JP4514233B2 - Immunochromatography test kit - Google Patents

Description

  The present invention relates to an immunochromatographic test kit for use in immunochromatographic analysis.

  As an immunological analysis method for immunologically detecting or quantifying a test substance in a sample using the specificity of an antigen-antibody reaction, there are various measurement methods such as a radioimmunoassay method, an enzyme immunoassay method, and an agglutination method. It is used. The immunochromatography method is an inspection method that utilizes the fact that the test substance moves on each solid support by capillary action and is captured by the determination unit, because it has high detection sensitivity and is easy to operate. It is used for the determination of prostate cancer and the like that require rapid determination in colon cancer screening, outpatient examination, etc. that handle many samples at once, such as influenza virus antigen specific examination and mass screening that require rapid examination. In addition to those for doctors, it is also used as a diagnostic reagent kit for general users such as pregnancy diagnostic reagents.

  The immunochromatography method includes a sample addition part for adding a test substance, a label substance holding part for holding a labeled substance labeled with a specific binding substance that reacts specifically with the test substance, and a reaction specifically for the test substance. A determination unit to which a capture substance that captures a labeled substance labeled with a specific binding substance is immobilized, so that a test piece formed on a chromatography medium and an added specimen sample are developed on the test piece. The basic composition is composed of the developing solution. In this configuration, the labeling substance labeled with the specific binding substance includes insoluble particles such as gold colloid (metal colloid, colored resin particles, dye colloid, colored liposome, etc.) corresponding to the test substance, such as antibody and antigen. A substance labeled with a substance is used, and as a capture substance of the determination unit, an antibody, an antigen or the like corresponding to the test substance is immobilized. In such an immunochromatography method, the specimen dropped into the sample addition part can move from the labeling substance holding part to the judgment part by the capillary action using the developing solution as a mobile phase, and the detection of the test substance can be performed in a short time. Is one possible immunological assay.

  By the way, the immunochromatography method has conventionally been pointed out as a problem that the detection site determination unit develops color even in the absence of a test substance because of impurities such as proteins mixed in the specimen. This non-specific reaction not only lowers the S / N ratio during detection, but can also cause false positives.

As a method for preventing color development due to non-specific reaction and preventing a decrease in antigen detection sensitivity, Patent Document 1 discloses an immunoassay for antibodies against non-specific factors present in a sample, specifically natural antibodies derived from humans. By adding to the system, the one that suppresses non-specific reactions caused by non-specific factors is presented. Patent Document 2 discloses an immunoassay reagent comprising an antibody corresponding to a substance to be measured (antigen or antibody) or an insoluble carrier carrying an antigen and γ-globulin. This reagent prevents non-specific reaction by adding γ-globulin as an inhibitor of non-specific reaction and increases sensitivity.
Japanese Patent Laid-Open No. 11-287801 JP 2002-181822 A

  However, the method disclosed in Patent Document 1 uses human-derived innate immunity antibodies, but human-derived innate immunity antibodies are obtained from various antigens that can be contacted by humans. When used without appropriate treatment such as purification by purpose, depending on the antibody compatibility and the amount of the compound, the target nonspecific factor and appropriate reactivity cannot be obtained, and the nonspecific reaction suppression effect is I can't expect much. On the other hand, the reagent disclosed in Patent Document 2 can also be expected to prevent nonspecific reaction to some extent, but depending on the amount of the reagent, it may conversely cause a nonspecific reaction. Therefore, the sensitivity of the conventional immunoassay is not satisfactory, and a more excellent sensitivity is required.

  In view of the above, the present invention suppresses a nonspecific reaction caused by a nonspecific factor that causes a false positive at the time of detection and improves the detection sensitivity of the test substance so that the test substance in the sample can be accurately and It is an object of the present invention to provide an immunochromatographic test kit that can be rapidly detected.

  The present inventors have intensively studied and introduced into the detection system a substance that binds preferentially to the labeled substance with respect to the protein or the like that causes a nonspecific reaction. Attempts were made to suppress nonspecific reactions. And, as such a substance, an immunoglobulin having the same subclass derived from the same production animal species as the capture substance (antibody, antigen) immobilized on the determination part (hereinafter sometimes abbreviated as immunoglobulin) is applied. It was decided to. In addition, as a specific form of introduction of the immunoglobulin into the detection system, there are two forms of immunochromatography, one in which the immunoglobulin is mixed in a developing solution added together with the sample and the other in which the immunoglobulin is held in advance in the test piece. It was a test kit for graphy.

  According to a first aspect of the present invention, a sample addition unit for adding a test substance, a label substance holding unit for holding a label substance labeled with a specific binding substance that specifically reacts with the test substance, and a test substance A test piece formed on a chromatographic medium, and a test piece on which a capture substance that captures a labeled substance labeled with a specific binding substance that specifically reacts is fixed, and the test substance are developed on the test piece An immunochromatography test kit comprising: a developing solution for causing an immunoglobulin having the same subclass derived from the same production animal species as the capture substance of the determination unit to 1 mL of the developing solution; The immunochromatographic test kit is characterized by containing 5 to 2000 μg.

Further, the second invention according to the present application includes a sample addition unit for adding a test substance, a labeling substance holding unit for holding a labeled substance labeled with a specific binding substance that specifically reacts with the test substance, A determination part on which a capture substance that captures a labeled substance labeled with a specific binding substance that specifically reacts with the substance is fixed; a test piece formed on a chromatography medium; and the test substance on the test piece An immunochromatographic test kit comprising: a sample addition unit and / or a labeling substance holding part of the test piece derived from the same production animal species as the capture substance fixed to the determination part Imunokuroma immunoglobulins to the sample addition part and / or surface area 1 cm ² of the labeling substance holding portion, characterized in that it is 0.6~250μg retained with it and the same subclass by It is a chromatographic test kit.

  The present invention is characterized in that, as described above, an immunoglobulin derived from the same production species as the capture substance (antibody or the like) fixed to the determination part of the test piece and having the same subclass is introduced into the detection system. And Here, it should be noted that the immunoglobulin producing animal species and subclass to be introduced consider only the capture substance of the determination part, and antibodies held and fixed at other sites such as the labeling substance holding part, It is not based on antigen subclasses. The reason for this is not clear, but when an immunoglobulin having the same subclass as the subclass of the antibody held on the labeling substance held in the labeling substance holding part other than the determination part, for example, is applied, This is because it has been confirmed that the detection sensitivity is inferior to that of the invention. By selecting an immunoglobulin based on the capture substance of the determination unit of the present invention, false positives due to non-specific reactions can be suppressed and detection sensitivity can be improved.

  Immunoglobulins include human, mouse, rat, rabbit, goat, horse and the like as production animal species, and each has a predetermined range of immunoglobulins. In humans, there are five classes of immunoglobulins: IgG, IgM, IgA, IgD, and IgE. IgG has four subclasses, IgG1 to IgG4, and IgA has two subclasses, IgA1 and IgA2. Also in the case of mice, there are five classes of immunoglobulins, IgG, IgM, IgA, IgD, and IgE, but only IgG has subclasses, and there are four subclasses of IgG1, IgG2a, IgG2b, and IgG3. . However, since the production animal species and subclass of the capture substance of the detection unit differ depending on the detection target of the immunochromatographic test piece, the present invention is limited to any one of the above production animal species and subclasses. It is not a thing.

  The first and second inventions according to the present application are different only in the site where the immunoglobulin is mixed and retained, and are not different in other configurations. Hereinafter, the contents of the developing solution and the test piece of the immunochromatography test kit according to the present invention will be described, but the differences are described in order to avoid duplication.

  The developing solution is a liquid serving as a mobile phase for developing the test substance in the specimen sample on the test piece, and includes a solvent and an appropriate buffer. As the solvent, water is generally used, and preferable examples of the buffering agent include phosphate, trishydroxymethylaminomethane, Good's buffering agent, and the like as conventional ones. Further, additives other than these can be contained, and like conventional ones, protein components such as bovine serum albumin (hereinafter referred to as BSA), denaturants (for example, urea, guanidine hydrochloride, thiocyanate, etc., polymers) A polymer (for example, a soluble cellulose derivative such as carboxymethyl cellulose, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, etc.) and a basic compound (for example, spermine, spermidine, protamine sulfate, etc.) may optionally be included.

  And when adding an immunoglobulin in a developing solution (the 1st form of this application), the addition amount shall be 5-2000 micrograms with respect to 1 mL of developing solutions as above-mentioned. About addition amount, 10-1000 micrograms is preferable and 50-500 micrograms is more preferable.

  The test pieces that make up the kit together with the developing solution are the sample addition part on the chromatography medium, the labeling substance holding part that holds the labeling substance labeled with the specific binding substance that specifically reacts with the test substance, and the capture substance What is formed by the fixed determination unit is a basic configuration. The chromatographic medium is not particularly limited as long as it can absorb and flow the specimen sample by capillary action. For example, nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose and the like are selected from these polymers.

  The sample adding part is a part that absorbs the specimen sample and the developing solution and sends them to the communicating labeling substance holding part. As a sample addition part, what is necessary is just to affix the pad of the material similar to the above-mentioned chromatography medium on a chromatography medium.

  The labeling substance labeled with the specific binding substance held in the labeling substance holding part is composed of a specific binding substance such as an antibody or an antigen that can specifically react with the test substance, and a labeling substance for labeling this. A labeling complex consisting of Examples of antigens that can be specifically bound include various antigens such as Prostate Specific Antigen (prostate specific antigen, PSA) and Helicobacter pylori. The antibody may be either a monoclonal antibody or a polyclonal antibody. Specific examples include anti-E. Coli antibodies, anti-human albumin antibodies, anti-human immunoglobulin antibodies, anti-microglobulin antibodies, and anti-influenza antibodies. These antibodies and antigens may be used alone or in combination of several kinds.

  Examples of labeling substances include metal colloids such as gold colloids, non-metal colloids such as selenium colloids, insoluble particulate substances such as colored resin particles, dye colloids and colored liposomes, and metal colloids, particularly gold colloids are preferred. . The labeling substance is preferably 100 nm or less from the viewpoint of immobility of the specific binding substance or BSA, dispersibility of the labeling substance, reagent sensitivity when an immunochromatographic test piece is used, and the specific binding substance or BSA From the viewpoint of ease of purification when held in a labeling substance, the thickness is preferably 10 nm or more. The labeled complex is formed by immobilizing (holding) an antibody, an antigen or the like on a labeling substance, and can be immobilized by dropping and drying a suspension of the immune complex on a solid support. Alternatively, a pad that is fixed by immersing the suspension in an immune complex may be attached to the chromatography medium.

  The capture substance immobilized on the determination unit is a test substance-label substance complex formed by the reaction between the test substance and the labeled substance labeled with the specific binding substance in the label substance holding part. Capturing is performed by a reaction through a bond between a substance and a capturing substance. The presence or absence of the test substance in the sample can be determined by observing the degree of coloration based on the reaction of the captured substance with the naked eye. The capture substance is an antibody or antigen that can react specifically with the test substance, and is fixed by applying and drying a solution containing the antibody and antigen on the chromatographic membrane medium.

  In addition, in the structure of a test piece, in addition to the sample addition part, the labeling substance holding part, and the judgment part, it comprises an absorption part for capturing unreacted labeling substance that has passed through the judgment part. In addition, a backing sheet that does not absorb an aqueous solution is usually used for the immunochromatography medium that is the base material of the test piece.

  When the immunoglobulin is held on the test piece (second form of the present application), the immunoglobulin is contained in the sample addition part or the labeling substance holding part. Since the reaction of the labeled substance labeled with the immunoglobulin and specific binding substance to the protein in the sample is competitive, in order to react with the immunoglobulin preferentially, at the same time or immediately after the sample addition. It is necessary to contact the immunoglobulin. In order to hold the immunoglobulin, the solution is applied to the holding site and dried.

In addition, the amount of immunoglobulin retained in the sample addition part or labeling substance holding part of the immunochromatographic test piece in this way is as described above relative to the surface area of 1 cm ^{2 of the} sample addition part and / or labeling substance holding part. 0.6 to 250 μg. About this holding | maintenance amount, 1.3-125 micrograms is preferable and 6.3-62.5 micrograms is more preferable. In addition, when immunoglobulin is added to the test piece in this way, it may be added to either the sample addition part or the labeling substance holding part, but it may be added to both.

  The method of using the immunochromatography test kit of the present invention is not fundamentally different from the method of using the conventional immunochromatography test kit. For detection, a sample diluent (sample suspension) obtained by appropriately diluting the specimen sample is dropped onto the sample addition portion of the immunochromatographic test piece. At this time, the developing solution of the kit is dropped simultaneously. However, the developing solution may be mixed (preliminarily) with the sample diluent to prepare a sample solution, which may be dropped. After dropping the developing solution and sample diluent, start the reaction, and after a suitable time has elapsed, detect or quantify the test substance by visually observing the determination site or measuring with a color development measuring device. .

  The test substance using the kit of the present invention is not particularly limited as long as it can form a test substance-labeled substance complex by an immunochemical reaction. For example, viruses such as bacteria and influenza viruses, or tumor marker antigens can be mentioned. The specimen is not limited and includes whole blood, serum, plasma, urine, saliva, sputum, sweat, mucosal scrapings, and the like.

  As described above, the present invention introduces an immunoglobulin having the same production animal species and subclass as the capture substance immobilized on the determination unit into the detection system. According to the present invention, it is possible to suppress a non-specific reaction that occurs during an immune reaction, prevent a false positive reaction, and improve detection sensitivity in the determination unit.

  In the following, preferred embodiments of the present invention will be described together with comparative examples. In the present embodiment, two types of immunochromatographic test kits are prepared in which the capture substance of the determination unit is identical to the production animal species and subclass, and the immunoglobulin is added to the developing solution and the test piece. It was.

1st Embodiment : This embodiment manufactures a test kit for immunochromatography in a form in which immunoglobulin is mixed with a developing solution (first form), and examines its detection sensitivity.

[Example 1]
Preparation of test piece : In the following procedure, an immunochromatographic test piece was prepared using a sheet made of nitrocellulose (trade name: HF120, manufactured by Millipore) as a chromatography medium.

A. Preparation of determination part on chromatography medium Dilute anti-mouse PSA monoclonal antibody (subclass IgG1) with phosphate buffer solution (pH 7.4) containing 5% by weight of isopropyl alcohol to a concentration of 1.0 mg / ml did. This solution was applied onto a chromatography medium and dried at 50 ° C. for 30 minutes. After drying, it was immersed in 30 ml of a phosphate buffer solution (pH 7.4) containing 0.5 wt% casein (manufactured by Wako Pure Chemical Industries, Ltd.) at 30 ° C. for 30 minutes for blocking. After blocking, it was washed with a washing solution containing 0.05% by weight of Tween 20, and dried overnight at room temperature. With the above procedure, a determination unit using an anti-mouse PSA monoclonal antibody as a capture substance was prepared on a chromatography medium.

B. Preparation of labeling substance holding part Gold colloid dispersion (AU Colloid Solution-LC (particle size 60 nm) manufactured by Tanaka Kikinzoku Kogyo Co., Ltd.) 0.5 ml, phosphate buffer (pH 7.4) to a concentration of 0.1 mg / ml 0.1 ml of the diluted anti-PSA monoclonal antibody was added and allowed to stand at room temperature for 10 minutes. Next, 0.1 ml of a phosphate buffer (pH 7.4) containing 10% by weight of bovine serum albumin was added and stirred sufficiently, followed by centrifugation at 8000 rpm for 15 minutes. After removing the supernatant, 0.1 mL of a phosphate buffer solution (pH 7.4) containing 1% by weight of BSA was added. The labeling substance solution carrying the anti-PSA monoclonal antibody on the colloidal gold insoluble carrier particles was prepared by the above procedure. Next, this labeling substance solution was uniformly added to a glass fiber pad, and then dried with a vacuum dryer to prepare a labeling substance holding part.

C. Preparation of test piece Next, on the base material composed of a backing sheet, a labeling substance holding part (pad) on the chromatography medium having the judgment part prepared above, a versatile sample pad as a sample addition part, and a developed sample And an absorption pad for absorbing the labeling substance. And it cut | judged so that a width | variety might be set to 5 mm with the cutting machine, and it was set as the test piece for immunochromatography.

Preparation of developing solution Mouse IgG1 having the same subclass as the capture substance of the determination unit described above was mixed with phosphate buffer solution (pH 7.4) containing 250 μg, 1 wt% BSA, 50 mM sodium chloride per 1 mL of developing solution. A developing solution was prepared. Then, a plurality of sample liquids having different test substance (PSA) concentrations were mixed with this developing solution to prepare a plurality of sample liquids.

[Comparative Example 1]
Instead of adding an immunoglobulin having the same production animal species and subclass as the capture substance of the determination unit in Example 1 to the developing solution, an immunoglobulin having a subclass different from the determination unit (subclass, IgG2a) is used as the developing solution. A test kit for immunochromatography was prepared in the same manner as in Example 1 except that it was added. The test piece was the same as in Example 1.

[Comparative Example 2]
Instead of adding an immunoglobulin having the same production animal species and subclass as the capturing substance of the determination part in Example 1 to the developing solution, the procedure was performed except that no immunoglobulin was added to the developing solution. A test kit for immunochromatography was prepared in the same manner as in Example 1. The test piece was the same as in Example 1.

Detection test of detection sensitivity The detection sensitivity of the immunochromatography test kit prepared as described above was compared and examined. The detection of the test substance was performed by dropping the developing solutions having different PSA concentrations on the sample addition portion of the test piece and developing the sample, and visually judging after 15 minutes. The test line that can confirm the red line is “+”, the red line can be confirmed, but the light color is “±”, and the red line that cannot be confirmed is “−”. Table 1 shows the results.

  From Table 1, it can be seen that in Comparative Example 2 in which no immunoglobulin was introduced into the developing solution, a false positive showing a positive (+) was generated even for a sample not containing PSA. Moreover, even if an immunoglobulin is introduced, the detection sensitivity is inferior in Comparative Example 1 in which the determination part and the subclass are not shared. On the other hand, when using a developing solution in which an immunoglobulin having the same subclass as the capture substance immobilized on the determination unit in Example 1 was used, no false positive due to nonspecific reaction was observed, and the detection sensitivity was subclassed. It turns out that it improved about 4 times when not considering.

[Example 2]
In the same configuration as in Example 1, a developing solution was prepared in which the concentration of immunoglobulin to be mixed was 50 μg per 1 ml of the developing solution. Except this, the same detection as in Example 1 was performed. In addition, about the test piece, the same thing as Example 1 was applied. The results are shown in Table 2.

[Reference Example 2]
In the same configuration as in Example 1, a developing solution was prepared in which the concentration of immunoglobulin to be mixed was 500 μg per 1 ml of the developing solution. Except this, the same detection as in Example 1 was performed. In addition, about the test piece, the same thing as Example 1 was applied. The results are shown in Table 2.

[Comparative Example 3]
In the same configuration as in Example 1, a developing solution was prepared in which the concentration of immunoglobulin to be mixed was 1 μg per 1 ml of the developing solution. Except this, the same detection as in Example 1 was performed. In addition, about the test piece, the same thing as Example 1 was applied. The results are shown in Table 2.

[Comparative Example 4]
In the same configuration as in Example 1, a developing solution was prepared in which the concentration of immunoglobulin to be mixed was 4000 μg per 1 ml of the developing solution. Except this, the same detection as in Example 1 was performed. In addition, about the test piece, the same thing as Example 1 was applied. The results are shown in Table 2.

  If the concentration of the immunoglobulin mixed in the developing solution is low (1 μg per 1 mL of the developing solution), the effect of preventing the nonspecific reaction cannot be expected. On the other hand, if it is too high (4000 μg per mL of developing solution), the detection sensitivity may be insufficient. Therefore, by adding the immunoglobulin concentration in the developing solution in the range of 5 to 2000 μg per 1 mL of the developing solution, particularly 50 to 500 μg as in the present example, it is possible to accurately detect the test object.

[Reference Example 1]
In Example 1, the subclass of the anti-mouse PSA monoclonal antibody immobilized on the determination part of the test piece was IgG2a, and the immunoglobulin added to the developing solution was mouse IgG2a, except for two points, Example 1 Similarly, a test kit for immunochromatography was prepared. The determination was performed in the same manner as in Example 1. The results are shown in Table 3 .

Example 5
In Example 1, immunochromatography was performed in the same manner as in Example 1 except that the subclass of the anti-mouse PSA monoclonal antibody in the determination part of the test piece was IgG2b and the immunoglobulin added to the developing solution was mouse IgG2b. A test kit was prepared. The determination was performed in the same manner as in Example 1. The results are shown in Table 3.

  From Table 3, good detection sensitivity is obtained in any combination of subclasses. Therefore, it was confirmed that a test kit for immunochromatography having good detection sensitivity can be obtained by blending the capture substance in the determination part of the test piece with the immunoglobulin that has the same origin and subclass from the producing animal in the developing solution. It was.

Second Embodiment : Here, an immunochromatographic test kit was manufactured by preparing a test substance label holding part of a test piece, which is a second form of the present application, and examining the detection sensitivity.

Example 6
In Example 1, except that the immunoglobulin was not added to the developing solution, and the immunoglobulin was added to the labeling substance holding part after the labeling substance solution was added to the test piece and dried. A test kit for immunochromatography was prepared in the same manner as in Example 1. The amount of immunoglobulin retained at this time was 31 μg per 1 cm ^{2 of the} labeling substance retaining part. Next, as in Example 1, developing solutions having different PSA concentrations (but not containing immunoglobulin) were dropped to examine the detection sensitivity. The results are shown in Table 4.

As is clear from Table 4, even if an immunoglobulin having the same subclass as the capture substance immobilized on the determination part is held in the labeling substance holding part on the test piece in advance, that is, it specifically reacts with the test substance. Any form of immunoglobulin is present in the developing solution as long as the immunoglobulin is present in the detection system before the labeling substance labeled with the binding substance is captured by the capturing substance of the determination unit. It was confirmed that the same effect as the structure which mix | blended the immunoglobulin is acquired.

Claims (1)

Sample addition part for adding a test substance, a labeled substance holding part for holding a labeled substance labeled with a specific binding substance that reacts specifically with the test substance, a specific binding substance that reacts specifically with the test substance A determination part to which a capture substance that captures a labeled substance is fixed, a test piece formed on a chromatography medium,
In an immunochromatography test kit comprising: a developing solution for developing the test substance on the test piece;
The developing solution contains 50 to 250 μg of immunoglobulin per 1 mL of the developing solution,
A test kit for immunochromatography, wherein the immunoglobulin is derived from the same production animal species as the capture substance of the determination unit and has the same subclass as that of only the capture substance of the determination unit.