively, a case where the amount is measured will be described as an example.

  The chromatographic measurement method of the present embodiment is performed using a chromatographic measurement apparatus 1 including a chromatographic device 20 including an insoluble carrier 21 as shown in FIG.

  More specifically, in the chromatographic measurement method of the present embodiment, there is a possibility that the insoluble carrier 21 having the line-shaped detection portion T on which an antibody exhibiting specific binding to the antigen is immobilized contains the antigen. The sample solution is dropped, and the sample solution is developed on the insoluble carrier 21 while dissolving the light-absorbing label having a light-absorbing property in the wavelength band of the ultraviolet light L and disposed on the insoluble carrier 21 in the sample solution. The solution is brought into contact with the detection unit T, and the light-absorbing label is brought into contact with the detection unit T so that the detection unit T forms an antibody-antigen-absorption label sandwich structure. One surface of the inspection site 21a of the insoluble carrier 21 is irradiated with ultraviolet light L absorbed by the light-absorbing label, and the autofluorescence Lf of the insoluble carrier 21 generated from the inspection site 21a when excited by the ultraviolet light L is applied to the other surface. The contrast of the autofluorescence Lf generated between the detection portion T and the surroundings due to the light-absorbing property of the light-absorbing label is measured through the filter 16, and the presence and / or amount of the antigen is determined by this contrast. The measurement is performed.

  Specifically, the measuring apparatus 1 shown in FIG. 1 includes a chromatographic device 20 including an insoluble carrier 21, a transparent installation base 18 on which the device is installed, and an inspection site of the insoluble carrier 21 in the installed device 20. The light source 10 that irradiates the ultraviolet light L to 21a, the filter 16 that transmits the fluorescence Lf generated from the examination site 21a and blocks the irradiated ultraviolet light L, and the side opposite to the side where the light source 10 is present with respect to the device 20 The contrast of the fluorescence Lf generated between the arranged photodetector 12 and the detection portion T due to the light absorption characteristics of the labeling substance and its surroundings is measured, and the presence / absence and / or amount of the test substance is measured by this contrast. And measuring means 14 for performing the above.

(Chromatograph device)
The chromatographic device 20 includes, for example, an insoluble carrier 21 and a device housing 22 that houses the insoluble carrier 21. 2A and 2B are a schematic top view and a schematic bottom view showing the external appearance of the device, respectively, and FIG. 2C is a schematic cross-sectional view taken along the line II in FIG. 2A.

  As shown in FIGS. 2A, 2B, and 2C, the device housing 22 of the chromatographic device 20 includes an inlet 23 for dropping a sample such as a sample solution onto the insoluble carrier 21, and an insoluble carrier 21 in the device housing. An irradiation window 24a opened so as to be able to irradiate ultraviolet light L to the inspection site 21a, and an observation window 24b opened so that the inspection site 21a can be observed from the outside of the device housing. An information display unit 25 is provided on the surface of the housing 22.

  As shown in FIG. 3, the insoluble carrier 21 exhibits specific binding properties to the sample addition pad 81, which is a portion where the sample is dropped, in the direction in which the sample such as the sample solution is developed (direction of arrow A), and the antigen. Absorbs the labeled substance holding pad 82 to which the light-absorbing label is fixed, the chromatographic carrier 83 having the line-shaped detection portion T (test line) to which the antibody showing the specific binding property to the antigen is fixed, and the sent sample. It has the absorption pad 84 and the transparent sheet 85 which supports these whole. Although one test line T is provided in the inspection site 21a of the present embodiment, the number of test lines T is not limited to this and may be two or more. By using multiple test lines T and fixing different antigens, it is possible to efficiently measure the presence and / or quantity of multiple antigens at once when the specimen solution contains multiple antigens. Become. The material of the insoluble carrier 21 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. However, in the present embodiment, in order to measure the contrast of the autofluorescence Lf generated from the examination site 21a, at least the chromatographic carrier 83 forming the test line T is selected from materials that emit autofluorescence. The shape of the detection unit is not limited to a line shape, and the shape of the insoluble carrier itself is not limited to a plate shape as shown in FIG.

  In the chromatographic carrier 83, a control line for judging the end of measurement is prepared as desired. The antibody may be directly immobilized on a part of the chromatographic carrier 83 by physical or chemical bonding, or the antibody is physically or chemically bonded to fine particles such as latex particles, and the fine particles are chromatographed. It may be trapped on a part of the graph carrier 83 and fixed.

  The labeling substance holding pad 82 is prepared by preparing a suspension containing the aforementioned light-absorbing label, applying the suspension to an appropriate pad (for example, a glass fiber pad), and then drying the suspension. is there. As a result, when the sample solution is injected into the insoluble carrier 21, the light absorbing label is bound together with the antigen while being developed together with the antigen, and flows to the test site 21 a.

  The sample addition pad 81 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 the measurement, the sample addition pad 81 is preliminarily subjected to nonspecific adsorption prevention treatment in order to prevent the antigen in the sample solution from nonspecifically adsorbing to the material of the sample addition portion and reducing the measurement accuracy. Sometimes used.

  The absorption pad 84 is a part that absorbs and removes unreacted substances that are not insolubilized in the test line T of the chromatographic carrier 83 while the added sample is physically absorbed by the chromatographic movement. The chromatographic speed after the chromatophore tip of the added sample reaches the absorption pad 84 differs depending on the material and size of the absorbent material of the absorption pad 84, so the speed suitable for antigen measurement is set by the selection. can do.

  The information display unit 25 displays information related to the inspection by handwriting or sticker attachment. Information related to the test includes, for example, information about the patient from whom the test substance was collected (name, age, gender, etc.) and information about the sample / reagent used for the test (name of the test substance to be tested, cleaning solution, amplification solution, etc., And the insoluble carrier 21 to be used and the type of the applied fluorescent treatment).

(Irradiation means)
The light source 10 of the present embodiment constitutes an irradiating means for emitting the ultraviolet light L absorbed by the light absorbing label of the present invention, and a UV lamp, a semiconductor laser (LD), or the like can be used. The light source 10 is preferably a variable type capable of changing the wavelength of the ultraviolet light L in accordance with the light absorption label. The wavelength of the ultraviolet light L is not particularly limited, but is determined from the viewpoint of the light absorption characteristics of the light absorbing label, the insoluble carrier 21 being excited by the autofluorescence Lf, and the like, and the center wavelength is preferably 100 to 400 nm. In particular, when using silver fine particles described later as an absorption label, the central wavelength is preferably 300 to 400 nm in consideration of the absorbance of the silver fine particles. The wavelength of the ultraviolet light L is provided, for example, separately by a wavelength control unit, for example, is determined according to a preset value, is determined based on designated light absorption label information, or is first calibrated during actual measurement, It is determined after calculating the optimum value. The peak of the wavelength distribution of the ultraviolet light L is not necessarily one, and may be two or more.

  Further, since ultraviolet light has a bactericidal effect, it can be expected that a sample solution or the like attached to the chromatographic device 20 can be sterilized to ensure the safety of the user. It is said that 260 nm is the most effective for sterilization. Therefore, you may use the ultraviolet light L which has two peaks of 260 nm which has the wavelength absorbed by the light absorption label, and a bactericidal effect. The ultraviolet light L is irradiated from the irradiation window 24a of the device housing 22 to a predetermined region including the test line T of the insoluble carrier 21 and at least a region wider than an inspection site 21a described later. Further, an optical system such as a lens, a mirror, and a filter can be used in combination with the light source 10.

(Detection means)
The photodetector 12 and the filter 16 of the present embodiment constitute the detection means of the present invention that detects the fluorescence Lf generated from the inspection site 21a of the insoluble carrier 21. The fluorescence Lf is detected through the installation base 18 that is transparent to the fluorescence Lf. The photodetector 12 may be a one-dimensional array type or a two-dimensional array type. In the case of using the one-dimensional array type photodetector 12, the array is arranged in a direction perpendicular to the test line T. As the photodetector 12, a CCD, PD (photodiode), photomultiplier, c-MOS, or the like can be used as appropriate. In particular, from the viewpoint of detection sensitivity, it is preferable to use a cooled CCD. In the present embodiment, the photodetector 12 converts information on the detected light intensity of the fluorescence Lf into an electrical signal for each pixel, and transmits this electrical signal to the measuring means 14 that measures contrast. Further, a spectroscopic means such as an optical filter or a spectroscope is used according to the detection conditions. In the present embodiment, by detecting the fluorescence Lf through the filter 16 that transmits the fluorescence Lf generated from the examination region 21a and blocks the irradiated ultraviolet light L, the ultraviolet light L that becomes noise can be removed, and the contrast is further improved. It becomes possible to do. As a preferred light source and photodetector, for example, LAS-3000 (trade name) manufactured by FUJIFILM Corporation can be mentioned.

(Measuring means)
The measuring means 14 receives the electrical signal transmitted by the photodetector 12, and based on this electrical signal, for example, converts the intensity graph of the fluorescence Lf corresponding to the one-dimensional array type electrical signal or the two-dimensional array type electrical signal. A gradation image of the corresponding fluorescence Lf is generated. Thereafter, the measuring means 14 selects a region including the test line T and the periphery of the test line T from, for example, an intensity graph or a gradation image, extracts the test site 21a, and absorbs the labeling substance from the test site 21a. Therefore, the contrast of the fluorescence Lf generated between the test line T and the periphery thereof is measured. The selection of the region in the extraction of the examination site 21a may be performed automatically by the measurement unit 14, or may be selected by the measurer via the user interface.

  In the present invention, since the light absorbing label that absorbs the ultraviolet light L is used, the ultraviolet light L is absorbed by the light absorbing label, and in the test line T on which the light absorbing label is fixed, the ultraviolet light L is autofluorescence of the insoluble carrier 21. Does not contribute to the excitation of. As a result, since autofluorescence is not detected from the test line T when the antigen is present in the sample solution, the test line T appears as a dark line in the examination site 21a and the measurement accuracy is improved. The detailed mechanism of the improvement in measurement accuracy is unknown, but light generated from the inspection site when irradiated with ultraviolet light L (reflected or scattered light of ultraviolet light and fluorescence provided by membrane or fluorination treatment) (Fluorescence from the mechanism) occurs, and the ultraviolet light L transmitted through the detection portion T decreases due to the absorption characteristics of the light-absorbing label. In the case of fluorescence, even if the ultraviolet light L is converted into fluorescence Lf in the detection unit T, it is considered that scattering or absorption occurs due to the presence of the labeling substance.

  When the fluorescence Lf is detected by the two-dimensional array type photodetector 12, it can be considered that this gradation image is a collection of a plurality of gradation images detected by the one-dimensional array type in the two-dimensional direction. Therefore, the subsequent processing can be simplified by taking the average value of the pixels arranged in the length direction of the test line T and converting it to a one-dimensional gradation image. As a result, when the coloring is uneven or when the test line surface has dust or scratches, the influence can be reduced as compared with the case where the influence is detected by the one-dimensional photodetector.

(Sample solution)
The sample solution that can be measured is not particularly limited as long as it may contain a test substance (in addition to an antigen, a physiologically active substance such as a natural product, a toxin, a hormone, or an agricultural chemical, or an environmental pollutant). For example, biological samples, especially body fluids of animals (particularly humans) (eg blood, serum, plasma, spinal fluid, tears, sweat, urine, pus, runny nose or sputum), excrement (For example, feces), organs, tissues, mucous membranes and skin, an over-extracted specimen (swab) or gargle liquid that is considered to contain them, or animals and plants themselves or their dried bodies diluted with a diluent described later, etc. Can be mentioned.

  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.

(Absorption label)
In the present invention, the labeling substance is a substance (absorbing label) having absorption characteristics in the wavelength band of the ultraviolet light L. This absorption characteristic does not have to be over the entire wavelength band of the ultraviolet light L, and only needs to have an absorption characteristic in a part of the wavelength band of the ultraviolet light L. The material for such a light-absorbing label is not particularly limited, but preferably contains silver fine particles because it has a high absorption coefficient for ultraviolet light L. For example, when the light-absorbing label is silver fine particles, the method for fixing the silver fine particles to the test line T is that the second antibody against the antigen (the epitope differs from that of the first antibody fixed to the test line T). The surface of the silver fine particles is modified and the complex of the silver fine particles and the second antibody formed thereby is fixed to the test line T by the sandwich method, or the metal fine particles with the second antibody against the antigen The composite of the metal fine particles and the second antibody formed thereby is fixed to the test line T by the sandwich method, and then a silver ion-containing compound is supplied to the test site 21a. Examples include a method of fixing silver fine particles to the test line T by silver deposition on metal fine particles by a reduction reaction of silver ions as a catalyst. The former method of fixing the silver fine particles corresponds to a method of fixing silver fine particles to the test line T as an ordinary labeling substance in the sandwich method, and the latter is a method of fixing silver ions using metal fine particles fixed to the test line T as a catalyst. This corresponds to the case of performing signal amplification (silver sensitization) using a reduction reaction. The latter will be described in detail in a third embodiment of a chromatographic measurement method described later.

  For example, when the light-absorbing label is silver fine particles, the particle size can be appropriately set from the viewpoint of the extinction coefficient of the ultraviolet light L.

(Substance exhibiting specific binding property to test substance)
In the present embodiment, the substance (specific binding substance) exhibiting specific binding to the test substance is an antibody against the antigen, but such a specific binding substance is not limited thereto. That is, when the test substance is one of a pair of substances exhibiting specific binding properties, the other substance may be selected as the specific binding substance. For example, when the test substance is a protein, metal ion or low molecular weight organic compound, an aptamer for them, and when the test substance is a nucleic acid such as DNA or RNA, DNA or RNA having a complementary sequence thereto Or a biotin when the test substance is avidin, and a complex that specifically binds to the test 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 an affinity for the test substance as described above can be used as the specific binding substance.

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

  As described above, in the chromatographic measurement method according to the present embodiment, the labeling substance having an absorption characteristic in the wavelength band of the ultraviolet light L is brought into contact with the detection unit T, and then the ultraviolet light that is absorbed by the labeling substance at the inspection site 21a. The light L is irradiated, the light generated from the test site 21a due to the irradiation of the ultraviolet light L is detected, and the presence or absence of the test substance and / or the Alternatively, the quantity is measured. As a result, in the chromatographic measurement method, since the ultraviolet light is absorbed by the light absorbing label, the measurement system can be improved.

  Furthermore, since the chromatographic measurement method according to the present embodiment does not require an insoluble carrier subjected to a low fluorescence treatment, the measurement can be performed at low cost.

  Furthermore, the chromatographic measurement method according to the present embodiment can ensure the safety of the user by sterilizing the sample solution or the like that has adhered to the chromatographic device 20 by the sterilizing effect of ultraviolet light. .

<Design change of the first embodiment>
In the first embodiment, the fluorescence is detected as light generated from the examination site. However, the present invention is not limited to this mode, and the reflected or scattered light of the ultraviolet light at the examination site is detected. It may be. Note that when detecting reflected or scattered light of ultraviolet light, a filter for removing the ultraviolet wavelength region is unnecessary.

<Second embodiment of chromatographic measurement method>
A chromatographic measurement method according to the second embodiment will be described. In the present embodiment, the presence or absence of the antigen is determined by a sandwich method using a test substance as an antigen, a substance exhibiting specific binding to the test substance as an antibody, and a labeling substance (absorption label) that absorbs ultraviolet light. Alternatively, a case where the amount is measured will be described as an example. The chromatographic measurement method of the present embodiment has substantially the same configuration as the chromatographic measurement method of the first embodiment, but the first is that an insoluble carrier subjected to a fluorescence treatment is used as an insoluble carrier. This is different from the chromatographic measurement method of the embodiment. Therefore, detailed description of the same components as those in the chromatographic measurement method of the first embodiment is omitted unless particularly required.

  The chromatographic measurement method of the present embodiment is performed using a chromatographic measurement apparatus including a chromatographic device 20 including an insoluble carrier 28 (FIG. 4) subjected to a fluorescence treatment. The insoluble carrier 28 is provided with a fluorescence mechanism that emits fluorescence Lf when excited by the ultraviolet light L by fluorescence treatment.

  More specifically, in the chromatographic measurement method of the present embodiment, there is a possibility that the insoluble carrier 28 having the line-shaped detection part T on which an antibody exhibiting specific binding to the antigen is immobilized contains the antigen. The sample solution is dropped, and the sample solution is developed on the insoluble carrier 28 while dissolving the light-absorbing label having a light absorption property in the wavelength band of the ultraviolet light L and disposed on the insoluble carrier 28 in the sample solution. The solution is brought into contact with the detection unit T, and the light-absorbing label is brought into contact with the detection unit T so that the detection unit T forms an antibody-antigen-absorption label sandwich structure. One surface of the inspection site of the insoluble carrier 28 is irradiated with ultraviolet light L absorbed by the light-absorbing label, and the autofluorescence Lf of the insoluble carrier 28 generated from the other surface of the inspection site is excited by the ultraviolet light L. and Alternatively, the fluorescence Lf from the fluorescence mechanism provided on the insoluble carrier 28 is detected through the filter 16, and the fluorescence Lf (including autofluorescence) generated between the detection portion T and the surroundings due to the light absorption characteristics of the light absorbing label. And the presence / absence and / or amount of the antigen is measured based on the contrast.

  The measuring apparatus shown in FIG. 1 is the same as that of the first embodiment except for the chromatographic device.

(Chromatograph device)
As shown in FIG. 4, the insoluble carrier 28 in the chromatographic device of the present embodiment is fixed with a sample addition pad 81 which is a portion where a sample such as a sample solution is dropped, and an absorption label indicating specific binding to an antigen. A labeled substance holding pad 82, a chromatographic carrier 83 having a line-shaped detection portion T (test line) on which an antibody exhibiting specific binding to an antigen is fixed, an absorption pad 84 for absorbing the fed sample, and A fluorescent sheet 86 (fluorescent layer) that supports the whole and emits fluorescence Lf when excited by ultraviolet light L is provided. This fluorescent sheet 86 plays a role as a fluorescent mechanism of the present invention. For example, the fluorescent sheet 86 contains a fluorescent substance (fluorescent dye or fluorescent fine particles) excited by the ultraviolet light L emitted from the light source. The fluorescent substance contained in the fluorescent sheet 86 is not particularly limited as long as it has an absorption characteristic (absorption spectrum) similar to that of the absorption label, and can be appropriately selected. Further, it is preferable to use a substance having a large Stokes shift so that the fluorescent substance Lf and the ultraviolet light L can be easily separated. For example, JP 2009-510198 teaches a dye compound excited at 400 nm and having a Stokes shift of about 80 nm or more.

  The fluorescent mechanism is not limited to the aspect in which the fluorescent layer is provided as described above. For example, the fluorescent substance may be added to the chromatographic carrier 83 itself. However, if a fluorescent substance is added to the chromatographic carrier 83 on which the sample migrates, there is a possibility of affecting the characteristics and migration of the sample. A layer 86 is preferably provided.

  As described above, in the chromatographic measurement method according to the present embodiment, the labeling substance having the light absorption characteristic in the wavelength band of the ultraviolet light L is brought into contact with the detection unit T, and then the ultraviolet light that is absorbed by the labeling substance at the examination site. L is irradiated, and the light generated from the examination site due to the irradiation of the ultraviolet light L is detected, and the presence and / or amount of the test substance is detected by the dark part of the examination site relating to the light caused by the light absorption characteristics. It is characterized by measuring. As a result, the same effects as those of the first embodiment are obtained.

  Further, since the chromatographic measurement method according to the present embodiment uses the insoluble carrier 28 provided with the fluorescence mechanism excited by the ultraviolet light L, the fluorescence Lf is also generated from this fluorescence mechanism, and the measurement accuracy is further improved. Can be improved. In addition, since it does not depend on the condition that the autofluorescence of the insoluble carrier 28 is caused, the selection range of the type of light-absorbing label and the wavelength of the ultraviolet light L is widened, and the measurement conditions can be selected more easily.

  Further, the insoluble carrier for chromatography and the chromatographic measurement apparatus according to the present invention are provided with a fluorescence mechanism that emits fluorescence when excited by ultraviolet light, so that the detection target is made fluorescent in the chromatographic measurement method. In this case, fluorescence can be generated more efficiently, and further improvement in measurement accuracy can be realized.

<Third embodiment of chromatographic measurement method>
A chromatographic measurement method according to the third embodiment will be described. In the present embodiment, the presence or absence of the antigen is determined by a sandwich method using a test substance as an antigen, a substance exhibiting specific binding to the test substance as an antibody, and a labeling substance (absorption label) that absorbs ultraviolet light. Alternatively, a case where the amount is measured will be described as an example. The chromatographic measurement method of the present embodiment is the first embodiment particularly in that the measurement is performed using the chromatographic measurement apparatus 2 having label contact means for supplying a labeling substance having an absorption characteristic in the wavelength band of the ultraviolet light L. This is different from the chromatographic measurement method. More specifically, in this embodiment, the surface of the metal fine particles is modified with the second antibody against the antigen, and the complex of the metal fine particles and the second antibody formed thereby is put on the test line T by the sandwich method. After the fixation, a silver ion-containing compound is supplied to the inspection site, and silver fine particles are fixed to the test line T by silver deposition on the metal fine particles by a silver ion reduction reaction using the metal fine particles as a catalyst. Therefore, detailed description of the same components as those in the chromatographic measurement method of the first embodiment is omitted unless particularly required.

  The chromatographic measurement method of the present embodiment is performed using a chromatographic measurement apparatus 2 as shown in FIGS. 5 and 6.

  First, the configuration of the chromatographic measurement apparatus 2 according to this embodiment will be described. FIG. 5 is a schematic perspective view showing the appearance of the chromatograph measuring apparatus 2 according to the present embodiment, and FIG. 6 is a schematic view showing the interior when the immunochromatographic device 20 is loaded and cut along the line II-II in FIG. FIG.

  As shown in FIG. 5 and FIG. 6, the chromatograph measurement device 2 performs an operation of the menu displayed on the device housing 70, the screen display unit 71 disposed on the surface of the device housing 70, and the screen display unit 71. A menu operation unit 72 and a power switch 73 for performing the operation, a device loading unit 74 for loading the device 20 in the apparatus, and a first measurement unit for acquiring information from the device 20 constituting the apparatus 2 40, the second measurement unit 50, the amplification solution supply unit 60 that develops the amplification solution on the insoluble carrier 21 in the device 20, the first measurement unit 40, the second measurement unit 50, and the amplification solution supply unit 60. And a control unit 30 for controlling. In addition, the control part 30 also has a function as a measurement means of the chromatographic measurement apparatus according to the present invention.

(Chromatograph device)
The chromatographic device 20 of the present embodiment is the same as that described in the first embodiment except for the labeling substance holding pad 82. That is, the labeling substance holding pad 82 of the chromatographic device 20 of the present embodiment generates a light-absorbing label on the test line T by silver deposition on the metal fine particles, and therefore holds a labeling substance made of metal fine particles instead of the light-absorbing label. is doing.

(Measurement part)
The first measurement unit 40 functions as a detection unit of the chromatograph measurement apparatus according to the present invention that measures the coloration state of the examination site 21a through the observation window 24b of the device 20, and acquires optical information in the examination site 21a. Is. As shown in FIG. 6, the first measurement unit 40 includes a photodetector 42 and a filter 44 that transmits the fluorescence Lf generated from the examination site 21 a and blocks the irradiated ultraviolet light L, and the device 20 is a measurement apparatus. These are arranged below the device 20 so as to be opposed to the observation window 24b when loaded in 2. And based on the optical information in the test | inspection site | part 21a acquired by the 1st measurement part 40, the fluorescence Lf which arises from the test | inspection site | part 21a as a coloration state of the test | inspection site | part 21a is detected. The photodetector 42 has a configuration in which, for example, 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 photodetector 42 is a band extending in the longitudinal direction of the device 20.

  The second measurement unit 50 functions as an irradiation unit of the chromatographic measurement apparatus 2 according to the present invention that irradiates the examination site 21a with the ultraviolet light L from the irradiation window 24a of the device 20. Further, the second measurement unit 50 irradiates the information display unit 25 of the device 20 with illumination light (if the information display unit 25 can read the light, it is not limited to white light), and the information displayed on the information display unit 25 is displayed. Is obtained by the photodetector 52. As shown in FIG. 6, the second measurement unit 50 includes a photodetector 52 and a light source 54, and when the device 20 is loaded in the measurement apparatus 2, these are arranged above the device 20 and are chromatographed. It is configured to be movable so that it can face the irradiation window 24 a and the information display unit 25 of the graph device 20.

  The light source 54 is a module in which, for example, an LED is built, and is configured to emit illumination light and ultraviolet light L. When the light source 54 includes a plurality of modules, a plurality of modules that emit monochromatic light having different wavelengths can be used. The light emitted from the light source 54 can illuminate the longitudinal direction of the device 20.

  The method for acquiring information is not particularly limited, and the information display unit 25 can be imaged as it is or read from bar-coded information. In particular, information on the insoluble carrier 21 to be used and the type of the fluorescent treatment performed, which is obtained from the information display unit, is used when the control unit 30 appropriately sets the wavelength of the ultraviolet light L. And the information regarding the test | inspection acquired by the 2nd measurement part 50 and a test result are linked | related and managed. The photodetector 52 is the same as the photodetector 42 described above.

(Amplification solution supply unit)
The amplification solution supply unit 60 includes an amplification solution storage pot 62 for storing an amplification solution containing a silver ion-containing compound, an amplification solution injection nozzle 62a for injecting the amplification solution from the amplification solution storage pot 62, and silver contained in the amplification solution. A reducing agent storage pot 64 for storing a reducing agent for reducing ions and a reducing agent injection nozzle 64a for injecting the reducing agent from the reducing agent storage pot 64 are provided in each of the amplification solution storage pot 62 and the reducing agent storage pot 64. The stored liquid can be injected into the injection port 23 of the loaded device 20 through the respective injection nozzles 62a and 64a. In the present embodiment, the amplification solution supply unit 60 functions as a label contact means in the chromatographic measurement apparatus of the present invention. The liquid may be injected, for example, by driving the amplification solution supply unit 60 by a driving device and sequentially or alternately injecting each of the injection nozzles, or by combining two nozzles at the tip part. You may make it inject | pour simultaneously the liquid supplied from the storage pot.

  The amplification solution supply unit 60 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 2. For example, a storage pot may be provided outside the measurement apparatus (housing 70), and the solution may be injected into the injection port 23 of the device 20 by operating only the injection nozzle.

  The chromatographic measurement method of the present embodiment is likely to contain the antigen in the insoluble carrier 21 (chromatographic device) having the line-shaped detection portion T to which an antibody exhibiting specific binding to the antigen is fixed. A sample solution is dropped, the chromatographic device 20 is inserted into the above-described chromatographic measurement apparatus 2, and the insoluble carrier 21 is dissolved in the sample solution while dissolving the labeling substance composed of metal fine particles placed on the insoluble carrier 21 in a dry state. The sample solution is spread out, the sample solution is brought into contact with the detection unit T, and the labeling substance is brought into contact with the detection unit T to form an antibody-antigen-labeled substance sandwich structure in the detection unit T. The test line T is cleaned by developing a reducing agent (where the reducing agent also functions as a cleaning liquid) on the insoluble carrier 21 by the supply unit. (Washing treatment), by spreading the amplification solution on the insoluble carrier 21, silver fine particles formed by reduction of silver ions are deposited on the labeling substance to amplify the colored state (amplification treatment). One surface of the inspection site 21a of the insoluble carrier 21 including the periphery of the detection unit T is irradiated with ultraviolet light L absorbed by the silver fine particles deposited on the labeling substance, and excited by the ultraviolet light L to be inspected by the ultraviolet light L. The autofluorescence Lf of the insoluble carrier 21 resulting from the above is detected through the filter 44, and the contrast of the autofluorescence Lf generated between the detection portion T and the surroundings due to the light absorption characteristics of the silver fine particles is measured. Is used to measure the presence and / or amount of antigen.

(Labeling substance)
As the material of the metal fine particles of the labeling substance, 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, gold colloids, silver colloids, platinum colloids, iron colloids, aluminum hydroxide colloids, composite colloids thereof, and the like are preferable. Gold colloids, silver colloids, platinum colloids, and composite composite colloids thereof are preferable. It is desirable to be. In particular, gold colloid and silver colloid are preferable. By using the gold colloid, the amplification process using the silver ion-containing compound can be easily performed. The average particle size of the metal colloid is preferably about 1 to 500 nm, more preferably 1 to 100 nm.

(Amplification solution)
The amplification solution is a silver ion solution that causes precipitation of metallic silver by physical development on fine metal particles. Details include general books in the field of photographic chemistry (for example, “Basics of Revised Photo Engineering-Silver Salt Photography 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.

(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 as silver 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. preferable.

(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 Fe ²⁺ , V ²⁺ , and Ti ³⁺ . 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 Fe ²⁺ as a reducing agent, a complex of Fe ³⁺ that is an oxide can be formed using citric acid or ethylenediaminetetraacetic acid, and can be rendered harmless. In this system, it is preferable to use such an inorganic reducing agent, and more preferable is a metal salt of Fe ²⁺ .

  As described above, in the chromatographic measurement method according to the present embodiment, the labeling substance having an absorption characteristic in the wavelength band of the ultraviolet light L is brought into contact with the detection unit T, and then the ultraviolet light that is absorbed by the labeling substance at the inspection site 21a. The light L is irradiated, the light generated from the examination site 21a due to the irradiation of the ultraviolet light L is detected, and the presence / absence of the test substance is detected by the dark part of the test site relating to the light caused by the light absorption characteristics. The quantity is measured. As a result, the same effects as those of the first embodiment are obtained.

  Furthermore, since the chromatographic measurement method according to the present embodiment can increase the light absorption characteristics of the labeling substance by silver sensitization, the measurement accuracy can be further improved.

<Design change of the third embodiment>
The insoluble carrier 21 according to the third embodiment has been described with respect to the case where the fluorescent treatment is not performed, but naturally the insoluble carrier subjected to the fluorescent treatment can also be used in the third embodiment.

  In the third embodiment, the amplification solution supply unit functions as a label contact unit. However, the label contact unit of the present invention is not limited to such a mode. For example, a light absorbing label may be stored as a normal labeling substance instead of an amplification solution or a reducing agent in a storage pot of the amplification solution supply unit, and supplied to an insoluble carrier in which the labeling substance is not placed in a dry state.

<Example>
Measurement was actually performed on the chromatographic measurement method of the first embodiment. First, an insoluble carrier made of nitrocellulose was prepared, a specimen solution containing influenza virus was developed on the insoluble carrier, and the dried metal colloid was fixed to the test line as a label. Thereafter, silver fine particles were fixed to the test line T by performing silver sensitization using the metal colloid as a catalyst and a compound containing silver and a reducing agent for silver ions. Details regarding this silver sensitization are described in JP-A-2008-286590. Next, using LAS-3000 (trade name) manufactured by FUJIFILM Corporation, self-generated from the insoluble carrier through a filter that irradiates ultraviolet light of 312 nm from one side of the insoluble carrier and transmits light having a wavelength longer than 320 nm. Fluorescence was detected. A fluorescence signal is detected by the CCD from the other surface of the insoluble carrier, a two-dimensional gradation image is generated based on the fluorescence signal, and an examination site including a test line and its surroundings is extracted from the gradation image, With respect to this inspection site, the contrast of the autofluorescence generated between the test line and the surroundings due to the light absorption characteristics of the light absorbing label was measured.

<Comparative example>
The same procedure as in the example was performed until the silver fine particles were fixed to the test line. Then, using LAS-3000 manufactured by FUJIFILM Corporation, the insoluble support was irradiated with white light (natural light), and the reflected light was detected. A reflected light signal is detected by the CCD, a two-dimensional gradation image is generated based on the reflected light signal, an inspection part including a test line and its surroundings is extracted from the gradation image, and the inspection commission part is related. The contrast of the reflected light generated between the test line and the surrounding due to the light absorption characteristics of the light absorbing label was measured.

<Measurement results>
FIG. 7A and FIG. 7B are diagrams showing the gradation images obtained in the example and the comparative example, respectively. 8A and 8B average the pixel values in the direction perpendicular to the length direction of the insoluble carrier for the examination sites (region X and region Y, respectively) in the gradation images obtained in the examples and comparative examples. FIG. 5 is a graph showing brightness values in which the average values are arranged one-dimensionally in the length direction of the insoluble carrier.

  From the graphs of FIG. 8A and FIG. 8B, it is more preferable to detect the fluorescence generated by irradiating the insoluble carrier with ultraviolet light and being excited by the ultraviolet light, rather than irradiating white light and detecting the reflected light. It was found that the contrast between the test line and its surroundings increased.

DESCRIPTION OF SYMBOLS 1, 2 Chromatographic measuring apparatus 10 Light source 12 Photo detector 14 Measuring means 16 Filter 18 Installation stand 20 Chromatograph device 21, 28 Insoluble carrier 21a Inspection site 22 Device housing 23 Inlet 24a Irradiation window 24b Observation window 25 Information display part 30 Control Unit 40 Measurement Unit 50 Measurement Unit 60 Amplification Solution Supply Unit 70 Device Housing 81 Sample Addition Pad 82 Labeled Substance Holding Pad 83 Chromatographic Carrier 84 Absorption Pad 85 Transparent Sheet 86 Fluorescence Mechanism (Fluorescence Layer)
L Ultraviolet light Lf Fluorescence (in-house firefly)
T detector (test line)

Claims (11)

A sample solution that may contain the test substance is developed on an insoluble carrier having a detection part on which a substance exhibiting specific binding to the test substance is fixed, and the sample solution is brought into contact with the detection part, In a chromatographic measurement method for measuring the presence and / or amount of the test substance immobilized on the detection unit via a substance exhibiting specific binding properties,
While bringing the sample solution into contact with the detection unit, contacting the detection unit with a labeling substance having an absorption characteristic in the wavelength band of ultraviolet light,
Irradiating the test site of the insoluble carrier including the detection unit and the periphery of the detection unit with ultraviolet light absorbed by the labeling substance,
Detecting light generated from the examination site due to irradiation of the ultraviolet light,
A chromatographic measurement method, wherein the presence / absence and / or amount of the test substance is measured by a dark part of the test site relating to light generated from the test site, which is caused by the light absorption characteristics of the labeling substance.   2. The chromatographic measurement method according to claim 1, wherein fluorescence generated by the ultraviolet light when excited by the ultraviolet light is detected as the light generated from the inspection site.   3. The chromatographic measurement method according to claim 2, wherein the insoluble carrier is one that has been subjected to a fluorescence treatment so as to emit fluorescence when excited by the ultraviolet light.   The fluorescent treatment is characterized in that a fluorescent layer that emits fluorescence when excited by the ultraviolet light is formed in a portion of the examination site that is shaded with respect to the irradiated ultraviolet light. 3. The chromatographic measurement method according to 3.   The chromatographic measurement method according to claim 2, wherein the fluorescence is detected through a filter that transmits the fluorescence and blocks ultraviolet light.   The chromatographic measurement method according to claim 1, wherein silver fine particles are used as the labeling substance.   A chromatograph characterized in that it is an insoluble carrier having a detection part to which a substance exhibiting specific binding to a test substance is fixed, and is provided with a fluorescence mechanism that emits fluorescence when excited by ultraviolet light. Insoluble carrier.   The fluorescence mechanism is excited by ultraviolet light, which is formed in a part of the inspection site of the insoluble carrier including the detection part and the periphery of the detection part, which is shaded with respect to the irradiated ultraviolet light. The insoluble carrier for chromatography according to claim 7, which is a fluorescent layer that emits fluorescence. A dropping unit for dropping the sample solution;
9. The insoluble carrier for chromatography according to claim 7 or 8, comprising a labeling substance that is placed dry between the dropping part and the inspection site. A fluorescence mechanism that has a detection unit to which a substance exhibiting specific binding to a test substance is fixed and emits fluorescence when excited by ultraviolet light is provided at an examination site including the detection unit and the periphery of the detection unit An insoluble carrier;
A label contact means for contacting a labeling substance having an absorption characteristic in the wavelength band of ultraviolet light with the inspection site;
A light source that emits ultraviolet light that is absorbed by the labeling substance, and irradiation means that irradiates the ultraviolet light to the examination site;
A detector that includes a photodetector and detects fluorescence generated by the ultraviolet light in a direction parallel to the irradiation direction of the ultraviolet light by being excited by the ultraviolet light;
Measuring means for measuring presence / absence and / or amount of the test substance by using a dark part of the examination site relating to fluorescence detected by the detection means, which is caused by the light absorption characteristics of the labeling substance. A characteristic chromatograph measuring device.   The fluorescent mechanism is a fluorescent layer that is formed in a portion of the inspection site that is shaded with respect to the irradiated ultraviolet light and emits fluorescence when excited by the ultraviolet light. Chromatographic measurement device.