JP2009133740A - Immunochromatographic specimen - Google Patents

Abstract

To provide a test piece for immunochromatography capable of suppressing non-specific reactions caused by various non-specific factors without reducing detection sensitivity.
[MEANS FOR SOLVING PROBLEMS] At least an insoluble carrier carrying a specific binding substance that reacts specifically with an object to be detected, and a specific binding substance that reacts specifically with the object immobilized on the membrane. In an immunochromatographic test piece comprising
A test piece for immunochromatography, wherein the insoluble carrier is blocked with bovine serum albumin and the membrane is blocked with casein.
[Selection figure] None

Description

  The present invention relates to a test piece constituting an immunochromatography kit used for immunochromatography 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 a test method using a membrane due to capillary phenomenon, and it has high detection sensitivity and is easy to operate. Therefore, rapid determination in influenza virus antigen specific test, outpatient test, etc. that require rapid test is possible. It is used to determine required prostate cancer. In addition to those for doctors, they are also used as diagnostic reagent kits for general users such as pregnancy diagnostic reagents.

  In the following, the principle of immunochromatography will be described using the influenza virus antigen detection test method as an example. When the specimen suspended in the antigen extract is added to the sample addition part of the solid support, the antibody-sensitized gold colloid in the insoluble carrier holding part forms an immune complex with the influenza virus antigen in the specimen. This immune complex moves on the solid support by capillary action, is captured by the anti-influenza virus antibody fixed at each holding portion on the support, and exhibits a red color. When this red color is visually confirmed, it can be recognized that the influenza virus antigen is present in the sample. Further, the unreacted antibody-sensitized gold colloid is captured by the antibody that reacts with the antibody sensitized by the gold colloid, and exhibits a red color. This shows that the reaction has proceeded normally. Thus, detection by immunochromatography is widely used because the detection method is simple and does not require a large-scale apparatus.

  By the way, the immunochromatography apparatus test piece has a problem that coloring of a detection site occurs even in the absence of a test substance due to adsorption of non-specific factors such as proteins mixed in the sample. This non-specific adsorption (also referred to as non-specific reaction) not only lowers the S / N during detection but can also cause false positives. From this situation, it is desired to develop an immunochromatography apparatus that suppresses nonspecific adsorption without lowering the measurement sensitivity.

  As a method for suppressing color development due to a non-specific substance, which has been a problem in the past, for example, an immunochromatography apparatus in which tannin (see Patent Document 1) and amino acids are blended (see Patent Document 2) as a blocking agent is used. Proposed.

Japanese Patent Laid-Open No. 06-289025 JP 2001-289852 A

The invention disclosed in Patent Document 1 contains tannin in a measurement system to suppress a nonspecific reaction during urine measurement in which blood or occult blood is advanced, and the nonspecific reaction substance is hemolyzed hemoglobin. It is limited to the case. The invention disclosed in Patent Document 2 is a product in which amino acids which are bipolar substances are blended in a developing agent. The present invention is intended to prevent false positive reaction by suppressing electrostatic interaction between an insoluble carrier and a solid-phased antibody (antigen), which is a cause of non-specific color development. However, there is a problem that the detection sensitivity is lowered when an amino acid is added to an amount capable of sufficiently absorbing the non-specific factor.

  In view of the above, an object of the present invention is to provide an immunochromatographic test strip that can suppress non-specific reactions caused by various non-specific factors without reducing detection sensitivity.

  The present inventors have intensively studied and applied an optimum blocking agent to each of the labeling substance and the chromatographic membrane constituting the detection system, thereby causing a reduction in detection sensitivity of nonspecific reactions due to adsorption of nonspecific substances. The inventors have found out that the present invention can be achieved without any problem, and have arrived at the present invention.

  That is, the present invention comprises at least an insoluble carrier on which a specific binding substance that specifically reacts with an object to be detected is supported and a specific binding substance that specifically reacts with an object to be detected immobilized on a membrane. An immunochromatographic test strip, wherein the insoluble carrier is blocked with bovine serum albumin and the membrane is blocked with casein.

  As a means for suppressing the adsorption of non-specific substances, the application of a blocking agent itself is also known from the above background art, but the present invention applies a different blocking agent to one test piece. In addition, only bovine serum albumin (hereinafter referred to as BSA) is selected as the insoluble carrier, and only casein is selected as the chromatographic membrane, and the optimum blocking agent corresponding to the application site is clearly indicated. By doing so, the adsorption of non-specific substances is suppressed and the detection sensitivity is maximized.

  In this regard, when only the membrane is blocked, non-specific adsorption of an insoluble carrier carrying a specific binding substance that reacts specifically with the detected substance may occur depending on conditions. Non-specific adsorption is related not only to the membrane but also to the insoluble carrier side on which the specific binding substance is supported. By blocking the surface of the insoluble carrier, non-specific adsorption is surely prevented. can do. Moreover, even if the insoluble carrier side is blocked with casein and the membrane side is blocked with BSA, the detection sensitivity also decreases. Furthermore, even if the same blocking agent such as a combination of BSA and BSA or a combination of casein and casein is combined, good sensitivity cannot be obtained.

  Hereinafter, the configuration of the immunochromatographic test piece according to the present invention will be described. In the present invention, the “object to be detected” is not particularly limited as long as it can form a complex with a specific binding substance by a specific reaction such as an immunochemical reaction (that is, an antigen-antibody reaction). For example, antigens derived from bacteria, antigens derived from viruses, tumor marker antigens and the like can be mentioned.

  The specific binding substance supported on the insoluble carrier that binds to the detection target may be any substance that binds to the detection target by a specific reaction such as an antibody antigen reaction, such as an antigen, a hapten, an antibody, etc. Is mentioned. In the present invention, the specific conjugate is preferably an immunochemical component such as an antigen or an antibody. Antigens include tumor markers such as PSA (prostate specific antigen), AFP (α-fetoprotein), CEA (carcinoembryonic antigen), virus-derived proteins such as adenovirus, influenza virus, hepatitis C virus, β2-microglobulin And blood-derived proteins such as human albumin and human hemoglobin. The antibody may be any of IgG, IgM, IgA, IgE, and IgD, and may be any of a monoclonal antibody and a polyclonal antibody. Specifically, anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-hepatitis C virus antibody, anti-β2-microglobulin antibody, anti-human albumin antibody, anti-human hemoglobin antibody Etc. One or more antibodies may be used against one type of antigen, but it is more preferable to use two types of antibodies having different antigen recognition sites. Further, the detected object is not limited to one type and may be two or more types.

  As the insoluble carrier, it is only necessary to fix a specific binding substance and BSA on the surface thereof. For example, metal colloid such as gold colloid, non-metal colloid such as selenium colloid, colored resin particles, dye colloid and colored liposome Insoluble particulate materials, and the like, metal colloids, particularly gold colloids are preferred. When using colloidal gold as an insoluble carrier, the particle size is preferably from the viewpoints of immobilization of specific binding substances and BSA, dispersibility of the insoluble carrier, reagent sensitivity when used as a test piece for immunochromatography, etc. The thickness is 100 nm or less, and preferably 10 nm or more from the viewpoint of ease of purification when a specific binding substance or BSA is supported on an insoluble carrier.

Here, the suitable addition amount of BSA which is a blocking agent for the insoluble carrier is 0.01 to 50 mg, preferably 0.01 to 5 mg per 1 cm ² of the surface area of the insoluble carrier. If it is less than 0.01 mg, the components mixed in the sample adsorb nonspecifically to the insoluble carrier and easily cause false positives. If it exceeds 50 mg, the reagent sensitivity when used as a test piece for immunochromatography is lowered. However, it is considered that a desired effect cannot be obtained.

  On the other hand, the membrane is not particularly limited as long as it can absorb and flow the sample specimen by capillary action. For example, nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose, or an artificial polymer composed of a mixed fiber thereof is selected.

The amount of casein that is a blocking agent for blocking the membrane is 0.1 to 100 mg, preferably 0.1 to ²⁰ mg per 1 cm ² of the surface area of the chromatographic membrane. If it is less than 0.1 mg, an insoluble carrier carrying a component or a specific binding substance mixed in the sample is non-specifically adsorbed to the membrane and easily causes false positives. Reagent sensitivity when used as a test piece decreases.

  As a method for blocking each site in the present invention, the blocking of the insoluble carrier may be performed after the specific binding substance is supported on the insoluble carrier. The specific binding substance is supported by adding the specific binding substance (antibody, antigen) to the solution (suspension) of the insoluble carrier and stirring or standing for a specific time to specifically bind the insoluble carrier. After the specific binding substance is supported on the insoluble carrier, the insoluble carrier is blocked with BSA by adding a BSA solution (for example, a phosphate buffer containing BSA) to the solution system. A suspension of specific binders can be obtained. Alternatively, after carrying the specific binding substance on the insoluble carrier, centrifuging to remove the specific binding substance not carried on the insoluble carrier, and then adding the BSA solution (for example, phosphate buffer containing BSA) By doing so, a suspension of the specific binding substance in which the insoluble carrier is blocked with BSA can be obtained. In preparing the test piece, it may be dropped on a chromatography medium and dried, or a suspension prepared by applying a suspension to an appropriate pad may be attached to the chromatography medium.

  The membrane can be blocked by immersing the membrane in a casein solution (for example, a phosphate buffer containing casein) and drying it. After dipping in the casein solution, it may be washed with a buffer solution containing a surfactant. The amount of casein added can be adjusted by the concentration of the casein solution and the immersion time.

  In immunochromatographic test strips, in addition to an insoluble carrier holding part holding an insoluble carrier to which a specific binding substance is immobilized, a specific binding substance for capturing a detected substance-specific binding substance complex. Is composed of a determination unit (test line) in which is fixed, an absorption unit for capturing the insoluble carrier that has passed the determination unit, and the like. 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.

  As described above, the immunochromatographic test strip of the present invention has a detection sensitivity by blocking both the insoluble carrier and the membrane using BSA as the blocking agent and casein on the membrane side. Nonspecific adsorption can be suppressed without incurring a decrease in.

  In the following, preferred embodiments of the present invention will be described together with comparative examples.

[Example 1]
Preparation of test piece : A test piece for immunochromatography was prepared according to the following procedure.

A. Membrane blocking and determination part preparation A sheet made of nitrocellulose (trade name: HF120, 300 mm × 25 mm, manufactured by Millipore) was used as the membrane. The mouse-derived anti-PSA monoclonal antibody was diluted with a phosphate buffer (pH 7.4) containing 5% by weight of isopropyl alcohol to a concentration of 1.0 mg / ml. 150 μL of this solution was applied on the membrane so as to be 300 mm × 1 mm, and dried at 50 ° C. for 30 minutes. After drying, it was immersed in 100 ml of a phosphate buffer solution (pH 7.4) containing 0.5% by weight of casein (manufactured by Wako Pure Chemical Industries, Ltd.) at room temperature for 30 minutes for blocking. After blocking, it was washed with a phosphate buffer (pH 7.4) containing 0.05% by weight of Tween 20 and dried overnight at room temperature. At this time, the amount of the blocking agent added was 6.7 mg per 1 cm ^{2 of} the membrane surface area.

B. Preparation of antibody on insoluble carrier, blocking and preparation of label immobilization part Gold colloid dispersion (Tanaka Kikinzoku Kogyo Co., Ltd .: Au colloid solution-LC) 0.5 ml, phosphate buffer (pH 7.4) 0.1 mg 0.1 ml of mouse-derived anti-PSA monoclonal antibody diluted to a concentration of / ml was added and allowed to stand at room temperature for 10 minutes. Next, 0.1 ml of a phosphate buffer solution (pH 7.4) containing 10% by weight of BSA was added and stirred sufficiently, followed by centrifugation at 8000 × g for 15 minutes. After removing the supernatant, 0.1 ml of a phosphate buffer (pH 7.4) containing 1% by weight of BSA was added. A labeled substance obtained by labeling the anti-PSA monoclonal antibody with colloidal gold 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 an insoluble carrier holding part. The addition amount of the blocking agent at this time was 0.5 mg per 1 cm ² of the surface area of the gold colloid as the addition amount of BSA.

C. Preparation of test piece Next, the base material composed of a backing sheet is made to absorb the membrane having the determination part prepared above, the soluble carrier holding part, the sample pad as the sample addition part, the developed sample and the insoluble carrier. The pads were pasted together. 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.

Measurement test of detection sensitivity The detection sensitivity of the immunochromatographic test piece of Example 1 produced as described above was compared and examined. For detection, a developing solution in which a phosphate buffer solution (pH 7.4) containing 1% BSA and 50 mM sodium chloride is mixed is prepared, and a plurality of samples having different test substance (PSA) concentrations are mixed with 100 μL each. A plurality of developing solutions were prepared. And the developing solution from which PSA density | concentration differs was dripped at the sample addition of the test piece, it was made to develop, and visual determination was carried out 15 minutes afterward. 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.

[Example 2]
A test piece for immunochromatography was prepared in the same manner as in Example 1 except that the concentration of casein blocking the membrane in Example 1 was 1.5% and the amount of BSA blocking gold colloid was 2 ml. . At this time, the amount of the blocking agent added to the membrane was 20.1 mg per cm ² of the membrane, and the amount of the blocking agent added to the gold colloid was 10 mg per cm ² of the gold colloid. The measurement test and determination of detection sensitivity were performed in the same manner as in Example 1. The results are shown in Table 1.

Example 3
A test piece for immunochromatography was prepared in the same manner as in Example 1, except that the concentration of casein blocking the membrane in Example 1 was 0.05% and the concentration of BSA blocking gold colloid was 1%. did. At this time, the amount of the blocking agent added to the membrane was 0.7 mg per cm ² of the membrane, and the amount of the blocking agent added to the gold colloid was 0.05 mg per cm ² of the gold colloid. The measurement test and determination of detection sensitivity were performed in the same manner as in Example 1. The results are shown in Table 1.

Example 4
In Example 1, a test piece for immunochromatography was prepared in the same manner as in Example 1 except that the concentration of casein blocking the membrane was 5%. At this time, the amount of the blocking agent added to the membrane was 67 mg per 1 cm ² of the membrane (the amount of the blocking agent added to the gold colloid was the same as in Example 1). The measurement test and determination of detection sensitivity were performed in the same manner as in Example 1. The results are shown in Table 1.

[Comparative example]
Test specimens for immunochromatography were prepared in the same manner as in the Examples except that the blocking treatment was performed with various combinations of blocking agents instead of the membrane blocking agents and colloidal gold blocking agents in the examples. The sample was measured in the same manner as in the example. The amount of blocking agent added was the same as in Example 1. Moreover, the manufacturing process of the test piece was also performed in the same manner as in Example 1.

  As is clear from Table 1, only the test piece according to the example blocked with a combination using BSA on the gold colloid side and casein on the membrane side could detect the PSA antigen. Detection was not possible when the colloidal gold side was blocked with casein and the membrane side was blocked with BSA, the so-called reverse configuration. Furthermore, even with the same protein combination, such as a combination of BSA and BSA and a combination of casein and casein, a reaction that appears to be caused by a non-specific reaction appeared, and good results were not obtained. In addition, specific detection was impossible when gelatin was used instead of BSA. However, when the amount of blocking with BSA on the membrane side is increased (Example 4), there is a slightly inferior sensitivity.

Claims (3)

In an immunochromatographic test strip comprising at least an insoluble carrier carrying a specific binding substance that specifically reacts with a detection substance and a specific binding substance that specifically reacts with the detection substance immobilized on the membrane. ,
A test piece for immunochromatography, wherein the insoluble carrier is blocked with bovine serum albumin and the membrane is blocked with casein. ^2. The immunochromatographic test strip according to claim 1, wherein the amount of bovine serum albumin that blocks the insoluble carrier carrying the specific binding substance is 0.01 to 50 mg per 1 cm ² of the surface area of the insoluble carrier. The immunochromatographic test piece according to claim 1, wherein the amount of casein blocking the membrane is 0.1 to 100 mg per 1 cm ² of surface area of the membrane.