Specimen extract for plant immunochromatographic inspection and kit for immunochromatographic inspection

Abstract

To provide a kit for immunochromatographic inspection that reduces the possibility that a green line does not appear in a detection zone, which leads to a misjudgement, in immunochromatographic inspection using plant tissues (leaves, leaf stalks, stems and the like) as specimens.SOLUTION: A specimen extract includes: a sodium phosphate buffer or a potassium phosphate buffer in an amount of 3-95 mM and having a pH of 7.0-8.5; and as a surfactant, (1) an amphoteric surfactant or a polyoxyethylene alkyl phenyl ether-based nonionic surfactant in an amount of 0.4-2.5%(v / v), or (2) a mixture of an amphoteric surfactant in an amount of 0.05-0.3%(v / v) and a polyoxyethylene sorbitan-based nonionic surfactant in an amount of 0.2-0.7%(v / v).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a specimen extract for immunochromatographic testing of plants and an immunochromatographic testing kit. [Background technology]

[0002] Immunochromatography is a simple testing method that uses antibodies, and its major advantage is that it can be performed by any user and at any testing location due to its simplicity and speed of operation. The labeled antibodies used in immunochromatographic test kits are mainly labeled with metal colloids or colored latex particles. If the test substance is present in the sample, the labeled antibodies will accumulate in the detection zone, causing a colored test line to appear, which can be visually confirmed as a positive result. In immunochromatographic testing using plant tissues (leaves, petioles, stems, etc.) as specimens, green lines can appear when plant tissue components or chlorophyll get caught in the detection area when the specimen homogenate is developed. In this case, there is a risk that people with little experience in immunochromatographic testing may mistakenly give a positive result, which is an issue. To solve the above problems, for example, Patent Document 1 proposes to improve the accuracy of the determination by maintaining a glycosylase in a dry state upstream of the detection zone to decompose cellulose, etc.; however, this still leaves room for issues such as an increase in the number of manufacturing procedures and ensuring the stability of the glycosylase. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2012-207944 Summary of the Invention [Problem to be solved by the invention]

[0004] The labeled antibodies used in immunochromatographic test kits are mainly labeled with metal colloids or colored latex particles. In immunochromatographic tests using plant tissues (leaves, petioles, stems, etc.) as specimens (hereinafter referred to as "immunochromatographic tests"), plant tissue components or chlorophyll may become trapped in the detection zone when the specimen homogenate is developed, causing a green line to appear. In this case, there is a problem that people with little experience in immunochromatographic tests may mistakenly determine a positive result. The present invention aims to provide an immunochromatographic test kit that prevents the appearance of a green line in the detection zone and reduces the possibility of erroneous determination in such immunochromatographic tests using plant tissues (leaves, petioles, stems, etc.) as specimens. [Means for solving the problem]

[0005] The inventors have discovered that by extracting a test substance (antigen) from a specimen of plant tissue (leaf, petiole, stem, etc.) using a specimen extractant containing 3 to 95 mM sodium phosphate buffer or potassium phosphate buffer solution at a pH of 7.0 to 8.5 and either (1) 0.4 to 2.5% (v / v) of an amphoteric surfactant or a polyoxyethylene alkylphenyl ether-based nonionic surfactant or (2) a mixture of 0.05 to 0.3% (v / v) of an amphoteric surfactant and 0.2 to 0.7% (v / v) of a polyoxyethylene sorbitan-based nonionic surfactant, a green line no longer appears in the detection area in a plant immunochromatographic test, and the test substance can be detected with little possibility of erroneous determination.

[0006] That is, the present invention is as follows. [1] A specimen extract for immunochromatographic testing of plants, comprising a 3 to 95 mM sodium phosphate buffer or potassium phosphate buffer solution at a pH of 7.0 to 8.5, and, as a surfactant, (1) 0.4 to 2.5% (v / v) of an amphoteric surfactant or a polyoxyethylene alkylphenyl ether-based nonionic surfactant, or (2) a mixture of 0.05 to 0.3% (v / v) of an amphoteric surfactant and 0.2 to 0.7% (v / v) of a polyoxyethylene sorbitan-based nonionic surfactant. [2] The sample extract according to [1] above, wherein the sample is a leaf, petiole, or stem of a plant. [3] The specimen extract according to [1] or [2], wherein the surfactant is selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CAS Registry Number 75621-03-3: CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CAS Registry Number 82473-24-3: CHAPSO), polyoxyethylene octylphenyl ether, a mixture of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) and polyoxyethylene sorbitan monolaurate (Tween 20), and a mixture of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and polyoxyethylene sorbitan monolaurate (Tween 20). [4] An immunochromatographic test kit comprising the specimen extract according to any one of [1] to [3] above and an immunochromatographic test piece. [5] An immunochromatographic testing method comprising: extracting a test substance (antigen) from a specimen using the specimen extract liquid described in any one of [1] to [3] above; spreading the specimen liquid containing the extracted test substance (antigen) on an immunochromatographic test piece; and detecting the test substance (antigen) from the immunochromatographic test piece. [Effects of the Invention]

[0007] By using the specimen extract of the present invention, no green line appears in the detection zone in the immunochromatographic test of plants, and the test substance can be detected with less possibility of erroneous determination. [Brief explanation of the drawings]

[0008] [Figure 1] Schematic diagram of immunochromatographic test strip DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention provides a specimen extract for immunochromatographic testing of plants, which contains a 3 to 95 mM sodium phosphate buffer or potassium phosphate buffer solution having a pH of 7.0 to 8.5 and, as a surfactant, (1) 0.4 to 2.5% (v / v) of an amphoteric surfactant or a polyoxyethylene alkyl phenyl ether-based nonionic surfactant, or (2) a mixture of 0.05 to 0.3% (v / v) of an amphoteric surfactant and 0.2 to 0.7% (v / v) of a polyoxyethylene sorbitan-based nonionic surfactant.

[0010] Immunochromatography testing is a type of immunoassay that uses immunochromatography. There are several types, but the sandwich method is the most common, and the mechanism for detecting the test substance is as follows: A test immunochromatograph prepared in the shape of a strip is generally called a test strip. An antibody specific to the test substance is immobilized in the detection area (4 in Figure 1) of the membrane of the test strip (3 in Figure 1). In addition, a detection reagent such as a dye-labeled antibody is held in the conjugate pad (2 in Figure 1). The sample is ground in the sample extract, which elutes the analyte into the sample extract. When the sample extract is dropped onto the sample pad (1 in Figure 1) of the test strip, it spreads (from 1 to 6 in Figure 1) along with the detection reagent, forming a complex of immobilized antibody, analyte, and dye-labeled antibody in the detection zone (4 in Figure 1). As a result, a signal appears in the detection zone, allowing the analyte to be detected.

[0011] In the present invention, the specimen refers to a plant tissue, that is, a specimen collected from a part of a plant body, such as a leaf, petiole, stem, flower, petal, etc. Preferably, it is a leaf, petiole, or stem.

[0012] In the present invention, the test substance (antigen) is contained in plant tissue and is, for example, a plant pathogen, a pest, a partial protein thereof, or a chemical substance such as a pesticide. Examples of plant pathogens include Cucurbit chlorotic yellows virus (CCYV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), and Tomato yellow leaf curl virus (TYLCV).

[0013] In the present invention, antibodies are labeled as needed for detection in immunochromatographic testing. Labeling can be performed by conventional methods. Labeling substances used for labeling include colored particles such as colloidal metal particles (colloidal particles of gold, silver, copper, iron, platinum, palladium, and mixtures thereof (e.g., a mixture of gold and platinum, a mixture of gold and silver, or a mixture of palladium and platinum)). Among colloidal metal particles, gold colloidal particles are preferred because of their ease of use.

[0014] A specimen extract is a liquid reagent added to a sample for immunochromatographic testing when it is ground, to prevent protein denaturation due to friction or pH changes and to develop the sample during testing. Generally, a buffer solution with buffering capacity in a near-neutral pH range is used, and may contain sugars or surfactants. Examples of buffer solutions with buffering capacity near neutral include phosphate buffer, Tris·HCl buffer, and citrate buffer. In the present invention, a sodium phosphate buffer or potassium phosphate buffer with a pH of 7.0 to 8.5 is used. The pH is preferably 7.0 to 8.4, more preferably 7.4 to 8.2, and even more preferably 7.6 to 8.0. The concentration of the buffer solution affects the accuracy of the results of immunochromatographic testing, and using a buffer solution with an extremely low or extremely high concentration is unsuitable because it may result in false positives. In the present invention, a 3 mM to 95 mM sodium phosphate buffer solution or potassium phosphate buffer solution is used. The concentration is preferably 5 mM to 90 mM, more preferably 7 mM to 80 mM, and even more preferably 30 mM to 75 mM.

[0015] In the specimen extract of the present invention, a surfactant is added to solubilize proteins that are poorly soluble in water or proteins that are difficult to extract, and to efficiently extract the analyte (antigen) from the specimen.

[0016] In the specimen extract of the present invention, the surfactant is (1) 0.4 to 2.5% (v / v) of an amphoteric surfactant or a polyoxyethylene alkylphenyl ether-based nonionic surfactant, or (2) a mixture of 0.05 to 0.3% (v / v) of an amphoteric surfactant and 0.2 to 0.7% (v / v) of a polyoxyethylene sorbitan-based nonionic surfactant. Examples of amphoteric surfactants include 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CAS registration number 75621-03-3: CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CAS registration number 82473-24-3: CHAPSO), decyldimethyl(3-sulfopropyl)ammonium hydroxide inner salt (Zwittergent3-10), and dodecyldimethyl(3-sulfopropyl)ammonium hydroxide inner salt (Zwittergent3-12). Examples of polyoxyethylene alkylphenyl ether-based nonionic surfactants include polyoxyethylene octylphenyl ether and polyoxyethylene nonylphenyl ether. Octylphenol ethoxylate, polyethylene glycol-t-octylphenyl ether, and the like are also synonymous with polyoxyethylene octylphenyl ether. Polyoxyethylene octylphenyl ether may be a mixture of ortho-, para-, and m-isomers represented by CAS Registry Number 9002-93-1, or the para-isomer represented by CAS Registry Number 9036-19-5. Specifically, commercially available surfactants such as Triton X-100 (registered trademark) and IGEPAL CA-630 (registered trademark) can be used. Examples of polyoxyethylene sorbitan-based nonionic surfactants include polyoxyethylene sorbitan monolaurate (CAS Registry Number 9005-64-5: Tween 20®), polyoxyethylene sorbitan monopalmitate (CAS Registry Number 9005-66-7: Tween 40®), polyoxyethylene sorbitan monostearate (CAS Registry Number 9005-67-8: Tween 60®), polyoxyethylene sorbitan monooleate (CAS Registry Number 9005-65-6: Tween 80®), and polyoxyethylene sorbitan trioleate (CAS Registry Number 9005-70-3: Tween 85®), and preferably polyoxyethylene sorbitan monolaurate (CAS Registry Number 9005-64-5: Tween 20®). In the specimen extract of the present invention, the surfactant is preferably selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CAS Registry Number 75621-03-3: CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CAS Registry Number 82473-24-3: CHAPSO), polyoxyethylene octylphenyl ether, a mixture of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) and polyoxyethylene sorbitan monolaurate (Tween 20), and a mixture of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and polyoxyethylene sorbitan monolaurate (Tween 20). Polyoxyethylene octylphenyl ether is more preferably Triton X-100 (registered trademark) or IGEPAL CA-630 (registered trademark). The concentration of the surfactant in the specimen extract of the present invention is preferably 0.45 to 2% (v / v), more preferably 0.5 to 2% (v / v), when the surfactant is an amphoteric surfactant or a polyoxyethylene alkylphenyl ether-based nonionic surfactant; and when the surfactant is a mixture of an amphoteric surfactant and a polyoxyethylene sorbitan-based nonionic surfactant, the concentration is preferably 0.1 to 0.25% (v / v) of the amphoteric surfactant and 0.25 to 0.5% (v / v) of the polyoxyethylene sorbitan-based nonionic surfactant, more preferably 0.15 to 0.2% (v / v) of the amphoteric surfactant and 0.3 to 0.4% (v / v) of the polyoxyethylene sorbitan-based nonionic surfactant.

[0017] The specimen extract of the present invention may further contain a reducing agent, which is preferably added to the specimen extract since the reducing agent can prevent a decrease in extraction efficiency. Considering safety and storage stability, examples of reducing agents include α-tocopherol, β-tocopherol, γ-tocopherol, σ-tocopherol, BHA (butylhydroxyanisole), BHT (dibutylhydroxytoluene), L-ascorbate, and isoascorbate, which are already used in foods and cosmetics. The concentration of the reducing agent in the specimen extract is preferably 0.005 to 7% (w / v), more preferably 0.005 to 1.5% (w / v).

[0018] The specimen extract of the present invention may further contain a preservative, which is preferably added to the specimen extract to prevent the invasion, growth, and proliferation of microorganisms in the buffer solution and to minimize spoilage. Any preservative commonly used in foods and cosmetics can be used without any particular limitation. Specific examples include sodium benzoate, calcium propionate, sodium propionate, and Proclin 300 (Sigma-Aldrich), with Proclin 300 being preferred. The concentration of the preservative in the specimen extract is preferably 0.01 to 0.05% (w / v), more preferably 0.015 to 0.035% (w / v).

[0019] The specimen extract of the present invention may further contain a chelating agent, which is preferably added to the specimen extract because it has the property of capturing metal ions that promote oxidation. The chelating agent can be any component that is typically incorporated into foods and cosmetics for the purpose of preserving their quality, and examples include EDTA (ethylenediaminetetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriaminepentaacetic acid), etc. The concentration of the chelating agent in the sample extract is preferably 0.1 to 0.7 mM, more preferably 0.5 mM.

[0020] The immunochromatographic test kit of the present invention is characterized by comprising a sample extract and an immunochromatographic test strip. In the immunochromatographic test strip included in the immunochromatographic test kit, an antibody against the test substance (antigen) is preferably immobilized on a solid phase support. For example, the immunochromatographic test strip may be in the form of a type in which the antibody-immobilized immunochromatographic membrane and other components are laminated to a backing sheet, and the upper surface of the immunochromatographic test strip is covered with a sheet-like film, or a type in which the immunochromatographic test strip is housed in a molded plastic container that takes into consideration the sample drop position and the antibody immobilization position on the membrane. The immunochromatographic test kit of the present invention may also comprise a standard sample of the test substance (antigen) protein, a detection reagent, a dilution buffer, a microtube and a homogenizer pestle, a zip-lock bag (trituration bag) for sample trituration, a dropper for dropping the sample trituration liquid, a moisture absorbent, an instruction manual, etc.

[0021] The immunochromatographic test method of the present invention is characterized by comprising extracting a test substance (antigen) from a test specimen using the above-mentioned specimen extract, spreading the test specimen liquid containing the extracted test substance (antigen) on an immunochromatographic test strip, and detecting the test substance (antigen) from the immunochromatographic test strip. The immunochromatographic test of the present invention can be carried out according to conventional methods, except for spreading the test specimen liquid containing the test substance (antigen) extracted using the above-mentioned specimen extract on an immunochromatographic test strip and detecting the test substance (antigen) from the immunochromatographic test strip.

[0022] In the immunochromatographic testing method of the present invention, the method for extracting the test substance (antigen) from the specimen can be carried out according to conventional methods except for using the above-mentioned specimen extract, and any suitable method such as conventionally used crushing, emulsification, stirring, shaking, or boiling can be used alone or in combination. For example, methods include crushing with a homogenizer or mixer, grinding with a mortar and pestle, using an ultrasonic crusher, placing the specimen, specimen extract and grinding net in a zip-lock bag and rubbing the bag with a stick over the top to grind, placing the specimen and specimen extract in a 1.5 ml tube and grinding with a grinding stick, placing the specimen, specimen extract and stainless steel beads in a 2 ml microtube and putting it through a bead crusher, etc.

[0023] In the immunochromatographic testing method of the present invention, the test substance (antigen) can be detected by a conventional method. For example, antibodies labeled with colored particles such as gold colloids accumulate in the detection zone of the immunochromatographic test strip, causing a colored test line to appear, which can be visually confirmed. Alternatively, the absorbance of the test line can be measured using an immunochromatographic reader. [Example]

[0024] EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0025] <Test method (immunochromatography test using plant tissue samples)> Using an immunochromatographic test strip for detecting Cucurbit chlorotic yellows virus (hereinafter referred to as CCYV), it was confirmed that the appearance of a green line in the detection area can be suppressed by optimizing the composition of the sample extract. 1 mg of healthy, CCYV-uninfected leaves (freeze-dried) or CCYV-infected leaves (freeze-dried) was added to 200 μl of each sample extract and ground to prepare the sample solution. 100 μl of each sample solution was dropped onto the sample pad of a CCYV detection immunochromatographic test strip, and after 30 minutes, the presence or absence of a test line or green line in the detection zone was visually determined. Note that the immunochromatographic test strip used in this test uses an antibody labeled with gold colloid, so a reddish-purple line appears in the case of a positive result. The test results are shown in each table. The presence or absence of a reddish-purple test line and a green line is indicated by + / -, and the judgement results based on these are indicated by + for a positive, ± for a false positive, and - for a negative. If a test line appears, the green line overlaps with the test line, making it impossible to judge, so the presence or absence of a green line is not indicated for infected leaves. If a test line appears on a healthy leaf, it is a false positive due to a non-specific reaction of the labeled antibody, and the green line is considered unreadable.

[0026] <Test Example 1 (Study of types of buffer solution)> The test was carried out according to the above test method using a specimen extract having the composition shown in Table 1. The results show that when sodium phosphate or potassium phosphate was used in the buffer and 1% (v / v) CHAPS was added as a surfactant, the appearance of the green line was suppressed, and false positive results were not detected. Table 1 TIFF2025119291000001.tif73157 Test line / green line: + yes, - no Judgment: +positive, ±false positive, -negative

[0027] <Test Example 2 (Study on pH of buffer solution)> The pH of the 1% (v / v) CHAPS-containing sodium phosphate buffer solution of the sample extract was varied in the range of pH 6.0 to pH 8.4 as shown in Table 2, and the results were determined according to the test method. When the pH of the sodium phosphate buffer was in the range of 7.0 to 8.4, the appearance of the green line was suppressed, and false positive results were not detected. Table 2 TIFF2025119291000002.tif83145 Test line / green line: + yes, - no Judgment: +positive, ±false positive, -negative *Contains 1% (v / v) CHAPS as a surfactant.

[0028] <Test Example 3 (Investigation of buffer concentration)> The sodium phosphate concentration of the sample extract in 1% (v / v) CHAPS-containing sodium phosphate buffer (pH 7.8) was varied in the range of 0 mM to 500 mM as shown in Table 3, and the results were determined according to the test method. When the sodium phosphate concentration was in the range of 5 mM to 90 mM, the appearance of the green line was suppressed, and there were no false positive results. Table 3 TIFF2025119291000003.tif62155 Test line / green line: + yes, - no Judgment: +positive, ±false positive, -negative *Contains 1% (v / v) CHAPS as a surfactant.

[0029] <Test Example 4 (Study of surfactants)> Tests were performed according to the test method using a sample extract prepared by dissolving the surfactants listed in Table 4 in 50 mM sodium phosphate buffer (pH 7.8). When 0.5-2% (v / v) of CHAPS, CHAPSO, IGEPAL CA-630, or Triton X-100 was used, or when a mixture of 0.1-0.25% (v / v) CHAPS and 0.25-0.5% (v / v) Tween 20 was used, the appearance of green lines was suppressed, eliminating false positives and preventing false positives. Table 4 TIFF2025119291000004.tif224170 TIFF2025119291000005.tif126170 Test line / green line: + yes, - no Judgment: +positive, ±false positive, -negative *Dissolved in 50 mM sodium phosphate buffer (pH 7.8). [Explanation of symbols]

[0030] 1 Sample pad (sample drop area) 2 Conjugate pad (dye-labeled antibody holding area) 3. Membrane (carrier) 4 Test line (detection area) 5. Control Line 6 Absorbent pad (sample liquid absorption part)

Claims

1. A specimen extract for immunochromatographic testing of plants, comprising a 3 to 95 mM sodium phosphate buffer or potassium phosphate buffer solution having a pH of 7.0 to 8.5, and, as a surfactant, (1) 0.4 to 2.5% (v / v) of an amphoteric surfactant or a polyoxyethylene alkylphenyl ether-based nonionic surfactant, or (2) a mixture of 0.05 to 0.3% (v / v) of an amphoteric surfactant and 0.2 to 0.7% (v / v) of a polyoxyethylene sorbitan-based nonionic surfactant.

2. The specimen extract according to claim 1, wherein the specimen is a leaf, petiole or stem of a plant.

3. 2. The specimen extract according to claim 1, wherein the surfactant is selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), polyoxyethylene octylphenyl ether, a mixture of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) and polyoxyethylene sorbitan monolaurate (Tween 20), and a mixture of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and polyoxyethylene sorbitan monolaurate (Tween 20).

4. An immunochromatographic test kit comprising the specimen extract according to any one of claims 1 to 3 and an immunochromatographic test piece.

5. An immunochromatographic testing method comprising: extracting a test substance (antigen) from a specimen with the specimen extract according to any one of claims 1 to 3; spreading the specimen liquid containing the extracted test substance (antigen) on an immunochromatographic test piece; and detecting the test substance (antigen) from the immunochromatographic test piece.

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