JP2013181935A - Chromatographic method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chromatographic method for improving reproducibility of a detection value without being affected by fluctuation derived from variation in size of particles.SOLUTION: There is provided a chromatographic method for developing a test substance, and a labeling substance containing metal modified by a first bondable substance to the test subject on an insoluble carrier at a state that a complex of them is formed, capturing the test substance and the labeling substance at a reaction part on the insoluble carrier having a second bondable substance to the test substance, after that, performing amplification by using a reducer for chemical compounds containing metal and metal ions, and measuring the number of labeling particles after the amplification per unit area of the reaction part to detect the test substance.

Description

  The present invention relates to a chromatographic method for performing a signal amplification operation for increasing detection sensitivity.

  Among immunoassay methods, the immunochromatography method is generally used because it is easy to operate and can be measured in a short time. Competitive reactions or sandwich-type reactions are widely used as immune reactions used in immunochromatography. Among them, the sandwich type reaction is mainly used in the immunochromatography method, and in the typical example, the following operation is performed in order to detect the test substance composed of the antigen in the sample. First, it has a reaction site by immobilizing fine particles sensitized with an antibody against the test substance antigen as a solid phase fine particle on a chromatographic carrier, or by immobilizing this antibody itself directly on the chromatographic carrier. A chromatographic carrier is prepared. On the other hand, sensitized labeled microparticles are prepared by sensitizing an antibody capable of specifically binding to the test substance to the labeled microparticles. The sensitized labeled fine particles are moved chromatographically on the chromatographic carrier together with the sample. By the above operation, the immobilized antibody becomes an immobilization reagent at the reaction site formed on the chromatographic carrier, and the sensitized labeled microparticles specifically bind to the immobilized reagent via the antigen as the test substance. As a result, the sensitized labeled microparticles are captured at the reaction site, and the presence or amount of the test substance in the sample can be measured by visually determining the presence or degree of the signal generated thereby. it can.

  In some immunochromatography methods, a detection signal is amplified in order to avoid the problem that the antigen is not detected due to low sensitivity (false negative). As a signal amplification method, an enzyme such as alkaline phosphatase or peroxidase may be used as a label, but a compound containing silver in a label selected from the group consisting of a metal colloid label and a metal sulfide label, and a reducing agent for silver ions In some cases, detection is performed by sensitization using silver (silver amplification). An immunochromatograph using amplification as described above is described in Patent Document 1 and the like. On the other hand, it is known to detect labels such as metal particles with an optical microscope in the field of biochemistry (Patent Document 2).

Japanese Patent Laid-Open No. 2002-202307 JP 2007-178439 A

  In the immunochromatography method described in Patent Document 1, after amplifying and increasing the size of the labeled particles for high sensitivity, the reflected light is visually measured or an appropriate measuring device (for example, a reflected light measuring device or a scanner). The labeled particles are detected by analyzing with. However, since the size of the particles varies, there is a problem that the amount of light shielded by the particles varies, and the reproducibility of the optical density detection result is deteriorated.

  This invention made it the subject which should be solved to provide the chromatographic method which can improve the reproducibility of a detected value, without receiving the fluctuation | variation resulting from the dispersion | variation in the magnitude | size of particle | grains.

  As a result of intensive studies to solve the above problems, the present inventor improves the reproducibility of detected values by performing measurement by measuring the number of particles after amplification in a chromatographic method for performing signal amplification operation. In particular, the present invention has been completed.

  That is, according to the present invention, a test substance and a labeling substance containing a metal modified with a first bondable substance for the test substance are developed on an insoluble carrier in a state where these complexes are formed, A chromatograph that captures the test substance and the labeling substance at a reaction site on an insoluble carrier having a second substance capable of binding to the test substance, and then amplifies using a metal-containing compound and a reducing agent for metal ions There is provided a chromatographic method as described above, wherein the test substance is detected by measuring the number of labeled particles after amplification per unit area of the reaction site.

Preferably, the insoluble carrier after amplification is infiltrated with a solvent for clarifying the insoluble carrier.
Preferably, the refractive index difference Δn between the solvent for clarifying the insoluble carrier and the insoluble carrier is −0.1 or more and 0.1 or less.
Preferably, the refractive index difference Δn between the solvent for clarifying the insoluble carrier and the insoluble carrier is −0.02 or more and 0.02 or less.

Preferably, amplification is performed using an amplification solution containing a compound containing silver and a reducing agent for silver ions.
Preferably, the amplified labeled particles are detected by an optical instrument using a lens, and the number of labeled particles per unit area of the reaction site is measured.
Preferably, the optical apparatus using a lens is a microscope, a scanner, a camera, or a loupe.

  According to the chromatographic method of the present invention, the reproducibility of the detected value can be improved by measuring by measuring the number of particles after amplification.

FIG. 1 shows the device used in the examples. FIG. 2 shows a second device component of the devices used in the example.

1. Detection of Particles and Measurement of Number of Particles The chromatographic method of the present invention is characterized in that the number of labeled particles after amplification per unit area of a reaction site is measured to detect a test substance.
As a means for detecting particles at the reaction site on the insoluble carrier, it is preferable to use an optical device (for example, a microscope, a scanner, a camera, a loupe, or the like) using a lens capable of enlarging an observation target. In the case of a scanner, a camera, or a loupe, a high-resolution one that can detect minute particles to be observed is preferable. In the case of a microscope, the type thereof is not particularly limited as long as particles can be detected. For example, a stereoscopic microscope, an optical microscope, a phase contrast microscope, a differential interference microscope, an electron microscope, and the like can be used. However, in the case where the measurement sensitivity is lowered due to suspended matters in the air as in an electron microscope, vacuum drying must be performed as a pretreatment, and there is a limitation that the number of measurement is limited. In the present invention, it is particularly preferable to use an optical microscope from the viewpoint that sample pretreatment is not necessary and measurement can be performed easily in a short time.

  In the present invention, amplification is performed using a metal-containing compound and a reducing agent for metal ions. In this way, the particles enlarged by amplification can be easily detected from a magnification of about 10 times. Therefore, the magnification in the case of using a microscope, a scanner, a camera, or a loupe is preferably 10 times or more, and the upper limit is not particularly limited. Therefore, it may be 800,000 times that can be used with an electron microscope or the like. However, as the magnification increases, the imaging range becomes narrower, so it takes time to image the area required for measurement. In order to perform measurement in a shorter time, the magnification is preferably 10 to 1000 times, more preferably 10 to 100 times, further preferably 10 to 50 times, and particularly preferably 10 to 30 times.

  As a method of measuring (counting) the number of particles, there is a measurement of the number of particles by image analysis. For example, an image taken with a microscope, a scanner, or a camera can be read by a particle number measurement software (Image J, NIH Image (Scion Image), UTHSCSA Image Tool, etc.) and the number of particles can be automatically measured. However, as long as particles can be measured, a method of visual measurement may be used, and the method is not limited to using automatic measurement by an image analysis method. For example, the number of particles may be measured visually using a loupe.

2. Clarification of Insoluble Carrier According to a preferred embodiment of the present invention, it is possible to take a means for clarifying the insoluble carrier before measuring the number of particles. This makes it easier to detect particles present in the entire thickness direction of the insoluble carrier. As a means for clarifying the insoluble carrier, a solvent for clarifying the insoluble carrier (hereinafter, also referred to as a carrier clarifying solvent) can be permeated into the insoluble carrier after amplification.
Moreover, the same effect as the case where the above-mentioned solvent is permeated can be achieved by reducing the thickness of the insoluble carrier instead of permeating the above-mentioned solvent. However, if the thickness of the insoluble carrier is reduced, the development amount of the test substance is also restricted, and measurement is restricted.

  In general, in an immunochromatography method, a membrane bundled with nitrocellulose (refractive index 1.48) fibers having a relatively high refractive index of about 0.1 μm to several μm in both pore diameter and thread diameter is often used as an insoluble carrier. . Nitrocellulose membrane (refractive index 1.48) has a large difference in refractive index from air or solvent (refractive index 1.33 in the case of water) that fills the voids in the membrane, so part of the light is scattered As a result, the portion becomes opaque and cannot be detected. Therefore, by permeating a solvent having a refractive index close to that of a nitrocellulose membrane (a carrier-clearing solvent), the light can be removed without being scattered, and the detectable region can be increased. The refractive index referred to in the present invention is a value measured at 20 ° C. and 589 nm.

  In the insoluble carrier having a thickness of 1 mm, the light scattered from the label in the lowest layer is scattered several hundred times at the interface between the void and the fiber and transmitted upward. From the result of simulating the dependence of the refractive index difference Δn on the medium filled in the gap, in order to obtain a transmittance of 80% or more, the refractive index difference Δn is −0.1 ≦ Δn ≦ 0.1, 90%. In order to obtain the above light transmittance, it is known that it is necessary to select a combination of an insoluble carrier having a refractive index satisfying −0.07 ≦ Δn ≦ 0.07 and a solvent. In addition, the smaller the refractive index difference Δn between the insoluble carrier and the solvent, the closer to the transmittance 100%, and the refractive index of the insoluble carrier and the refractive index of the solvent become equivalent. When −0.02 ≦ Δn ≦ 0.02, the light is almost transmitted. An excellent visibility with a rate of 100% can be obtained (see JP 2009-257819 A).

  That is, as a condition of the solvent having a refractive index close to that of the insoluble carrier, the refractive index difference Δn with the insoluble carrier is −0.1 ≦ Δn ≦ 0.1, and preferably −0.07 ≦ Δn ≦. 0.07, and more preferably −0.02 ≦ Δn ≦ 0.02.

As the carrier clarification solvent, a solvent having a boiling point of 70 ° C. or higher is preferable, and a solvent having a boiling point of 90 ° C. or higher is more preferable.
Solvents that can be used as the carrier clarification solvent include dimethyl sulfoxide (refractive index 1.48), glycerin (refractive index 1.45) and aqueous solutions thereof, benzene (refractive index of about 1). .5) and similar compounds (for example, benzyl alcohol (refractive index 1.54)), paraffin oil (refractive index 1.48) and the like.

When selecting the carrier clarification solvent, it is possible to select the solvent so that −0.1 ≦ Δn ≦ 0.1 calculated from the refractive index of the insoluble carrier. It is possible to select a solvent that makes the carrier transparent by permeating such a solvent.
The transparency of the insoluble carrier here means that the insoluble carrier has a transmittance of 80% or more as measured by a transmittance measuring device using visible light, or a transparent in which the insoluble carrier and the insoluble carrier are placed through the insoluble carrier. This means that the number of metal particles having a size of 1 μm or more arranged between different substrates (such as a slide glass) can be identified.
A dimethyl sulfoxide solution is particularly preferable as a carrier clarification solvent when a nitrocellulose membrane is used as an insoluble carrier.

  When the carrier clarification solvent is permeated into the insoluble carrier, it can be permeated by substituting the aqueous solution after amplification if it is a hydrophilic solvent. In the case of a hydrophobic solvent, the aqueous solution in the insoluble carrier can be removed by washing and dried and then permeated. However, any method may be used as long as the carrier-clearing solvent can be permeated into the insoluble carrier, and the method is not limited to these methods.

3. Reduction of coefficient of variation According to the chromatographic method of the present invention, the reproducibility of the detected value can be improved by measuring by measuring the number of particles after amplification. Here, a coefficient of variation can be used as an index of variation in the measured value. The coefficient of variation is a standard deviation divided by an average value, and indicates a relative variation.

  Small variation means that accurate measurement can be performed, and that accurate measurement means that positive / negative determination can be made with higher accuracy in diagnosis using an influenza test kit. Therefore, the small variation is an advantageous factor in diagnosis.

4). Chromatograph In general, the chromatographic method is a method for determining, measuring, and measuring a test substance in a simple, rapid and specific manner by the following method. That is, at least one containing an immobilizing reagent that can bind to a test substance (corresponding to a second binding substance to the test substance or a binding substance to the first binding substance described below; specifically, an antibody, an antigen, etc.) A chromatographic carrier having one labeled substance capture region is used as the stationary phase. On the chromatographic carrier, the liquid containing the labeling substance modified by the first binding substance to the test substance is moved chromatographically in the chromatographic carrier as a moving layer, and the test substance and the labeling substance are It reaches the labeling substance capturing region while specifically binding. In the labeled substance capturing region, the complex of the test substance and the labeled substance specifically binds to the immobilized reagent, so that the labeled substance is concentrated in the immobilized reagent only when the test substance is present in the analysis solution. This is a technique for qualitatively and quantitatively analyzing the presence of a test substance in a liquid to be analyzed by visual inspection or using an appropriate device.

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

5. 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 a test substance. For example, a biological sample, particularly an animal. (Especially human) body fluids (eg blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, runny nose or sputum) or excrement (eg feces), organs, tissues, mucous membranes and skin, etc. A rubbed specimen (swab), gargle, or animals and plants themselves or their dried bodies may be mentioned. Examples of the test substance include natural products, toxins, hormones, physiologically active substances such as agricultural chemicals, environmental pollutants, viruses, antigens, antibodies, and the like.

6). 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

7). 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.
The chromatographic strip includes a sample addition pad, a labeling substance holding pad (for example, a colloidal gold antibody holding pad) having a labeling substance holding area, and a chromatographic carrier (for example, a labeling substance capturing area) from upstream to downstream in the developing direction. Antibody-immobilized membrane) and an absorption (water absorption) pad can be arranged on the adhesive sheet in this order. The chromatographic carrier has a labeling substance capturing area such as an area in which an antibody or antigen that specifically binds to the test substance is immobilized, and a control zone in which the control antibody or antigen is immobilized if desired. (It may be described as a control region).

  The labeling substance holding pad can be 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. .

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

7-1. Labeled substance As the labeling substance for detection, 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. 1-15 nm is particularly preferable. 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.).

  According to the present invention, in a chromatograph using a metal colloid label or a metal sulfide label, other metal alloy label (hereinafter sometimes referred to as a metal label), or a polymer particle label containing a metal as a label for detection. The signal of the metallic label can be amplified. Specifically, after forming a complex of a test substance and a label for detection, 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 ions 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 the test substance is analyzed. It can be carried out with high sensitivity. Therefore, in the chromatographic method of the present invention, the silver particles generated by the reducing action of silver ions by the reducing agent are used to carry out the reaction for deposition on the label of the immune complex, and the thus amplified signal is analyzed. Except for this point, conventionally known chromatographic 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 a test substance (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. Colloids or silver colloids and mixtures thereof can be mentioned. Examples of metal sulfide labels include iron, silver, palladium, lead, copper, cadmium, bismuth, antimony, tin, or mercury sulfides. Can be mentioned. In the chromatographic method of the present invention, one or more of these metal colloid labels and / or metal sulfide labels can be used as labels.

7-2. 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.

  The labeled substance capture region in the chromatographic kit of the present invention has (a) a second binding substance for the test substance, or (b) a binding substance for 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.), and the like. 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 binding substance for the first binding 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) are bound to each other. Such a compound corresponds to this.

Preferably, the first binding substance is an antibody and / or the second binding substance is an antibody.
More preferably, the first binding substance is an antibody, and the second binding substance is an antibody that binds to the first binding substance.

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

7-3. Chromatographic carrier The chromatographic carrier is preferably a porous carrier. In particular, nitrocellulose membrane (refractive index 1.48), cellulose membrane (refractive index 1.49), acetylcellulose membrane (refractive index 1.47), polysulfone membrane (refractive index 1.63), polyethersulfone membrane (Refractive index 1.65), nylon film (refractive index about 1.53), glass fiber (refractive index 1.43 to 2.14), non-woven fabric (refractive index depends on material), cloth (refractive index depends on material) ) Or yarn (refractive index depends on the material).

  Usually, the labeling substance capturing region is prepared by immobilizing the second binding substance for the test substance or the binding substance for the first binding substance on a part of the chromatographic carrier. The second binding substance to the test substance or the binding substance to the first binding substance may be directly fixed to a part of the chromatographic carrier by physical or chemical binding, or may be fine particles such as latex particles. The fine particles may be physically or chemically bound and trapped on a part of the chromatographic carrier to be immobilized. 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.

7-4. 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 test substance, but also has a function of filtering insoluble matter particles and the like in the sample. In addition, in order to prevent the test substance in the sample from adsorbing non-specifically to the material of the sample addition part and reducing the accuracy of the analysis, the material constituting the sample addition part must be non-specific in advance. It may be used after anti-adsorption treatment. The sample addition pad may also serve as a label holding pad described below.

7-5. 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.

7-6. Absorption (water 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. Water-absorbing materials such as filter paper, non-woven fabric, cloth, and cellulose acetate are used. Since the chromatographic speed after the chromatographic tip of the added sample reaches the absorber varies depending on the material, size, etc. of the absorbent material, the selection can set the speed suitable for the measurement of the test substance. .

8). Inspection Method 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 excess amount of the labeled first antibody is further contacted, if the test substance is present in the test sample, the immobilized second antibody and the test substance (antigen) An immune complex consisting of the labeled 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. The region where the immobilized second antibody is immobilized on the first insoluble carrier is observed as it is, and the labeled product is detected or quantified to determine the presence or absence or amount of the test substance in the test sample. In the present invention, for example, by supplying a metal ion and a reducing agent, a signal from the label of the labeled first antibody that has formed the immune complex is amplified and detected.

  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, An antigen-antibody reaction on the first insoluble carrier is caused by the test substance itself having a binding property to one antibody 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 the 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. The substance having binding ability to the first antibody in the first insoluble carrier can be observed as it is, and the amount of the labeled substance can be detected or quantified to determine the presence or absence of the test substance in the test sample or to determine the quantity. . In the present invention, for example, by supplying a metal ion and a reducing agent to the fixed region, a signal from the label of the labeled first antibody forming the immune complex is amplified and detected.

9. Amplification Solution An amplification solution is a solution that can cause signal amplification by producing a colored compound or light emission by the reaction of a contained drug through the action of a labeling substance or a test substance. For example, a silver ion solution that causes metal silver to be deposited by physical development on a metal label may be used. However, a solution containing metal ions (for example, platinum ions, gold ions, etc.) other than silver ions can be used as long as the metal can be deposited on the metal label by physical development.

  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.

  In the present invention, of the two types of amplification reagents used for amplifying the signal of the labeling substance captured in the labeling substance capturing region, the first amplification reagent is used as the first liquid, and the second amplification liquid is used as the second amplification reagent. It is preferable that amplification is performed by adding the first liquid and the second liquid in order. The first liquid is preferably added to a pad for feeding a reducing agent solution located upstream of the labeling substance holding pad and the sample addition pad.

  As a specific example of the amplification liquid, a combination of a first liquid containing a reducing agent for metal ions (eg, silver ions) and a second liquid containing a compound containing metal ions (eg, silver ions) is used. Can do.

  Hereinafter, a reducing agent for silver ions contained in the first liquid and a compound containing silver ions contained in the second liquid will be described.

(Compound containing silver ions)
As the silver ion-containing compound, an organic silver salt, an inorganic silver salt, or a silver complex can be used. Preferably, it is a silver ion-containing compound having high solubility in a solvent such as water, and examples thereof include silver nitrate, silver acetate, silver lactate, silver butyrate, and silver thiosulfate. Particularly preferred is silver nitrate. The silver complex is preferably a silver complex coordinated to a ligand having a water-soluble group such as a hydroxyl group or a sulfone group, and examples thereof include hydroxythioether silver.

The inorganic silver salt or silver complex is generally contained in an amount of 0.001 mol / m ^{2 to} 0.2 mol / m ² , preferably 0.01 mol / m ^{2 to} 0.05 mol / m ^{2 as} silver.

(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⁺ that 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 also 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, thioenolic type ascorbic acid and the like, and 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.

10. Other auxiliaries Other auxiliaries for the amplification solution may include buffers, preservatives such as antioxidants or organic stabilizers, and 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.

As a method of spotting the amplification reagent on the chromatographic kit, the reducing agent solution (first liquid) is spotted on the pad for feeding the reducing agent solution, and the silver ion solution (second liquid) is captured by the labeling substance A method of spotting from above is preferable.
As a method for incorporating the two types of amplification reagents in the chromatographic kit, there may be mentioned a method in which a pot containing a solution containing each amplification reagent is placed above the site where each amplification reagent is spotted. The reducing agent solution (first liquid) is placed on the upper part of the pad for feeding the reducing agent solution, and the pot containing the silver ion solution (second liquid) is placed immediately above the silver ion solution filling hole. preferable. By arranging in this way, the liquid flows by pushing each pot and can be spotted on a predetermined part.

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

(1) Preparation of influenza virus antigen detection immunochromatography kit (1-1) Preparation of anti-influenza A antibody-modified gold colloid 9 mL of 50 nm diameter gold colloid solution (EM.GC50, BBI) in 50 mM KH2PO4 buffer (pH 7.5) ) 1 mL of 160 μg / mL anti-influenza A type monoclonal antibody (Anti-Influenza A SPTN-5 7307, Medix Biochemica) solution was added to the gold colloid solution adjusted to pH by adding 1 mL 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. After that, it was dispersed in 20 mL of colloidal gold preservation solution (20 mM Tris-HCl buffer (pH 8.2), 0.05% PEG (Mw. 20000), 150 mM NaCl, 1% BSA), and again 8000 × g, 4 ° C. After centrifugation for 30 minutes, the supernatant was removed, leaving 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 anti-influenza A antibody-modified gold colloid holding pad The influenza A antibody-modified gold colloid prepared in (1-1) is applied to a gold colloid coating solution (20 mM Tris-Hcl buffer (pH 8.2), The solution was diluted with 0.05% PEG (Mw. 20000), 5% sucrose) and water, and diluted so that the OD at 520 nm was 0.04. 0.8 mL of this solution was uniformly applied to each glass fiber pad (Glass Fiber Conjugate Pad, Millipore) cut into 8 mm × 150 mm, and dried under reduced pressure overnight to obtain a colloidal gold antibody holding pad.

(1-3) Preparation of anti-influenza A antibody and anti-mouse IgG 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 HF180, Millipore) (refractive index: 1.48) cut to 25 mm × 300 mm. Line the anti-influenza A monoclonal antibody (Anti-Influenza A SPTN-5 7307, Medix Biochemica) solution prepared at a position 10 mm from the bottom with the long side of the membrane down to 1.5 mg / mL. It was applied to. Furthermore, an anti-mouse IgG antibody (anti-mouse IgG (H + L), rabbit F (ab ') 2, product number 566-70621, Wako Pure Chemical Industries) prepared to be 0.2 mg / mL at a position 15 mm from the bottom The 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 test 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 pad cut into 80 mm × 250 mm (CFSP223000, Whatman)) was overlaid on the upper side of the antibody-immobilized membrane so as to be overlapped by about 5 mm. By cutting these laminated members (immunochromatography main body member) in parallel with the short side so that the long side of the member is 7 mm wide, the guillotine cutter (CM4000, Nipple Technocluster) 7 mm A x55 mm immunochromatographic strip was prepared.

(1-5) Preparation of silver amplification solution
(1-5-1) Preparation of reducing agent solution 1mol / l iron nitrate aqueous solution 23.6 prepared by dissolving iron nitrate (III) nonahydrate (Wako Pure Chemicals, 095-00995) in water 290g. ml and citric acid (Wako Pure Chemicals, 038-06925) 13.1 g were dissolved. When all were dissolved, 36 ml of nitric acid (10% by weight) was added while stirring with a stirrer, and 60.8 g of ammonium iron (II) sulfate hexahydrate (Wako Pure Chemicals, 091-00855) was added to obtain a reducing agent solution.

(1-5-2) Preparation of silver ion solution To 66 g of water, 8 ml of silver nitrate solution (containing 10 g of silver nitrate) and 24 ml of 1 mol / l aqueous iron nitrate solution were added. Further, this solution, 5.9 ml of nitric acid (10% by weight), 0.1 g of dodecylamine (Wako Pure Chemicals, 123-00246), surfactant C ₁₂ H ₂₅ —C ₆ H ₄ —O— (CH ₂ CH ₂ O ) A solution prepared by dissolving 0.1 g of ₅₀ H in 47.6 g of water in advance was mixed to obtain a silver ion solution.

(1-6) Production of assay device As shown in FIG. 1, a test strip for immunochromatography was loaded on the first device component (made of polypropylene by injection molding) shown in FIG. Next, as shown in FIG. 2, glass fiber pads (Glass Fiber Conjugate Pad, Millipore) were loaded as the second insoluble carrier and the third insoluble carrier. 1 185 μL of the reducing agent solution prepared in (1-5-1) above is placed in the cleaning liquid storage pot shown in FIG. 1 and sealed with an aluminum laminate sheet (NewADM, Sunrise) by thermal lamination, and the second device component is mounted. It was fitted to the first device part. The pot receiving portion shown in FIG. 1 was charged with a liquid storage pot in which 95 μL of the silver ion solution prepared in the above (1-5-2) was placed and sealed with an aluminum laminate sheet.

(2) Evaluation (2-1) Spotting Simulated positive specimen (BD Flu Examan Control A + B- (Becton Dickinson)) diluted 128-fold or 512-fold with extract (PBS containing 1% BSA) Then, 140 μL was spotted on the specimen injection hole and allowed to stand for 10 minutes.

(2-2) Pressing the second and third insoluble carriers After developing the sample liquid for 10 minutes, press the pressing part of the second device part with a force of 10N to deflect the second device part. The insoluble carrier for liquid feeding (second and third insoluble carriers) was pressed against the membrane. The pressing amount at this time is limited by the contact between the first device component member and the second device component. The height of the member was adjusted so that the membrane surface of the strip for immunochromatography and the portion of the second device component contacting the membrane of the insoluble carrier for liquid feeding were crushed to 0.2 mm. Further, the shape of the protrusion of the second device component was adjusted so that the clearance between the membrane surface of the immunochromatographic strip and the pressing surface between the rib members of the second device component was 0.005 mm.

(2-3) Cleaning When 30 seconds have passed since the insoluble carrier for liquid feeding was pressed, push the cleaning liquid storage pot of the second device part from above to break through the aluminum sheet and soak the reducing agent solution into the insoluble carrier for washing. I didn't. Thus, the reducing agent solution was spread on an immunochromatographic strip and fed for 3 minutes. By this step, the immunochromatographic strip was immersed in the reducing agent solution, and the material not specifically adsorbed was washed.

(2-3) Signal amplification with amplification solution Push the liquid storage pot fitted into the pot receiving part loaded in the second device component, break the seal by the protruding part, and discharge the silver ion solution from the liquid storage pot for injection Poured into the hole. The silver ion solution was filled with the clearance between the membrane surface and the protruding portion of the second device component, and the gold colloid label adsorbed on the detection line was amplified for 1 minute. The membrane portion of the immunochromatographic strip after the sensitization treatment was cut out and washed thoroughly with water.

(3) Detection
(3-1) <Comparative Example> Optical density reading method The anti-influenza A antibody-immobilized membrane after washing with water was placed on a white plate and photographed with LAS-4000 (FUJIFILM). The absorbance difference (ΔO.D., unit mABS) between the line part and the background part was determined and used as the detection value.

(3-2) <Example> Particle Counting Method After washing with water, the anti-influenza A antibody-immobilized membrane was immersed in dimethyl sulfoxide (refractive index: 1.48) for 30 minutes. The membrane was placed on a slide glass, covered with a cover glass, and photographed with an inverted electric research microscope IX81 (Olympus). The difference (in units / 0.05 mm ² ) between the number of silver particles in the line portion and the background portion calculated by the particle count software Image J was used as the detection value.

(3-3) Calculation of coefficient of variation Measurement was performed 10 times using a solution obtained by diluting the antigen at each magnification, and an immunochromatographic strip after amplification treatment was prepared. The strip after amplification treatment was detected by each method, and the coefficient of variation was determined from the average value and standard deviation of each detected value. The results are shown in Table 1. Since the coefficient of variation of the example was lower than that of the comparative example, it was demonstrated that the reproducibility was good at each antigen concentration.

Claims (7)

A test substance and a labeling substance containing a metal modified with a first bindable substance for the test substance are developed on an insoluble carrier in a state in which these complexes are formed, and second bindable to the test substance is possible. A chromatographic method in which the test substance and the labeling substance are captured at a reaction site on an insoluble carrier having a substance, and then amplified using a compound containing a metal and a reducing agent for metal ions. The chromatographic method described above, wherein the test substance is detected by measuring the number of labeled particles after amplification per unit area. The chromatographic method according to claim 1, wherein a solvent for clarifying the insoluble carrier is permeated into the insoluble carrier after amplification. The chromatographic method according to claim 2, wherein the refractive index difference Δn between the solvent for clarifying the insoluble carrier and the insoluble carrier is −0.1 or more and 0.1 or less. The chromatographic method according to claim 2 or 3, wherein the refractive index difference Δn between the solvent for clarifying the insoluble carrier and the insoluble carrier is from -0.02 to 0.02. The chromatographic method according to any one of claims 1 to 4, wherein amplification is performed using an amplification solution containing a compound containing silver and a reducing agent for silver ions. The chromatographic method according to any one of claims 1 to 5, wherein the amplified labeled particles are detected by an optical instrument using a lens, and the number of labeled particles per unit area of the reaction site is measured. The chromatographic method according to any one of claims 1 to 6, wherein the optical instrument using the lens is a microscope, a scanner, a camera, or a loupe.

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