Vitamin A quantification method

Abstract

To provide an immunochromatographic method capable of quickly and easily quantifying concentration of vitamin A in blood at a raising site of beef cows or the like.SOLUTION: Provided is a quantifying method including: a) a step of mixing a biological sample, a competitive agent, and a specimen diluent liquid to prepare a biological sample diluent liquid; b) a step of dripping the biological sample diluent liquid on an immunochromatographic strip to develop it by a capillary phenomenon; and c) a step of measuring coloration of a test line of the immunochromatographic strip to quantify the vitamin A. In the method, the concentration of polyethylene glycol in the biological sample diluent liquid is 1 to 4 mass% and the concentration of sodium chloride is 1 to 3 mass%.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an immunochromatographic method for quantifying vitamin A in a biological sample. [Background technology]

[0002] In beef cattle, especially in the production of high-quality Wagyu beef, the meat is valued more highly by increasing the amount of fat marbling in the muscles. For this reason, a breeding management method known as vitamin control, which induces fat marbling by drastically reducing the amount of vitamin A fed, which has the function of promoting fat decomposition, has become mainstream in recent years. However, an extreme deficiency of vitamin A can cause health problems such as impaired liver function, and can also cause muscle edema and myositis, resulting in a decline in meat quality, which can be a major problem. For this reason, blood vitamin A concentrations are measured to understand the nutritional status of cattle and prevent the occurrence of the problems mentioned above.

[0003] High performance liquid chromatography (HPLC) is commonly used to measure vitamin A concentrations. However, in most cases, veterinarians take blood samples on-site, take them home, and then request a testing institution to measure them, so the test results are usually available several days later, which means that immediate action cannot be taken even if vitamin A is insufficient or in excess.

[0004] For example, Patent Documents 1 and 2 describe a method and a measuring device for quantifying vitamin A by irradiating vitamin A extracted from serum with an organic solvent with light of a specific wavelength. However, this method requires centrifuging the collected blood to separate the serum, removing serum proteins with ethanol, and extracting vitamin A components with heptane, and although it does not require a large device such as HPLC, the operation is complicated and time-consuming to carry out at a cattle breeding site. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2010-230447 [Patent Document 2] Patent Publication No. 2015-169627 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an immunochromatographic method capable of rapidly and simply quantifying the vitamin A concentration in blood at the site of breeding beef cattle or the like. [Means for solving the problem]

[0007] As a result of extensive investigations aimed at solving the above problems, the present inventors have found that the above problems can be solved by the means described below, and have arrived at the present invention.

[0008] That is, the present invention comprises the following configuration. (1) a) mixing a biological sample, a competitive reagent, and a sample diluent to prepare a biological sample diluent; b) dropping the diluted biological sample onto an immunochromatographic strip and allowing it to develop by capillary action; c) measuring the color of the test line of the immunochromatographic strip to quantify the vitamin A; A method for quantifying vitamin A by a competitive method, comprising: The method for quantifying, wherein the concentration of polyethylene glycol in the biological sample dilution solution is 1 to 4% by mass, and the concentration of sodium chloride is 1 to 3% by mass. (2) The quantitative method according to (1), wherein the competitive reagent contains a complex of vitamin A and a compound. (3) The method according to (2), wherein the compound is biotin. (4) The quantitative method described in any one of (1) to (3), characterized in that the immunochromatographic strip comprises a sample addition member, a membrane carrier, and an absorption member arranged in sequence, and the membrane carrier has a test line on which anti-vitamin A antibodies are fixed. (5) The quantitative method described in (4), wherein the immunochromatographic strip comprises an impregnated member disposed between a sample application member and a membrane carrier, and the impregnated member is impregnated with a label. (6) The quantitative method according to any one of (1) to (5), wherein the biological sample is any one of blood, plasma, and serum. Effect of the Invention

[0009] INDUSTRIAL APPLICABILITY The present invention provides a method for quantifying vitamin A, which enables rapid and simple measurement of the vitamin A concentration in blood at beef cattle breeding sites. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing an example of an immunochromatographic strip for carrying out the measurement method of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing an example of an immunochromatographic strip for carrying out the measurement method of the present invention housed in a housing case. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below.

[0012] The present invention provides a method for quantifying vitamin A by a competitive method, comprising the steps of: a) mixing a biological sample, a competitive reagent, and a specimen dilution solution to prepare a biological sample dilution solution; b) dropping the biological sample dilution solution onto an immunochromatographic strip and allowing it to develop by capillary action; and c) measuring the color of a test line on the immunochromatographic strip to quantify the vitamin A, wherein the biological sample dilution solution has a polyethylene glycol concentration of 1 to 4% by mass and a sodium chloride concentration of 1 to 3% by mass.

[0013] (Vitamin A) In the present invention, vitamin A refers to retinoids such as retinol, retinal, retinoic acid, retinyl ester, etc. Vitamin A may also be in the form of a complex with retinol-binding protein (RBP) or prealbumin.

[0014] (Biological samples) In the present invention, the target biological sample may be whole blood, serum, or plasma, and is not particularly limited. The blood of animals such as cattle, humans, horses, dogs, and cats can be measured.

[0015] (Competitive Reagent) In the present invention, the competitive reagent is not particularly limited as long as it can compete with free or protein-bound vitamin A in a biological sample and can be detected by a labeling substance. As the competitive reagent, a complex of vitamin A and a compound is preferably used. The complex stabilizes vitamin A and is preferable because antibodies with good performance are easily available. Examples of the compound include bovine serum albumin, ovalbumin, and biotin, and among these, biotin is preferably used. Although the detailed reason is unclear, it is speculated that the competitive principle works more easily with vitamin A in a biological sample in a complex of vitamin A and low molecular weight biotin than in a complex of vitamin A and a high molecular compound such as albumin. In addition, a complex of vitamin A and a low molecular weight compound has the advantage of being less expensive to manufacture than a complex of vitamin A and a protein such as bovine serum albumin.

[0016] Vitamin A is an unstable compound, and light and heat are likely to cause isomerization of double bonds. It also reacts easily with acids, air, and metal ions, and may be easily decomposed. Therefore, it is preferable to store the competitive reagent in a low temperature, dark place until use. The storage temperature is preferably 4°C or lower, more preferably -20°C or lower, and even more preferably -80°C or lower. Therefore, it is preferable to mix the competitive reagent with a biological sample and a specimen dilution solution at (before) use. In this case, the competitive reagent may be in a freeze-dried state or in a solution state. The competitive reagent may also be mixed with a specimen dilution solution in advance and stored at a low temperature before use.

[0017] (Immunochromatography development) It is preferable to arrange the sample addition member 6 so that when the biological sample dilution solution prepared by mixing the biological sample, the competitive reagent, and the specimen dilution solution is dropped on the sample dropping section 10 of the sample addition member 6, the label pre-impregnated in the impregnated member 2 connected to the upstream end of the membrane carrier 3 is mixed with the mixed solution and chromatographically developed on the membrane carrier 3. Alternatively, the label may be mixed with the biological sample and the specimen dilution solution in an appropriate container other than the immunochromatographic strip 8 to prepare a mixed solution, and then the mixed solution may be dropped on the sample addition member 6 of the immunochromatographic strip 8 to chromatographically develop on the membrane carrier 3. It is preferable to perform the measurement within 5 to 12 minutes after the start of development. If the measurement is performed during this period, the competitive reaction between vitamin A and the competitive reagent (complex of vitamin A and compound) occurs most effectively, and a change in the measured value according to the change in vitamin A concentration is obtained on the test line, and the difference in vitamin A concentration is accurately reflected in the measured value, which is preferable. That is, the vitamin A concentration in the biological sample can be accurately measured. If the test is left for less than 5 minutes, the reaction between the anti-vitamin A antibody on the test line and the vitamin A in the biological sample or the competing reagent (a complex of vitamin A and a compound) will be insufficient, resulting in a low measured value, and it may not be possible to obtain a measured value that accurately reflects the difference in the concentration of vitamin A in the biological sample. If the test is left for more than 12 minutes, the non-specific reaction to the anti-vitamin A antibody will increase, and it may not be possible to obtain an accurate measured value change corresponding to the concentration of vitamin A in the biological sample.

[0018] (Competition Law) In the present invention, vitamin A in a biological sample is preferably quantified by a competitive method. Since it is difficult to sandwich a low molecular weight compound such as vitamin A between two types of antibodies, a competitive method is preferably used. That is, a biological sample dilution obtained by mixing a biological sample, a competitive reagent (complex of vitamin A and a compound), and a specimen dilution solution is dropped on an immunochromatographic strip and developed, whereby vitamin A in the biological sample and the competitive reagent are competitively captured by the anti-vitamin A antibody forming a test line. The captured complex is colored by a labeling substance having an antibody against the compound, and quantification can be performed by obtaining a signal on the test line. It is preferable to add 10 ng to 10 μg of the competitive reagent per 1 ml of the biological sample. Since the concentration of vitamin A in the plasma of a biological sample, particularly beef cattle, is 200 ng / ml to 1 μg / ml, if the difference between the concentration of vitamin A in the biological sample and the amount of the competitive reagent added is too large, the competition does not go well and the quantitation may decrease.

[0019] (Specimen diluent) In the present invention, the specimen dilution solution is used as a developing solution for diluting a biological sample, hemolyzing red blood cells, and developing a biological sample. The specimen dilution solution contains a buffer, a surfactant, a competitive reaction regulator, etc.

[0020] In the present invention, any type of buffer may be used as long as it has sufficient buffering capacity in the desired pH range, and examples of such buffers include Tris, phosphoric acid, phthalic acid, citric acid, maleic acid, succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, Good's buffer (MES, ADA, PIPES, ACES, cholamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycineamide, bicine), and the like.

[0021] In the present invention, the surfactant preferably contains a nonionic surfactant that improves the spreading of the biological sample (diluent) on the immunochromatographic strip and does not affect the immune reaction. Examples of the nonionic surfactant include polyoxyethylene alkylphenyl ether (Triton (registered trademark) surfactants, etc.), polyoxyethylene alkyl ether (Brij (registered trademark) surfactants, etc.), polyoxyethylene sorbitan fatty acid ester (Tween (registered trademark) surfactants, etc.), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, sucrose fatty acid ester, etc. The surfactant may be used alone or in combination of two or more kinds. The concentration of the surfactant is preferably 0.001% by mass to 1.0% by mass, more preferably 0.005% by mass to 0.5% by mass, and even more preferably 0.01% by mass to 0.3% by mass. If the concentration is too low, the biological sample diluent may not spread easily, and if the concentration is too high, the signal intensity of the test line may be reduced.

[0022] In the present invention, it is preferable to use polyethylene glycol (PEG) as the competitive reaction regulator. As a result of the study, it was found that the competitive reaction can be efficiently carried out by adding polyethylene glycol to the specimen dilution solution for diluting the specimen. In addition, the concentration at which this effect is exerted is 1 to 4 mass% in the sample (biological specimen dilution solution) after diluting the specimen. In addition, the number average molecular weight of the polyethylene glycol used is preferably 2000 to 16000, more preferably 5000 to 10000. When the number average molecular weight is small, sufficient antigen-antibody reaction promotion suitable for the present invention may not be obtained. In addition, when the number average molecular weight is large, similarly, the antigen-antibody reaction promotion may not be obtained, or the viscosity of the biological specimen dilution solution may increase, resulting in a decrease in the spreadability on the immunochromatographic strip.

[0023] In the present invention, polyethylene glycol (PEG) is a polymeric compound (polyether) having a structure in which ethylene glycol is polymerized. Polyethylene glycols with number-average molecular weights of about 200 (PEG200) to about 20,000 (PEG20000) are commercially available, with those with number-average molecular weights of 600 or less being liquid and those with number-average molecular weights of 1,000 or more being solid. In addition, PEGs have properties such as low toxicity, the ability to arbitrarily mix those with different molecular weights, and excellent solubility in water and many organic solvents.

[0024] In the present invention, the specificity of the competitive reaction can be further increased by adding sodium chloride to the specimen dilution solution. Specifically, it is preferable to add sodium chloride to the biological sample dilution solution so that the concentration of sodium chloride in the biological sample dilution solution is 1 to 3 mass %.

[0025] In the present invention, the dilution ratio when diluting a biological sample with the specimen dilution solution is preferably 10 to 1000, more preferably 30 to 500, and even more preferably 50 to 300. By using the dilution ratio, the influence of impurities in the biological sample can be suppressed, and therefore vitamin A can be measured more accurately.

[0026] (Immunochromatographic Strips) A specific example of the immunochromatographic strip is an immunochromatographic strip 8 as shown in FIG. 1. In FIG. 1, 1 denotes an adhesive sheet, 2 denotes an impregnated member, 3 denotes a membrane carrier, 4 denotes a test line, 5 denotes a control line, 6 denotes a sample addition member, and 7 denotes an absorption member. The membrane carrier 3 is a 5 mm wide, 25 mm long, strip-shaped nitrocellulose membrane filter, which is attached to the center of the adhesive sheet 1, which is also 5 mm wide. On the membrane carrier 3, an anti-vitamin A antibody is immobilized in a linear shape at a position 3 to 15 mm downstream from the end of the starting point of the chromatographic development, that is, the left side of FIG. 1 (hereinafter referred to as the "upstream side"; the opposite right side is referred to as the "downstream side"), forming a test line 4 for competitively capturing vitamin A in a biological sample with a competitive reagent (a complex of vitamin A and a compound). In addition, a control line 5 is provided at a position 8 to 25 mm downstream from the end of the upstream side of the membrane carrier 3. The test line is preferably located upstream of the control line, and the distance between the test line and the control line is preferably 3 mm or more and less than 10 mm. The control line 5 is for confirming that the immunochromatographic reaction has been carried out regardless of the presence or absence of the analyte, vitamin A. The control line 5 can be formed, for example, by immobilizing an antibody against the antibody (IgG) bound to the label.

[0027] (Test Line) In the present invention, the membrane carrier is provided with a test line and a control line. The antibody immobilized on the test line may be an anti-vitamin A antibody capable of specifically binding to vitamin A, and may be a polyclonal or monoclonal antibody, but from the viewpoint of reaction specificity, a monoclonal antibody is preferable. Vitamin A is a low molecular weight compound that does not have sufficient complexity, and therefore cannot normally induce an immune response. For this reason, in order to make an immunized animal produce antibodies, it is necessary to use an immunogen in which vitamin A is chemically bound to a carrier protein such as ovalbumin. In addition, if an immunogen is mixed with an adjuvant and then injected, the strength of the immune response increases, and the possibility of obtaining good antibodies increases. Polyclonal antibodies can be obtained by purifying antisera obtained by immunizing rabbits, mice, etc. Monoclonal antibodies can be obtained, for example, by immunizing an animal such as a mouse with a conjugate of vitamin A and ovalbumin together with an appropriate adjuvant, fusing the spleen cells of the immunized animal with myeloma cells, culturing the fused cells in a selective medium in which only the fused cells can grow, and selecting the grown cells using the conjugate of vitamin A, for example, by enzyme-labeled immunosorbent assay.

[0028] (Control Line) The control line has an antibody immobilized thereon that specifically binds to the compound in the label. For example, it can be formed by immobilizing an anti-rabbit IgG antibody or an anti-mouse IgG antibody on the membrane carrier. By using the control line, it can be confirmed that the label has migrated to the most downstream part of the membrane carrier, i.e., that the immunochromatographic reaction has taken place (normally).

[0029] (Membrane carrier) The membrane carrier 3 is a nitrocellulose membrane filter, but any material can be used as long as it is capable of chromatographically developing vitamin A contained in a biological sample and of immobilizing substances such as antibodies that form the test line 4; other cellulose-based membranes, nylon membranes, glass fiber membranes, etc. can also be used.

[0030] (Impregnated material) The impregnated member 2 uses glass fiber, but is not limited thereto. For example, filter paper, nitrocellulose membrane, porous plastic nonwoven fabric such as polyethylene or polypropylene can also be used. The impregnated member 2 can be produced by impregnating a member such as the glass fiber with a suspension containing a labeling body described later and drying it. In the present invention, the impregnated member may or may not be used. When using an impregnated member, an impregnated member in which a labeling body in which a labeling substance is bound to an antibody against a competitive reagent can be placed between the sample addition member and the membrane carrier, but when a specimen dilution solution containing a labeling body in which a labeling substance is bound to an antibody against the competitive reagent is used, the impregnated member is not necessary.

[0031] (labeled substance) In the present invention, the immunochromatographic strip may or may not have an impregnated member. When an impregnated member is provided, it is preferable to impregnate the label. When an impregnated member is not provided, the label may be included in advance in a sample dilution solution or the like. The label can be obtained by binding a labeling substance to an antibody against a compound bound to vitamin A. The antibody may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody against a compound bound to vitamin A, but is preferably a monoclonal antibody from the viewpoint of reaction specificity.

[0032] The labeling substance is not particularly limited, and examples thereof include colored (including fluorescent) labeling substances and enzyme labeling substances, but colored labeling substances are preferred because test results can be obtained quickly. Examples of colored labeling substances include colloidal metals, colored latex particles, and colored cellulose particles. Representative examples of colloidal metals include platinum colloids, gold colloids, silver colloids, platinum colloids, palladium colloids, gold nanorods, gold nanoplates, and silver nanoplates. The size of colloidal metal particles is usually about 3 to 100 nm in diameter. Representative examples of colored latex include polystyrene latex, polymethyl methacrylate, and acrylic acid polymers colored with red and blue pigments. The particle size of the latex particles is not particularly limited, but a particle size of 25 to 500 nm is preferred. In addition, commercially available colored cellulose particles can also be used. The particle size of the cellulose particles is not particularly limited, but a particle size of 100 to 500 nm is preferred. Examples of fluorescent labeling substances include those made of materials such as polystyrene, polymethyl methacrylate, polyvinyl toluene, and silica, and examples of fluorescent dyes include fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives, and the like.

[0033] The color of the colored cellulose particles is not particularly limited, but may be, for example, red, blue, yellow, green, black, white, or fluorescent. Among these, blue and black are preferred, as they are less susceptible to the red color derived from hemoglobin in the background, and blue is more preferred. Examples of such cellulose-based colored particles include colored cellulose nanobeads (NanoAct (registered trademark)) manufactured by Asahi Kasei Corporation, and among these, Navy (BL1), Dark Navy (BL2), and Black (KR1) are preferred, and Navy (BL1) and Dark Navy (BL2) are more preferred.

[0034] In addition, it is preferable to treat the surface of the labeling substance with a blocking agent in order to suppress non-specific binding. The blocking agent is preferably polyethylene glycol or a protein. As the protein, blocking peptide fragment, bovine serum albumin (BSA), casein, etc. are preferable. These blocking agents may be used if they are commercially available, or may be produced by a known method. There is no particular restriction on the molecular size, but an average molecular weight of 100 kDa or less is preferable. In general, the smaller the molecular size of the blocking agent, the greater the amount of protein bound to one detection particle, and the higher the performance such as sensitivity.

[0035] (Sample Addition Material) The sample addition member 6 can be, for example, a sheet or film of a porous synthetic resin such as porous polyethylene or porous polypropylene, or a cellulose paper or nonwoven fabric such as filter paper or cotton cloth.

[0036] (Absorption material) The absorbent member 7 may be made of any material that can quickly absorb and retain liquid, and examples of such material include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., with filter paper being particularly suitable.

[0037] 1, the immunochromatographic strip 8 can be produced by adhering a membrane carrier 3 to the middle of an adhesive sheet 1, overlapping and connecting the downstream end of an impregnated member 2 onto the upstream end of the membrane carrier 3, and adhering the upstream portion of the impregnated member 2 to the adhesive sheet 1. Furthermore, a downstream portion of a sample dropping section (sample addition member) 6 is placed on the upper surface of the impregnated member 2, and the upstream portion of the sample addition member 6 is adhered to the adhesive sheet 1. Also, an upstream portion of an absorption member 7 is placed on the upper surface of the downstream portion of the membrane carrier 3, and the downstream portion of the absorption member 7 is adhered to the adhesive sheet 1, thereby forming the immunochromatographic strip 8.

[0038] The immunochromatographic strip may be housed in a plastic housing case 9 for protection and ease of handling (FIG. 2). This case preferably has a sample dropping section 10 and a determination section (determination window) 11 open above the sample application member 6, test line 4, and control line 5 of the immunochromatographic strip. EXAMPLES

[0039] (Preparation of competitive reagent) Vitamin A (retinal, MyBiosource.Inc., MBS6023224) was biotinylated with Biotin-hydrazide (Dojindo Chemical, B303) to prepare a vitamin A-biotin complex (competitive reagent). After preparation, the complex was stored at -30°C until use.

[0040] (Preparation of sample diluent) A sample dilution solution was prepared by adding and dissolving Triton X-100 (Sigma-Aldrich, 10789704001), polyethylene glycol #6000 (Nacalai Tesque, Code: 10200-25), and sodium chloride in phosphate buffered saline (pH 7.4, Nacalai Tesque, 27576-21).

[0041] (Preparation of anti-biotin antibody-bound cellulose particles) Cellulose particle liquid (Asahi Kasei, BL1, 1% by mass) was suspended in 10 mM Tris Buffer (PBS) at pH 7.0, and anti-biotin antibody (SIGMA, B3640) was added and mixed, and the mixture was left to stand at 37°C for 120 minutes to bind the antibody to the cellulose particle surface. Furthermore, in order to suppress non-specific binding to the cellulose particle surface, 1% by mass casein was added and the mixture was left to stand at 37°C for 60 minutes to perform a blocking treatment. After this, a washing operation was performed, and the mixture was suspended in PBS containing 1% by mass sucrose at pH 7.4 to prepare an anti-biotin antibody-bound cellulose particle liquid.

[0042] (Preparation of immunochromatographic strips) (1) Preparation of impregnated members A strip of 8 mm x 150 mm nonwoven glass fiber fabric was impregnated with 0.5 ml of the anti-biotin antibody-bound cellulose particle liquid obtained above, dried at room temperature, and then cut into a size of 8 mm x 5 mm to prepare an impregnated member.

[0043] (2) Preparation of membrane carrier Anti-vitamin A antibody (Cloud-Clone Corp., PAD051Ge01) was prepared to a concentration of 1 mg / ml and then applied linearly to a 25 mm x 300 mm nitrocellulose membrane filter at a volume of 1.0 μl / cm to prepare a test line. Next, anti-rabbit IgG antibody (MyBiosource.Inc., MBS539780) was adjusted to a concentration of 1 mg / ml and then applied in a line shape to the above-mentioned nitrocellulose membrane filter in an amount of 1.0 μl / cm to prepare a control line. After preparing the test line and control line, they were dried at 50° C. for 30 minutes and cut into a size of 25 mm×5 mm to form a membrane carrier.

[0044] (3) Preparation of immunochromatographic strips As shown in FIG. 1, a sample-receiving member 6, a membrane carrier 3, an impregnated member 2, and an absorbing member 7 were arranged on an adhesive sheet 1 to prepare an immunochromatographic strip.

[0045] (sensitivity) A bovine blood sample was diluted with a sample diluent so that the vitamin A concentration was 5 ng / ml. This sample was developed on the immunochromatographic strip, and the absorbance measurement value at the test line after 10 minutes was taken as (A5). Next, only the sample diluent was developed in the same manner, and the absorbance measurement value at the test line after 10 minutes was taken as (A0). The difference in absorbance between (A0) and (A5) was divided by the difference in concentration, i.e., ((A0)-(A5)) / (5-0), was calculated and used as the sensitivity. A concentration difference of 20 mAbs or more, which is sufficient to distinguish a concentration difference of 1 ng / ml, was rated as ◯ (good), 10 mAbs or more but less than 20 mAbs was rated as △ (average), and less than 10 mAbs was rated × (bad).

[0046] Example 1 10 μl of blood was diluted with 690 μl of specimen dilution solution, and 5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The final concentrations of PEG and NaCl were 1% by mass and 1% by mass, respectively. 100 μL of the obtained biological sample dilution solution was dropped onto the sample drop section of the prepared immunochromatographic test strip and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatographic reader (Hamamatsu Photonics C10060-10, measurement mode: blue line measurement mode).

[0047] Example 2 A biological sample dilution solution was prepared in the same manner as in Example 1, except that the final concentrations of PEG and NaCl were 2% by mass and 1.8% by mass, respectively. 100 μL of the obtained biological sample dilution solution was dropped onto the sample dropping section of the prepared immunochromatographic test strip, and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatographic reader.

[0048] Example 3 A biological sample dilution solution was prepared in the same manner as in Example 1, except that the final concentrations of PEG and NaCl were 4% by mass and 3% by mass, respectively. 100 μL of the obtained biological sample dilution solution was dropped onto the sample dropping section of the prepared immunochromatographic test strip, and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatographic reader.

[0049] Comparative Example 1 A biological sample dilution solution was prepared in the same manner as in Example 1, except that the final concentrations of PEG and NaCl were 0.1% by mass and 0.1% by mass, respectively. 100 μL of the obtained biological sample dilution solution was dropped onto the sample dropping section of the prepared immunochromatographic test strip, and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatographic reader.

[0050] Comparative Example 2 A biological sample dilution solution was prepared in the same manner as in Example 1, except that the final concentrations of PEG and NaCl were 5% by mass and 4% by mass, respectively. 100 μL of the obtained biological sample dilution solution was dropped onto the sample dropping section of the prepared immunochromatographic test strip, and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatographic reader.

[0051] Comparative Example 3 A biological sample dilution solution was prepared in the same manner as in Example 1, except that the final concentrations of PEG and NaCl were 2% by mass and 0.1% by mass, respectively. 100 μL of the obtained biological sample dilution solution was dropped onto the sample dropping section of the prepared immunochromatographic test strip, and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatographic reader.

[0052] Comparative Example 4 A biological sample dilution solution was prepared in the same manner as in Example 1, except that the final concentrations of PEG and NaCl were 0.1% by mass and 2% by mass, respectively. 100 μL of the obtained biological sample dilution solution was dropped onto the sample dropping section of the prepared immunochromatographic test strip, and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatographic reader.

[0053] [Table 1] [Industrial Applicability]

[0054] INDUSTRIAL APPLICABILITY The present invention makes it possible to provide a vitamin A immunochromatography kit that can quickly and easily measure the vitamin A concentration in blood at beef cattle production sites. [Explanation of symbols]

[0055] 1 Adhesive sheet 2 Impregnated material 3. Membrane carriers 4. Test Line 5. Control Line 6 Sample Addition Material 7 Absorbing material 8 Immunochromatographic Strips 9 Housing Case 10 Sample dropping section 11 Judgment section (judgment window)

Claims

1. a) mixing a biological sample, a competitive reagent, and a specimen diluent to prepare a biological sample diluent; b) dropping the diluted biological sample onto an immunochromatographic strip and allowing it to develop by capillary action; c) competitively capturing the competitive reagent and vitamin A in the biological sample on the test line of the immunochromatographic strip; d) measuring the color of a test line resulting from the labeling substance bound to the competitive reagent; A method for quantifying vitamin A by a competitive method, comprising: the labeling substance is a blue or black cellulose particle, A quantitative determination method characterized in that the concentration of polyethylene glycol in the biological sample dilution solution is 1 to 4 mass %, and the concentration of sodium chloride is 1 to 3 mass %.

2. 2. The method according to claim 1, wherein the competitive reagent comprises a complex of vitamin A and a compound.

3. The method according to claim 2, wherein the compound is biotin.

4. The quantitative method according to any one of claims 1 to 3, characterized in that the immunochromatographic strip comprises a sample addition member, a membrane carrier, and an absorption member arranged in sequence, and the membrane carrier has a test line to which anti-vitamin A antibodies are fixed.

5. 5. The method according to claim 4, wherein the immunochromatographic strip comprises an impregnating member disposed between a sample application member and a membrane carrier, and the label is impregnated into the impregnating member.

6. 6. The method according to claim 1, wherein the biological sample is any one of blood, plasma, and serum.

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