JP2010217064A - Chromatographic method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunochromatographic method capable of quantification in a low concentration range and a high concentration range by greatly expanding a specimen quantifiable range than before. <P>SOLUTION: The chromatographic method, detecting a test substance, includes the steps of (a) conducting a first optical concentration measurement for quantifying an acquired labeled substance, (b) amplifying the acquired labeled substance, (c) conducting a second optical concentration measurement for quantifying the amplified labeled substance, and (d) quantifying the test substance based on values of the first optical concentration measurement and/or the second optical concentration measurement. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a measurement method using an amplification chromatography method.

  There are very many physiologically active substances or environmental pollutants such as natural products, toxins, hormones, or agricultural chemicals that act in extremely small amounts. Therefore, instrumental analysis methods capable of highly sensitive analysis have been widely used for qualitative and quantitative measurement of these substances. However, the instrumental analysis method has low specificity, requires time for analysis including the sample pretreatment step, and is complicated in operation, and thus is inconvenient for the purpose of quick and simple measurement that has been required in recent years. On the other hand, immunological measurement methods have high specificity and are much easier to operate than instrumental analysis, so that they have gradually spread to the field of measurement of physiologically active substances or environmental pollutants. However, conventional immunoassay methods such as an enzyme immunoassay method using a 96-well plate and a latex agglutination method do not always satisfy the rapid convenience or detection sensitivity of the measurement.

  Another need is that, even in tests that currently use relatively invasive specimens such as swabs and blood, high sensitivity can be achieved, resulting in relatively low nasal discharge, gargle, urine, etc. Since it becomes possible to detect a test sample contained in a very small amount in an invasive specimen, it can be expected that an examination method with less burden on the patient will be possible.

  In recent years, a test kit using an immunochromatographic method (in the following description, referred to as an immunochromatographic kit) is often used for infectious diseases that require a particularly rapid diagnosis. With the widespread use of these kits, it is possible to identify infections in patients by a quick and simple method, and to perform subsequent diagnosis and treatment quickly and accurately. For example, in an immunochromatography method using a sandwich method, an insoluble thin film support (for example, a glass fiber membrane, a nylon membrane) in which a first antibody that specifically binds to an analyte (for example, an antigen) is immobilized in a specific region. A labeled second antibody that specifically binds to the analyte, and a sample solution that may contain the analyte, and the insoluble thin film support first On the region where one antibody is immobilized, an immune complex with the analyte can be formed, signals such as coloring or color development of the label can be detected, and the analyte can be measured. As the label, for example, an enzyme-containing protein, colored latex particles, metal colloid, or carbon particles can be used.

  The immunochromatography method does not require heavy equipment and equipment for its determination and measurement, is easy to operate, and is allowed to stand for about 5 minutes to 10 minutes after a sample solution that may contain an analyte is dropped. As it is quick enough to obtain measurement results, it is widely used in many situations, such as clinical tests in hospitals and laboratory tests in laboratories, as a simple, rapid, and highly specific determination and measurement technique. Yes.

  In addition, biologically active substances such as natural products, toxins, hormones, and agricultural chemicals, or environmental pollutants, and specimens in the early stages of viral infection are substances that act in extremely small amounts that cannot be detected by conventional general immunochromatography. In many cases, development of a rapid, simple and highly sensitive immunochromatographic method is required.

  On the other hand, by using the principle of immunochromatography and using a reader that optically measures the optical density of the detection site, the test substance can be quantified. In some cases, such as the Roche Diagnostics Cobas h232 series, the blood concentration of a protein called a myocardial marker is rapidly measured at about 15 minutes by quantification using an immunochromatographic method. is there.

However, conventionally, the measurable range in the quantitative immunochromatography method using colloidal gold as a label has a limit in the low concentration range from the limit of absorbance per gold fine particle. Further, when the gold colloid signal is amplified by silver amplification, there is a problem that the absorbance in the high concentration range is saturated because the shielding area does not change much even if the amount of silver increases.
Conventionally, high sensitivity methods using enzyme methods and silver amplification methods are known (Patent Documents 1 and 2). However, the enzymatic method is a system in which a signal is generated only by performing an enzyme reaction, and the signal before amplification cannot be quantified. In addition, in the conventional silver amplification method, since no washing was performed, a sufficient signal / noise ratio could not be obtained, and in fact, sufficient sensitivity could not be obtained in a low concentration range.

Japanese Patent No. 3309977 Japanese Patent Laid-Open No. 2002-202307

  As described above, the conventional immunochromatography method using colloidal gold has a problem that the quantitative measurement range is narrow. In addition, in order to enable quantification at a low concentration, it is difficult to quantitate the sample at a high concentration simply by measuring the signal after amplification. That is, the present invention has an object to be solved by providing an immunochromatographic method that enables quantification in a low concentration region and a high concentration region by greatly expanding the quantifiable range of a sample than before. .

  In the present invention, after measuring absorbance with a colloidal gold label, washing is performed to reduce noise due to non-specific adsorption, then the colloidal gold label is amplified, and the absorbance is measured again. The measurement method of measuring and quantifying enabled the quantification in the low concentration range and the high concentration range, and greatly expanded the quantifiable range of the sample than before.

That is, according to the present invention, in a chromatographic method for detecting a test substance,
(A) a step of performing a first optical density measurement for quantifying the captured labeling substance;
(B) a step of amplifying the captured labeling substance,
(C) a second optical density measurement step for quantifying the labeled substance after amplification, and (d) the amount of the test substance from the first optical density measurement and / or the second optical density measurement value. Is provided.

  Preferably, when the optical density value measured in the first optical density measurement is larger than a predetermined measurement switching optical density value, the amount of the test substance is calculated from the optical density value measured in the first optical density measurement. If the optical density value measured in the first optical density measurement is less than or equal to the predetermined measurement switching optical density value, the test is performed from the optical density value measured in the second optical density measurement after amplification. Quantify the amount of the substance.

  Preferably, a predetermined known amount of the test substance compared to the average value obtained by performing the first optical density measurement a plurality of times using a test solution that does not contain the test substance, plus two times the standard deviation A known amount such that the value obtained by subtracting twice the standard deviation from the average value obtained by performing the first optical density measurement multiple times using a test solution containing Set as the quantification limit concentration that can be quantified in the optical density measurement, the average value obtained by performing the first optical density measurement multiple times using a test solution containing the test substance at the quantification limit concentration and the measurement switching optical density value To do.

Preferably, the method of the present invention is an immunochromatographic method.
Preferably, the labeling substance modified with the first binding substance for the test substance is developed on an insoluble carrier in a mixed state, and the second binding substance for the test substance or the first binding for the test substance The test substance and the labeling substance are captured at a reaction site on an insoluble carrier having a substance capable of binding to the substance, and the test substance is detected.

Preferably, the insoluble carrier is a porous carrier.
Preferably, the first binding substance and / or the second binding substance is an antibody or a fragmented antibody.

Preferably, the method of the invention comprises between step (a) and step (c)
(E) a step of cleaning by developing a cleaning solution;
including.

Preferably, the amplification is performed using an amplification solution containing a compound containing silver and a reducing agent for silver ions.
Preferably, the reducing agent for silver ions includes divalent iron ions.

Preferably, the labeling substance is a metal colloid.
Preferably, the metal colloid is a gold colloid.
Preferably, the average particle size of the labeling substance after amplification when performing the second optical density measurement is a size of 1 μm or more and 20 μm or less.

  According to the chromatographic method of the present invention, the quantifiable range of the sample can be expanded as compared with the conventional method, and quantification in a low concentration region and a high concentration region is possible.

It is a longitudinal cross-sectional view which shows typically the longitudinal cross-section of the immunochromatography kit which can be used by this invention. 1 is a back adhesive sheet, 2 is a gold colloid antibody holding pad, 3 is an antibody-immobilized membrane, 3a is a capture site, 31 is a detection unit, 32 is a control unit, 4 is an absorption pad, 5 is a sample addition pad, and 6 is an increase A feeling sheet is shown.

Hereinafter, the present invention will be described more specifically.
A chromatographic method for detecting a test substance according to the present invention comprises:
(A) a step of performing a first optical density measurement for quantifying the captured labeling substance;
(B) a step of amplifying the captured labeling substance,
(C) a second optical density measurement step for quantifying the labeled substance after amplification, and (d) the amount of the test substance from the first optical density measurement and / or the second optical density measurement value. Quantifying. Preferably, the chromatographic method according to the present invention is an immunochromatographic method. In the chromatographic method according to the present invention, for example, a labeling substance modified with a first binding substance for a test substance is developed on an insoluble carrier in a mixed state, and a second binding substance for the test substance, or The test substance can be detected by capturing the test substance and the labeling substance at a reaction site on an insoluble carrier having a substance capable of binding to the first binding substance with respect to the test substance.

  Steps (b) and (c) may be omitted depending on the result of the first optical density measurement.

  As described above, in the present invention, the first optical density measurement is performed before the amplification step with the amplification solution, and the second optical density measurement is performed after the amplification step. The optical density measurement referred to in the present invention is photometry using an appropriate optical measurement device, and may be either transmission photometry or reflection photometry as long as the optical density reflecting the captured labeling substance can be measured. The wavelength to be measured may be a specific single wavelength, a plurality of wavelengths, or an optical density of light over a wide wavelength as long as the optical density reflecting the captured labeling substance can be measured. .

  Preferably, in step (d), when the optical density value measured in the first optical density measurement is larger than the predetermined measurement switching optical density value, the optical density value measured in the first optical density measurement is used. When the optical density value measured in the first optical density measurement is less than or equal to the predetermined measurement switching optical density value, the optical quantity measured in the second optical density measurement after amplification is quantified. The amount of the test substance can be quantified from the concentration value.

  By determining the measurement switching optical density value in advance as described above, the test substance concentration can be easily determined from the low concentration range to the high concentration range.

The method for determining the measurement switching optical density value is not particularly limited, but as an example, the average value obtained by performing the first optical density measurement a plurality of times using a test solution that does not contain the test substance. Compared to the value obtained by adding twice the standard deviation, it is twice the standard deviation from the average value obtained by performing the first optical density measurement a plurality of times using a test solution containing a predetermined known amount of the test substance. A known amount such that the value obtained by subtracting the value becomes larger is set as a quantification limit concentration that can be quantified in the first optical density measurement, and the first optical is measured using a test solution containing the test substance at the quantification limit concentration. An average value obtained by performing density measurement a plurality of times can be used as a measurement switching optical density value. That is, in the present invention, the following formula:
[Average value of signal measurement value in case of sample liquid not containing sample + 2 × SD (standard deviation)] <[Average value of signal measurement value in case of sample liquid containing sample of concentration A−2 × SD (standard) deviation)]
The concentration A that satisfies the above condition can be set as the limit of quantification (detection limit) that can be quantified in the first optical density measurement.

1. Chromatography In general, the chromatographic method is a method for determining and measuring an analyte simply, quickly, and specifically by the following method. That is, a chromatographic carrier having at least one reaction site containing an immobilization reagent (antibody, antigen, etc.) that can bind to an analyte is used as a stationary phase. On this chromatographic carrier, a dispersion liquid in which a labeling substance modified with a reagent capable of binding to the analyte is dispersed is used as a moving layer to move chromatographically in the chromatographic carrier, and the analyte and It reaches the reaction site while specifically binding to the labeling substance. In the reaction site, the complex of the analyte and the labeling substance specifically binds to the immobilization reagent, so that the immobilization reagent part is labeled only when the analyte is present in the analyte liquid. This is a technique for qualitatively and quantitatively analyzing the presence of a test substance in a liquid to be analyzed by using the fact that substances are concentrated and visually or using an appropriate instrument.

  The apparatus for performing the chromatographic method in the present invention may contain a compound containing silver and a reducing agent for silver ions, and a complex of the analyte and the labeling substance bound to the immobilization reagent is used as a nucleus. As a result, the signal can be amplified by an amplification reaction, and as a result, high sensitivity can be achieved. According to the present invention, a rapid and highly sensitive chromatograph can be performed.

2. Test sample The test sample that can be analyzed by the chromatographic method of the present invention is not particularly limited as long as it is a sample that may contain an analyte, for example, a biological sample, particularly Animal (especially human) body fluids (eg blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, runny nose or sputum) or feces (eg feces), organs, tissues, mucous membranes and skin, Mention may be made of squeezed specimens (swabs), gargles or animals and plants themselves or their dried bodies that are considered to contain them.

3. Pretreatment of test sample In the chromatographic method of the present invention, the test sample is used as it is, or in the form of an extract obtained by extracting the test sample with an appropriate extraction solvent, and further, the extraction The solution can be used in the form of a diluted solution obtained by diluting the solution with an appropriate diluent, or in the form obtained by concentrating the extract using an appropriate method. As the solvent for extraction, a solvent (for example, water, physiological saline, or buffer solution) used in usual immunological analysis methods, or direct antigen-antibody reaction by diluting with the solvent It is also possible to use a water-miscible organic solvent capable of

4). Configuration The chromatographic strip that can be used in the chromatographic method of the present invention is not particularly limited as long as it is a chromatographic strip that can be used in a normal chromatographic method. For example, FIG. 1 schematically shows a cross-sectional view of an example of the chromatographic strip of the present invention.

  The chromatographic strip of the present invention comprises a sample addition pad 5, a labeled substance holding pad (for example, a colloidal gold antibody holding pad) 2, and a chromatographic carrier (for example, an antibody-immobilized membrane) 3 from upstream to downstream in the developing direction. , And the absorbent pad 4 are arranged on the adhesive sheet 5 in this order.

  The chromatographic carrier 3 includes a detection zone (which may be referred to as a detection unit) 31 that has a supplementary site 3a and is an area in which an antibody or antigen that specifically binds to an analyte is immobilized. If desired, it further has a control zone (which may be referred to as a control part) 32 which is a region where a control antibody or antigen is immobilized.

  The labeling substance holding pad 2 is prepared by preparing a suspension containing the labeling substance, applying the suspension to an appropriate absorbent pad (for example, a glass fiber pad), and then drying the suspension. be able to.

  As the sample addition pad 1, for example, a glass fiber pad can be used.

4-1. Labeling substance As the labeling substance, colored particles used in the immunoaggregation reaction can be used. For example, a metal such as a metal colloid can be used. The average particle size of the carrier particles (or colloid) is preferably in the range of 0.001 to 1 μm. Liposomes and microcapsules containing pigments can also be used as colored particles. Any conventionally known colored metal colloid can be used as colored particles for labeling. Examples thereof include a gold colloid, a silver colloid, a platinum colloid, an iron colloid, an aluminum hydroxide colloid, and a composite colloid thereof, and a gold colloid, a silver colloid, a platinum colloid, and a composite colloid thereof are preferable. In particular, gold colloid and silver colloid are preferable in that the gold colloid is red and the silver colloid is yellow at appropriate particle sizes. The average particle size of the metal colloid is preferably about 1 nm to 500 nm, more preferably 1 to 50 nm. The metal colloid and the specific binding substance can be bound according to a conventionally known method (for example, The Journal of Histochemistry and Cytology, Vol. 30, No. 7, pp 691-696, (1982)). That is, a metal colloid and a specific binding substance (for example, an antibody) are mixed in an appropriate buffer at room temperature for 5 minutes or more. After the reaction, the target metal colloid-labeled specific binding substance can be obtained by dispersing the precipitate obtained by centrifugation in a solution containing a dispersant such as polyethylene glycol. When gold colloid particles are used as the metal colloid, commercially available products may be used. Alternatively, colloidal gold particles can be prepared by a conventional method, for example, a method of reducing chloroauric acid with sodium citrate (Nature Phys. Sci., Vol. 241, 20, (1973), etc.). Further, the size of the labeling substance at the time of detection is preferably 1 μm or more and 20 μm or less, more preferably 3 μm or more and 20 μm or less. As a method of changing the size before detection to the size at the time of detection, there is an amplification reaction using, for example, silver ions and a reducing agent.

  According to the present invention, in the immunochromatograph using a metal colloid label or a metal sulfide label, other metal alloy label (hereinafter sometimes referred to as a metal-based label), or a polymer particle label containing a metal as the labeling substance, The signal of the metallic label can be amplified. Specifically, after the complex of the analyte and the labeling substance is formed, a reducing agent is contacted for silver ions and silver ions supplied from a compound containing silver such as an inorganic silver salt or an organic silver salt, When silver particles are generated by reducing silver ions with a reducing agent, the silver particles are deposited on the metal label using the metal label as a nucleus, so that the metal label is amplified and analysis of the analysis object is performed. Can be implemented with high sensitivity. Therefore, in the immunochromatography method of the present invention, the reaction of depositing on the label of the immune complex is carried out using silver particles generated by the reducing action of silver ions by the reducing agent, and thus the amplified signal is analyzed. Except for, conventionally known immunochromatographic methods can be applied as they are.

  In the chromatographic method of the present invention, a metal colloid label or a metal sulfide label is used as a label used to label an antibody or antigen that specifically binds to an analyte (antigen or antibody) or a standard compound. The metal colloid label or the metal sulfide label is not particularly limited as long as it is a label that can be used in a usual chromatographic method. Examples of the metal colloid label include platinum colloid, gold colloid, and palladium. Colloid or silver colloid and mixtures thereof can be mentioned, and examples of metal sulfide labels include iron, silver, palladium, lead, copper, cadmium, bismuth, antimony, tin, or mercury sulfide. Can be mentioned. In the immunochromatographic method of the present invention, one or more of these metal colloid labels and / or metal sulfide labels can be used as labels.

4-2. Binding substance In the present invention, the labeling substance is modified with a first binding substance for the test substance. The first binding substance has an affinity for the test substance, such as an antibody against the test substance (antigen), an antigen to the test substance (antibody), an aptamer to the test substance (protein, low molecular weight compound, etc.), etc. Any compound can be used.

  In the present invention, the porous carrier has (a) a second binding substance for the test substance, or (b) a substance having binding properties to the first binding substance. The second binding substance to the test substance includes, for example, an antibody to the test substance (antigen), an antigen to the test substance (antibody), an aptamer to the test substance (protein, low molecular compound, etc.), etc. Any compound having an affinity for it may be used. Further, the second binding substance and the first binding substance may be different or the same. The substance having the binding property to the first binding substance with respect to the test substance may be the test substance itself or a compound having a site recognized by the first binding substance. For example, a derivative of the test substance and a protein (for example, BSA) And the like).

  Preferably, the first binding substance is an antibody and / or the second binding substance is an antibody. In the immunochromatographic method of the present invention, the antibody having specificity for the analyte is not particularly limited. For example, an antiserum prepared from the serum of an animal immunized with the analyte, An immunoglobulin fraction purified from the antiserum, a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with the analyte, or a fragment thereof [eg, F (ab ′) 2, Fab, Fab ′ or Fv] can be used. These antibodies can be prepared by a conventional method.

4-3. Chromatographic carrier The chromatographic carrier is preferably a porous carrier. In particular, a nitrocellulose film, a cellulose film, an acetylcellulose film, a polysulfone film, a polyethersulfone film, a nylon film, a glass fiber, a nonwoven fabric, a cloth, or a thread is preferable.

  Usually, a detection zone is prepared by immobilizing a detection substance on a part of a chromatographic carrier. The detection substance may be directly immobilized on a part of the chromatographic carrier by physical or chemical bonding, or the detection substance may be physically or chemically bonded to fine particles such as latex particles. The fine particles may be trapped and immobilized on a part of the chromatographic carrier. The chromatographic carrier is preferably used after immobilizing the detection substance and then subjecting it to nonspecific adsorption prevention treatment by treatment with an inactive protein or the like.

4-4. Sample addition pad Examples of the material of the sample addition pad include, but are not limited to, cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth having uniform characteristics. The sample addition unit not only accepts a sample containing the added analysis target, but also has a function of filtering insoluble matter particles and the like in the sample. In addition, in order to prevent the analyte in the sample from adsorbing nonspecifically on the material of the sample addition part and reducing the accuracy of the analysis, the material constituting the sample addition part is preliminarily nonspecific. In some cases, the anti-adsorption treatment is used.

4-5. Labeling substance holding pad Examples of the material of the labeling substance holding pad include cellulose filter paper, glass fiber, and non-woven fabric. The labeling substance holding pad is impregnated with a certain amount of the labeling substance prepared as described above and dried.

4-6. Absorption pad The absorption pad is a part that absorbs and removes unreacted labeling substances that are not insolubilized in the detection part of the chromatographic carrier while the added sample is physically absorbed by the chromatographic movement. Cellulose filter paper, non-woven fabric Water-absorbing materials such as cloth and cellulose acetate are used. 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 analyte. it can.

5). Hereinafter, the sandwich method and the competitive method, which are specific embodiments of the chromatographic method of the present invention, will be described.
The sandwich method is not particularly limited. For example, the test substance can be analyzed by the following procedure. First, a first antibody and a second antibody having specificity for a test substance (antigen) are prepared in advance by the method described above. The first antibody is labeled in advance. The second antibody may be immobilized on a suitable first insoluble carrier (for example, a nitrocellulose membrane, a glass fiber membrane, a nylon membrane, or a cellulose membrane) and contain a test substance (antigen). When contacted with a test sample (or an extract thereof), an antigen-antibody reaction occurs when a test substance is present in the test sample. This antigen-antibody reaction can be performed in the same manner as a normal antigen-antibody reaction. At the same time as or after the antigen-antibody reaction, when an excessive amount of the labeled first antibody is further contacted, if the test substance is present in the test sample, the immobilized second antibody, the test substance (antigen) and the label An immune complex consisting of the first antibody is formed.

  In the sandwich method, after the reaction between the immobilized second antibody, the test substance (antigen), and the first antibody is completed, the labeled first antibody that did not form the immune complex is removed. It is good to perform the first optical density measurement on the region where the immobilized second antibody is immobilized on the insoluble carrier, and the amount of the test substance in the test sample can be measured by quantifying the label. Then, by supplying a metal ion and a reducing agent, after amplifying a signal from the label of the labeled first antibody that has formed the immune complex, a second optical density measurement is performed, thereby performing the amplified label. The substance can be quantified and the amount of the test substance in the test sample can be measured.

  The competitive method is not particularly limited. For example, the test substance can be analyzed by the following procedure. The competition method is known as a technique for detecting an antigen of a low molecular weight compound that cannot be assayed by the sandwich method.

  First, a first antibody having specificity for a test substance (antigen) is prepared in advance. Further, the first antibody is previously labeled with a metal colloid or the like. A test substance itself having a binding property to the first antibody, or a compound having a site similar to the test substance and having the same epitope for the first antibody as the test substance is added to a suitable first insoluble carrier (for example, nitrocellulose) -A glass membrane, a glass fiber membrane, a nylon membrane, a cellulose membrane or the like). When contacted with a test sample (or an extract thereof) that may contain a test substance (antigen), if the test substance is not present in the test sample, the labeled first antibody and the first antibody The antigen-antibody reaction on the first insoluble carrier is caused by the test substance itself having a binding property to the test substance or a compound having the same epitope for the first antibody as the test substance. On the other hand, if the test substance is present, the test substance (antigen) binds to the labeled first antibody, and therefore has the binding ability to the subsequent first antibody, or similar to the test substance itself or the test substance The antigen-antibody reaction on the first insoluble carrier with a compound having a specific site and having the same epitope for the first antibody as the test substance is inhibited, and binding due to the antigen-antibody reaction does not occur.

  After the reaction between the immobilized product having binding property to the first antibody and the labeled first antibody is completed, the labeled first antibody that did not form the immune complex is removed. By performing the first optical density measurement on the substance having binding property to the first antibody in one insoluble carrier, the amount of the labeled substance can be quantified and the amount of the test substance in the test sample can be measured. Next, by supplying a metal ion and a reducing agent to the fixed region, amplifying a signal from the label of the labeled first antibody that formed the immune complex, and then performing a second optical density measurement The labeled substance after amplification can be quantified, and the amount of the test substance in the test sample can be measured.

6). Washing In the present invention, between the step of performing the first optical density measurement for quantifying the captured labeled substance and the step of performing the second optical density measurement for quantifying the amplified labeled substance, the washing liquid Cleaning can be performed by developing

(Cleaning solution)
The washing solution for removing the labeled first antibody that has not formed the immune complex may have any washing function.

  The washing solution is not particularly limited as long as it is a liquid for washing the labeling substance remaining in the chromatographic carrier other than the specific binding reaction, that is, nonspecifically remaining, and is not limited to mere water or A solvent such as ethanol alone may be used, and for example, a PBS buffer containing 1% BSA, a solution of a surfactant, or the like may be used. Further, as the cleaning liquid, a liquid containing silver ions, which will be described later, or a liquid containing a silver ion reducing agent can be used. Since the washing solution is developed while washing the non-specifically remaining labeling substance in the middle of the development, the washing solution is developed while containing the labeling substance. Use liquids that do not contain substances. In order to enhance the cleaning effect, the pH may be adjusted, or a cleaning solution containing a surfactant component, a protein such as BSA, or a polymer compound such as polyethylene glycol may be used.

(Development of cleaning liquid, direction)
After the sample liquid is developed, the washing liquid is added to the chromatographic strip, and the labeling substance other than that bound by the antigen-antibody reaction remaining on the chromatographic strip is washed. As a method of feeding the cleaning liquid, after the sample liquid is developed, it is added to the sample dropping portion as it is, or a cleaning liquid addition pad and a water absorption pad are attached to the strip in advance, and the cleaning liquid addition pad is attached to the strip. There are a method of adding and feeding the liquid in the direction of the pad of water absorption, a method in which a strip is previously provided with a site for adding a cleaning liquid, and a method of adding the cleaning liquid to the site of adding the cleaning liquid after spreading the sample liquid. In this method, after the liquid is spread on the strip, a cleaning liquid addition pad and a water absorption pad for feeding the cleaning liquid are attached to the strip, the cleaning liquid is supplied to the cleaning liquid addition pad, and the cleaning liquid is developed. As a method of supplying the cleaning liquid to the cleaning liquid addition pad, the cleaning liquid addition pad may be inserted into a pot containing the cleaning liquid, or the cleaning liquid may be dropped into the cleaning liquid addition pad.

  In this specification, the development direction of the liquid of the test substance is defined as a direction connecting the sample addition pad and the absorption pad, and the development direction of the cleaning liquid is a cleaning liquid addition pad and a water absorption pad for feeding the cleaning liquid. Is defined as the direction connecting

  When the angle between the development direction of the test substance liquid and the development direction of the cleaning liquid is 45 degrees to 170 degrees, the cleaning effect is increased. Furthermore, the angle formed by the developing direction of the test substance liquid and the developing direction of the cleaning liquid is preferably 60 degrees to 170 degrees, more preferably 60 degrees to 150 degrees.

The washing liquid addition pad (also referred to as a second insoluble carrier) may be anything as long as the washing liquid can be added, and a glass fiber pad, a cellulose membrane, a nitrocellulose membrane or the like can be used.
The water absorption pad may be any substance that can absorb water, and cellulose, nitrocellulose, glass fiber, a mixture thereof, and the like can be used.

7). Amplification solution An amplification solution is a solution that can cause amplification of a signal by producing a colored compound or luminescence, etc., when a contained drug reacts catalytically by the action of a labeling substance or a test substance. Examples thereof include a silver ion solution in which metallic silver is precipitated by physical development on a metal label, and a solution of a phenylenediamine compound and a naphthol compound that become a dye by the action of a peroxidase label and hydrogen peroxide.

  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 developer can be used, and the solution contains silver ions and the silver ions in the solution are developed. As long as it is a so-called physical developer that is reduced mainly with a metal colloid or the like that becomes the core of the above, it can be used as an amplification solution without any particular limitation.

  As a specific example of the amplification solution, an amplification solution containing a compound containing silver and a reducing agent for silver ions can be used. Hereinafter, the compound containing silver and the reducing agent for silver ions will be described.

(Compound containing silver (ion))
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. As a silver complex, the silver complex coordinated to the ligand which has water-soluble groups, such as a hydroxyl group and a sulfone group, is preferable, and hydroxythioether silver etc. are mentioned.

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.

(Reducing agent for silver ions)
As the reducing agent for silver ions, any inorganic or organic material or a mixture thereof can be used as long as silver ions can be reduced 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⁺ which is an oxide can be formed using citric acid or EDTA, and can be rendered harmless. In this system, it is preferable to use such an inorganic reducing agent, more preferably a metal salt of Fe ²⁺ .

  The developing agents used in wet silver halide photographic materials (for example, methyl gallate, hydroquinone, substituted hydroquinone, 3-pyrazolidones, p-aminophenols, p-phenylenediamines, hindered phenols, amidoximes , Azines, catechols, pyrogallols, ascorbic acid (or derivatives thereof, and leuco dyes), and other materials apparent to those skilled in the art, such as US Pat. No. 6,020,117 The materials described in 1) can also be used.

  As the reducing agent, an ascorbic acid reducing agent is preferable. Useful ascorbic acid reducing agents include ascorbic acid analogs, isomers and derivatives thereof, such as D- or L-ascorbic acid and sugar derivatives thereof (eg γ-lactoascorbic acid, glucoascorbic acid, fucoscorbic acid , Glucoheptascorbic acid, maltoascorbic acid), sodium salt of ascorbic acid, potassium salt of ascorbic acid, isoascorbic acid (or L-erythroascorbic acid), salts thereof (eg alkali metal salts, ammonium salts or the art) Salt), enediol type ascorbic acid, enaminol type ascorbic acid, thioenol type ascorbic acid and the like, particularly D, L or D, L-ascorbic acid (and Its alkali metal salt) Ku is isoascorbic acid (or alkali metal salts) is preferably, sodium salts are preferred salts. A mixture of these reducing agents can be used as necessary.

8). Dye amplification solution The dye amplification solution is preferably a chromogenic substrate for detection of horseradish peroxidase, as described in `` Staining using clinical tests Vol.41 no.9 1020 H ₂ O ₂ -POD system ''. It can be used. Further, the chromogenic substrate described in Japanese Patent Application No. 2007-332287 can be used particularly preferably.

9. Detection solution A detection solution means a solution in which the contained drug reacts with a labeling substance or a test substance to cause a change in color, generation of a colored compound, luminescence, etc. For example, it is complexed with calcium ions as the test substance. Examples thereof include ortho-cresolphthalein complexone that develops color, and a copper ion solution that reacts and changes color with a protein as a test substance. Also included are solutions of labeled complexes that specifically bind to the test substance. Examples thereof include labeled DNA and labeled RNA for detecting DNA and RNA by hybridization, antibody-sensitized particles and antibody-labeled enzyme for detecting antigen.

10. Other auxiliaries Other auxiliaries for the amplification solution may include buffers, preservatives such as antioxidants or organic stabilizers, rate regulators. As a buffering agent, for example, a buffering agent using acetic acid, citric acid, sodium hydroxide or any salt thereof, tris (hydroxymethyl) aminomethane, or a buffering agent used for general chemical experiments is used. Can do. These buffers can be used as appropriate to adjust the pH to the optimum value for the amplification solution. Alkylamine can be used as an additive as an antifoggant, and dodecylamine is particularly preferable.
In order to improve the solubility of these additives, a surfactant can be used, and C ₉ H ₁₉ —C ₆ H ₄ —O— (CH ₂ CH ₂ O) ₅₀ H is particularly preferable.

11. Calculation method of average particle size at the time of detection At the time of detection (after amplification), cut out the test line part, attach the back of the sample to the sample stage with carbon paste, cut the cross section, deposit carbon, and with a scanning electron microscope, Observe the shape and size. For example, with the FE-STEM S-5500 manufactured by Hitachi High-Technologies, the sample surface can be observed by reflected electrons at an acceleration voltage of 10 KV. Thereafter, 100 signal particles are selected, the equivalent circle diameter of the projected area of the particles is measured, the average value is calculated, and the average particle size at the time of detection is obtained.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

(1) Preparation of influenza A type B detection immunochromatography kit (1-1) Preparation of anti-influenza A type B antibody modified gold colloid (1-1-1) F (ab ') ₂ fragmented anti-influenza A virus antibody Preparation anti-influenza a virus antibody (Product No. 7307, Medix Biochemica) was used to prepared using ^{ImmunoPure R IgG1 Fab and F (ab} ') 2 Preparation Kit ( part No. 44880, Pierce).

(1-1-2) Preparation of F (ab ') _2- fragmented anti-influenza B virus antibody Anti-influenza B virus antibody (Part No. 1131 (Vyrostat)) and lysyl endopeptidase (Part No. 125-05061) Photopure drug) was diluted with 50 mM Tris-Hcl buffer (pH 8.5) so as to have a molar ratio of 1: 100, and reacted at 37 ° C. for 3 hours. Thereafter, the F (ab ′) ₂ antibody was purified with ImmunoPure (A) IgG Purification Kit (product number 44667, Pierce).

(1-1-3) Preparation of colloidal gold colloid modified with anti-influenza type A antibody By adding 1 mL of 50 mM KH2PO4 buffer (pH 7.5) to 9 mL of 50 nm diameter gold colloid solution (EM.GC50, BBI), pH 1 mL of a 160 μg / mL anti-influenza A type monoclonal antibody (Anti-Influenza A SPTN-5 7307, Medix Biochemica) solution was added to the colloidal gold solution prepared above and stirred. After standing for 10 minutes, 550 μL of 1% polyethylene glycol (PEG Mw. 20000, product number 168-11285, Wako Pure Chemical Industries) aqueous solution was added and stirred, followed by 10% bovine serum albumin (BSA FractionV, product number A-7906, SIGMA) 1.1 mL of aqueous solution was added and stirred. After centrifuging this solution at 8000 × g, 4 ° C. for 30 minutes (himacCF16RX, Hitachi), the supernatant was removed except for about 1 mL, and the gold colloid was redispersed with an ultrasonic washer. Then, it is dispersed in 20 mL of colloidal gold stock solution (20 mM Tris-HCl buffer (pH 8.2), 0.05% PEG (Mw. 20000), 150 mM NaCl, 1% BSA, 0.1% NaN3), and again 8000 × g After centrifugation at 4 ° C. for 30 minutes, the supernatant was removed except for about 1 mL, and the gold colloid was redispersed with an ultrasonic washer to obtain an antibody-modified gold colloid (50 nm) solution.

(1-1-4) Preparation of anti-influenza B antibody-modified gold colloid Adjust pH by adding 1 mL of 50 mM glycine buffer (pH 9.0) to 9 mL of 50 nm diameter gold colloid solution (EM.GC50, BBI). 1 mL of a 120 μg / mL anti-influenza B monoclonal antibody (MONOTOPE aby Influenza B Virus (nuclear) Purified 1131, ViroStat, Inc.) solution was added to the colloidal gold solution and stirred. After standing for 10 minutes, 550 μL of 1% polyethylene glycol (PEG Mw. 20000, product number 168-11285, Wako Pure Chemical Industries) aqueous solution was added and stirred, followed by 10% bovine serum albumin (BSA FractionV, product number A-7906, SIGMA) 1.1 mL of aqueous solution was added and stirred. After centrifuging this solution at 8000 × g, 4 ° C. for 30 minutes (himacCF16RX, Hitachi), the supernatant was removed except for about 1 mL, and the gold colloid was redispersed with an ultrasonic washer. Then, it is dispersed in 20 mL of colloidal gold stock solution (20 mM Tris-HCl buffer (pH 8.2), 0.05% PEG (Mw. 20000), 150 mM NaCl, 1% BSA, 0.1% NaN3), and again 8000 × g After centrifugation at 4 ° C. for 30 minutes, the supernatant was removed except for about 1 mL, and the gold colloid was redispersed with an ultrasonic washer to obtain an antibody-modified gold colloid (50 nm) solution.

(1-2) Preparation of colloidal gold antibody retaining pad Influenza A and B antibody-modified colloids prepared in (1-1) are mixed in a 1: 1 ratio, and then a colloidal gold coating solution (20 mM Tris-Hcl buffer) The solution was diluted with (pH 8.2), 0.05% PEG (Mw. 20000), 5% sucrose) and water, so that the OD at 520 nm was 3.0. 0.8 mL of this solution was uniformly applied to each glass fiber pad (Glass Fiber Conjugate Pad, Millipore) cut to 8 mm × 150 mm and dried under reduced pressure overnight to obtain a colloidal gold antibody holding pad.

(1-3) Preparation of antibody-immobilized membrane (chromatographic carrier) (1-3-1) Preparation of antibody-immobilized membrane (chromatographic carrier)
An antibody-immobilized membrane was prepared by immobilizing an antibody by the following method with respect to a nitrocellulose membrane (with plastic backing, HiFlow Plus HF120, Millipore) cut to 25 mm × 200 mm. Anti-influenza A monoclonal antibody (Anti-Influenza A SPTN-5 7307, Medix Biochemica) solution for immobilization, prepared at 1.5 mg / mL, 7 mm from the bottom, with the long side of the membrane down Was applied in a line shape having a width of about 0.7 mm using an inkjet type coating machine (BioDot). Similarly, at a position of 10 mm from the bottom, widen the anti-influenza B monoclonal antibody (MONOTOPE aby Influenza B Virus (nuclear) Purified 1131, ViroStat, Inc.) solution for immobilization prepared to 1.5 mg / mL. It was applied in a line of about 0.7mm. Similarly, at a position 13 mm from the bottom, an anti-mouse IgG antibody for control (anti-mouse IgG (H + L), rabbit F (ab ') 2, part number 566-70621) prepared to 0.5 mg / mL was prepared. , Wako Pure Chemicals) solution was applied in a line. The coated membrane was dried at 50 ° C. for 30 minutes with a hot air dryer. 500 mL of blocking solution (50 wm borate buffer (pH 8.5) containing 0.5 w% casein (milk-derived, product number 030-01505, Wako Pure Chemical Industries)) was placed in a vat and allowed to stand for 30 minutes. After that, transfer to 500 mL of washing / stabilizing solution (50 mM Tris-HCl (pH 7.5) buffer containing 0.5 w% sucrose and 0.05 w% sodium cholate) in the same vat, soak for 30 minutes. did. The membrane was removed from the solution and dried overnight at room temperature to obtain an antibody-immobilized membrane.

(1-4) Preparation of immunochromatographic strip The antibody-immobilized membrane prepared in (1-3) was attached to a back adhesive sheet (ARcare9020, Nipple Technocluster). At that time, the anti-influenza A antibody line side is the lower side of the membrane long side. Attach the gold colloid antibody holding pad prepared in 2 so that it overlaps about 2 mm below the antibody-immobilized membrane, and then add the sample addition pad (18 mm × 250 below the gold colloid antibody holding pad so that it overlaps about 4 mm. A glass fiber pad (Glass Fiber Conjugate Pad, Millipore) cut into mm was pasted and pasted. Further, an absorption pad (cellulose glass membrane (CF6, Whatman) cut to 80 mm × 250 mm) was overlaid on the upper side of the antibody-immobilized membrane so as to be overlapped by about 5 mm. By cutting these lap-bonded members (immunochromatography main body member) in parallel with the short side so that the long side of the member is 25 mm wide, the guillotine cutter (CM4000, NIPPN Technocluster Co., Ltd.) is 25 mm A x55 mm immunochromatographic strip was prepared. These were used as test immunochromatography kits.

(1-5) Washing solution A PBS buffer containing 1% BSA in which 1% by weight of BSA (Sigma) was dissolved in PBS buffer (Wako Pure Chemical Industries, Ltd.) was used as a washing solution.

(1-6) Preparation of silver amplification solution (1-6-1) Preparation of amplification solution A (1-6-1-1) Preparation of amplification solution A-1 Iron (III) nitrate nonahydrate in 325 g of water Product (Wako Pure Chemicals, 095-00995) dissolved in water, 40mL of 1mol / L iron nitrate aqueous solution, citric acid (Wako Pure Chemicals, 038-06925) 10.5g, dodecylamine (Wako Pure Chemicals, 123- 00246) 0.1 g of surfactant C ₉ H ₁₉ —C ₆ H ₄ —O— (CH ₂ CH ₂ O) ₅₀ H 0.44 g is dissolved. When all are dissolved, add 40 mL of nitric acid (10 wt%) while stirring with a stirrer. 80 mL of this solution was measured, and 11.76 g of ammonium iron (II) sulfate hexahydrate (Wako Pure Chemical Industries, Ltd., 091-00855) was added to obtain an amplification solution A-1.

(1-7-1-2) Preparation of amplification solution A-2 Add water to 10 mL of silver nitrate solution (containing 10 g of silver nitrate) to make the total volume 100 g, and then use amplification solution A-2 (10 wt% silver nitrate). Aqueous solution).

(1-7-1-3) Preparation of amplification solution A 40 mL of amplification solution A-1 was weighed, and 4.25 mL of amplification solution A-2 was added and stirred to obtain amplification solution A.

(2) Evaluation (2-1) Spotting To the sample addition pad of the test immunochromatography kit prepared in (1-4), add 500 μL of the sample liquid meter uniformly using an 8 pipette man. Left to stand.

(2-2) Cleaning The membrane was removed from the case, and the sample pad and water absorbing pad were removed. The straight line connecting the washing solution addition pad and the water absorption pad passes through the point between the two ends of the strip in the middle of the area between the two capture sites (TL). And the cleaning liquid addition pad (Glass Fiber Conjugate Pad, Millipore) 13mm x 25mm at the upstream end of the cleaning liquid development. Paste the back adhesive sheet (ARcare9020, NIPPN Technocluster Co., Ltd.) with a tape, and use a pad for water absorption (100mm x 25mm cellulose membrane (CF6, Whatman)) 95mm x Paste a back adhesive sheet (ARcare9020, Nipple Techno Cluster Co., Ltd.) cut to 25 mm, and add 7 mL of cleaning liquid to a rectangular parallelepiped container (9 mm long x 64 mm wide x 25 mm high) Doo is stood so immersed in the liquid, and washed the left 10 minutes.

  In this experiment, the height of the liquid surface depends on the shape of the cleaning liquid container at the time of cleaning, the shape and material of the sample addition pad of the immunochromatography kit, the experimental environment (temperature and humidity), the material and thickness of the absorption pad, and the absorption pad. Bonding between the nitrocellulose membrane and the nitrocellulose membrane is a factor that changes the water absorption speed and amount of the cleaning liquid, and it is necessary to keep it constant in the experiment. The water absorption speed and amount of the cleaning liquid are factors that influence the final cleaning effect (reduction of the remaining amount of gold fine particles). This experiment was conducted at an air temperature of 22 ± 3 ° C and humidity of 50 ± 15%.

(2-3) Pre-amplification measurement The reflection absorption of the detection line before amplification was measured using an immunochromatographic concentration meter ICA-1000 (Hamamatsu Photonics), and the difference in absorbance between the background and the line (ΔOD) ) Was evaluated.

(2-4) Signal amplification with amplification solution, background evaluation Remove the water absorbing pad, immerse the amplification solution A prepared in (1-6) in a plastic tray containing 40 mL, and shake with a shaker. Silver amplification was performed for minutes. The membrane was removed from the amplification solution and washed thoroughly with water for 3 minutes.

(2-5) Post-amplification measurement This was measured using an immunochromatographic concentration measuring instrument ICA-1000 (Hamamatsu Photonics), and the difference in absorbance (ΔOD, unit mABS) between the background and the line portion was determined and evaluated.

(3-1) Preparation of calibration curve BD Flu Examan Control A + B- (Becton Dickinson) was dissolved in a PBS buffer containing 1% BSA to prepare an antigen dilution solution. Using this antigen dilution, × 1, × 1/8, × 1/32, × 1/128, the test line concentration before amplification is measured (N = 3), and the test line concentration (mABS) is measured on the x-axis. The following calibration curve was established by taking the antigen concentration (BD positive control solution dilution ratio) on the y axis and linearly approximating them.
y = -0.011405 + 2.0795x (Formula 1)

Also, measure the test line concentration before amplification using the same antigen dilution solution, × 1/10, 1/100, × 1/1000, × 1/10000 (N = 3), and test line concentration (mABS) The x-axis and the antigen concentration (BD positive control solution dilution ratio) are on the y-axis, and the following calibration curve was drawn by approximating them to a quartic function.
y = 0.00067891 -0.06895x + 2.375x ^ 2 -17.261x ^ 3 + 42.734x ^ 4 (Formula 2)

(4) Results
(4-1) Mock specimen preparation BD Flu Examan Control A + B- (Becton Dickinson) was dissolved in PBS buffer containing 1% BSA, and diluted antigen solution (× 0.5, × 0.1, × 0.025, × 0.0125) , X0.00160, x0.0004, x0.0002, x0.0001).

<Comparative example> Quantification by measurement before amplification Only from the measurement of the test line concentration before amplification, the quantitative value of antigen: the calculated concentration quantitative value was determined using (Equation 1). Further, an error rate (((actual concentration−calculated quantitative value) / actual concentration) × 100) with the actual concentration was obtained. The results are shown in Table 1.
When it was defined that the calculated quantitative value was obtained and the error rate was 35% or less, it was possible to quantify the dilution rate from 0.5 to 0.025.

<Example> Quantification by measurement before and after amplification When the PBS buffer containing 1% BSA was used as a specimen, the first optical density value was measured 10 times, and the average value was 0.0015 and the standard deviation was 0.00022. Compared to the value 0.0019 obtained by adding the double, the first optical density value in the known concentration sample solution obtained by diluting BD Flu Examan Control A + B- to a concentration of 0.013 with PBS buffer containing 1% BSA A value of 0.0092 obtained by subtracting a value 0.002 that is twice the standard deviation 0.001 from the average value 0.0112 is 10 times larger, and the value is sufficiently larger. This analyte concentration x 0.013 is the quantification limit concentration, and the average value 0.0112 of the first optical density value at this time is the measurement switching optical density value. If the first optical measurement value is smaller than 0.0112, the first The amount of the test substance was calculated from the measurement value, and when the first optical measurement value was 0.0112 or more, the amount of the test substance was calculated from the second measurement value.

  From the concentration value before amplification, the concentration value before amplification, which is 0.0112 or more, to the antigen dilution ratio of 0.025, the quantitative value of the antigen: the calculated concentration quantitative value was determined using (Equation 1). The results are shown in Table 1.

  For the simulated sample that had a concentration value before amplification below that, the quantitative value of the antigen: the calculated quantitative value was determined from the concentration value after amplification using (Equation 2). When it was defined that the calculated quantitative value was obtained and the error rate was 35% or less, it was possible to quantify the dilution rate from 0.5 to 0.0002. Therefore, in the embodiment, the range is expanded by about two digits compared to the comparative example.

  In addition, when the sample after performing the second optical measurement in this experiment was SEM-observed by the method described in “11. Calculation method of average particle size at detection” in this specification, the average particle size was It was confirmed that it was in the range of 5 to 10 μm.

Claims (13)

In a chromatographic method for detecting a test substance,
(A) a step of performing a first optical density measurement for quantifying the captured labeling substance;
(B) a step of amplifying the captured labeling substance,
(C) a second optical density measurement step for quantifying the labeled substance after amplification, and (d) the amount of the test substance from the first optical density measurement and / or the second optical density measurement value. Quantifying. If the optical density value measured in the first optical density measurement is larger than the predetermined measurement switching optical density value, the amount of the test substance is quantified from the optical density value measured in the first optical density measurement. When the optical density value measured in the first optical density measurement is less than or equal to the predetermined measurement switching optical density value, the amount of the test substance is calculated from the optical density value measured in the second optical density measurement after amplification. The method according to claim 1, wherein A test containing a predetermined known amount of test substance, compared to the average value obtained by performing the first optical density measurement multiple times using a test solution that does not contain the test substance, plus two times the standard deviation. The first optical density measurement is carried out using a known amount such that a value obtained by subtracting twice the standard deviation from the average value obtained by performing the first optical density measurement a plurality of times using the liquid is larger. Set as the quantitation limit concentration that can be quantified in the measurement, the average value obtained by performing the first optical density measurement a plurality of times using a test solution containing the test substance of the quantification limit concentration is used as the measurement switching optical density value, Item 3. The method according to Item 2. The method according to claim 1, which is an immunochromatographic method. The labeling substance modified with the first binding substance for the test substance is developed on the insoluble carrier in a mixed state, and the second binding substance for the test substance or the first binding substance for the test substance is developed. The method according to any one of claims 1 to 4, wherein the test substance and the labeling substance are captured at a reaction site on an insoluble carrier having a binding substance to detect the test substance. 6. A method according to claim 5, wherein the insoluble carrier is a porous carrier. The measurement method according to claim 5 or 6, wherein the first binding substance and / or the second binding substance is an antibody or a fragmented antibody. Between step (a) and step (c),
(E) a step of cleaning by developing a cleaning solution;
The method according to claim 1, comprising: The method according to claim 1, wherein the amplification is performed using an amplification solution containing a compound containing silver and a reducing agent for silver ions. The method of claim 9, wherein the reducing agent for silver ions comprises divalent iron ions. The method according to any one of claims 1 to 10, wherein the labeling substance is a metal colloid. The method of claim 11, wherein the metal colloid is a gold colloid. The method according to any one of claims 1 to 12, wherein an average particle size of the labeling substance after amplification when performing the second optical density measurement is a size of 1 µm or more and 20 µm or less.