A method for determining vitamin A in whole blood

Abstract

To provide an immunochromatographic method capable of quickly and easily quantifying concentration of vitamin A in blood by using the whole blood specimen without separating serum and blood plasma at a raising site of beef cows or the like.SOLUTION: Provided is a method for quantifying vitamin A in the whole blood including: a step of obtaining a specimen diluent liquid by mixing the whole blood, a diluent liquid containing a surfactant, and a competitive agent; a step of developing the specimen diluent liquid on an immunochromatographic strip where an antivitamin A antibody is immobilized in advance, and competitively binding the vitamin A in the whole blood and the competitive agent to the antivitamin A antibody; and a step of providing coloration with an indicator body specifically binding to the competitive agent binding to the antivitamin A antibody. In the method, the concentration of the surfactant in the specimen diluent liquid is 0.05 mass% to 0.2 mass%.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an immunochromatographic method for quantifying vitamin A in a biological sample, particularly in whole blood. [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 send them to a testing institution for testing. The test results are usually available several days later, meaning 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 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] The present invention aims to provide an immunochromatographic method capable of quickly and simply quantifying the vitamin A concentration in blood using whole blood at beef cattle breeding sites without separating serum or plasma. [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) mixing whole blood, a diluent containing a surfactant, and a competitive reagent to obtain a specimen diluent; a step of spreading the sample dilution solution on an immunochromatographic strip on which an anti-vitamin A antibody has been immobilized in advance, and competitively binding the vitamin A in the whole blood and a competitive reagent to the anti-vitamin A antibody; a step of coloring with a label that specifically binds to the competitive reagent bound to the anti-vitamin A antibody; Including, A method for quantifying vitamin A in whole blood, wherein the concentration of the surfactant in the specimen dilution solution is 0.05% by mass to 0.2% by mass. (2) The quantitative method described in (1), wherein the surfactant is a nonionic surfactant. (3) The method according to (1) or (2), wherein the competitive reagent is a complex of vitamin A and biotin. (4) The method according to any one of (1) to (3), wherein the dilution rate of the whole blood is 30 to 300 times. (5) The quantitative method described in any one of (1) to (4), 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. (6) The quantitative method described in (5), 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. Effect of the Invention

[0009] According to the present invention, it is possible to rapidly and simply determine the vitamin A concentration in blood using whole blood without separating serum or plasma. [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 in whole blood, comprising the steps of: mixing whole blood, a diluent containing a surfactant, and a competitive reagent to obtain a specimen diluent; spreading the specimen diluent on an immunochromatographic strip to which an anti-vitamin A antibody has been immobilized in advance, thereby competitively binding vitamin A in the whole blood and the competitive reagent to the anti-vitamin A antibody; and coloring the sample with a label that specifically binds to the competitive reagent bound to the anti-vitamin A antibody, wherein the concentration of the surfactant in the specimen diluent is 0.05% by mass to 0.2% by mass.

[0013] (Whole blood sample) In the present invention, the whole blood used refers to blood that has not been subjected to separation or removal of blood cell components by procedures such as centrifugation, as in the case of serum or plasma, and may or may not contain an anticoagulant.

[0014] (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.

[0015] (Dilution solution) The diluent may be any type of buffering agent as long as it has sufficient buffering capacity in a given pH range, such as 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), etc. In the present invention, the given pH is 6 to 8.

[0016] In addition, the diluent preferably contains a nonionic surfactant that does not affect immune reactions in order to hemolyze red blood cells in whole blood and improve the development of the biological sample. Examples of nonionic surfactants include polyoxyethylene alkylphenyl ethers (Triton (registered trademark) surfactants, etc.), polyoxyethylene alkyl ethers (Brij (registered trademark) surfactants, etc.), polyoxyethylene sorbitan fatty acid esters (Tween (registered trademark) surfactants, etc.), polyoxyethylene fatty acid esters, sorbitan fatty acid esters, alkyl glucosides, sucrose fatty acid esters, etc. In addition, the surfactants may be used alone or in combination of two or more. In addition, the concentration of the surfactant in the specimen diluent is preferably 0.01% by mass to 0.2% by mass, more preferably 0.05% by mass to 0.2% by mass, and even more preferably 0.05% by mass to 0.15% by mass. If the concentration is too low, the biological sample diluent is difficult to develop, and if the concentration is too high, the signal of the test line is low.

[0017] In addition, the diluent preferably contains polyethylene glycol and sodium chloride to improve the efficiency, promotion, and specificity of the competitive reaction. The concentrations of polyethylene glycol and sodium chloride added to the diluent are preferably adjusted so that the final concentrations in the specimen diluent are 1% by mass to 4% by mass and 1% by mass to 3% by mass, respectively, taking into account the dilution rate of whole blood. The number-average molecular weight of the polyethylene glycol used is preferably 2000 to 16000, more preferably 5000 to 10000. If the number-average molecular weight is small, sufficient promotion of the antigen-antibody reaction suitable for the present invention may not be obtained. Similarly, if the number-average molecular weight is large, the promotion of the antigen-antibody reaction may not be obtained, or the viscosity of the specimen diluent may increase, resulting in a decrease in the development on the immunochromatographic strip.

[0018] (Dilution ratio) In the present invention, the dilution ratio of the biological sample is suitably 30 to 300 times. If the dilution ratio is too low, impurities in the biological sample may affect the quantitative value. If the dilution ratio is too high, the amount of vitamin A in the biological sample may be reduced, resulting in a decrease in the accuracy of the measurement.

[0019] (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 label (substance). The competitive reagent is preferably a complex of vitamin A and a compound. 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 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. The concentration of the competitive reagent in the specimen dilution solution is preferably 10 ng to 10 μg per 1 ml of biological sample (whole blood). If the concentration of the competitive reagent is too low, the signal during measurement is generally low and quantitativeness is poor. Moreover, if the concentration of the competing reagent is too high, non-specific signals will increase, also resulting in poor quantitation.

[0020] Vitamin A is an unstable compound, and light and heat can easily cause isomerization of double bonds. It also easily reacts with acids, air, and metal ions, and may easily decompose. 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, the competitive reagent may be added to the dilution solution at (before) use, or may be added to the dilution solution in advance, mixed, and stored at the above temperature until use.

[0021] (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, a competitive reagent (complex of vitamin A and a compound), and a specimen dilution solution are mixed to obtain a biological sample dilution solution, which 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 a signal is obtained on the test line, whereby the vitamin A can be quantified. It is preferable to add 10 ng to 10 μg of the competitive reagent per 1 ml of the biological sample.

[0022] (Anti-vitamin A antibody) In the present invention, the antibody immobilized on the test line of the membrane carrier may be an anti-vitamin A antibody capable of specifically binding to vitamin A, and may be a polyclonal or monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of reaction specificity. Vitamin A is a low molecular weight compound that does not have sufficient complexity, so it 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. In addition, polyclonal antibodies can be obtained by purifying antisera obtained by immunizing rabbits, mice, etc. On the other hand, monoclonal antibodies can be obtained by, for example, 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 them 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.

[0023] (labeled substance) In the present invention, the label is preferably one in which a labeling substance is bound 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.

[0024] 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.

[0025] The color of the colored cellulose particles is not particularly limited, but may be, for example, red, blue, yellow, green, black, white, or fluorescent. In the present invention, since a whole blood sample is measured, blue or black, which is less affected by the red color derived from hemoglobin, is preferred, and blue is more preferred. 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.

[0026] 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.

[0027] (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 middle of the adhesive sheet 1, which is also 5 mm wide. The membrane carrier 3 is formed with a test line 4 (with an anti-vitamin A antibody immobilized in a linear shape) for competitively capturing a competitive reagent (complex of vitamin A and a compound) and vitamin A in a biological sample at a position 3 to 15 mm from the end of the left side (upstream side) of FIG. 1 toward the right side (downstream side). Furthermore, 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 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 vitamin A, which is the substance to be analyzed.

[0028] (Test Line) In the present invention, an anti-vitamin A antibody that specifically binds to vitamin A is immobilized on the test line. From the viewpoint of reaction specificity, the anti-vitamin A antibody is preferably a monoclonal antibody.

[0029] (Control Line) In the present invention, an antibody that specifically binds to a compound in a label is immobilized on the control line. For example, the control line can be formed by immobilizing an anti-rabbit IgG antibody or an anti-mouse IgG antibody on a membrane carrier. By using the control line, it is possible to confirm that the label has migrated to the most downstream part of the membrane carrier as described above, that is, that the immunochromatographic reaction has been carried out (normally).

[0030] (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 whole blood and of immobilizing substances such as antibodies that form the test line 4; other cellulose membranes, nylon membranes, glass fiber membranes, etc. can also be used.

[0031] (Impregnated material) The impregnated member 2 uses glass fiber, but is not limited thereto, and filter paper, nitrocellulose membrane, porous plastic nonwoven fabric such as polyethylene or polypropylene, etc. 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 label 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 label in which a labeling substance is bound to an antibody against a competitive reagent is previously impregnated can be placed between the sample addition member and the membrane carrier, but when a diluent containing a label in which a labeling substance is bound to an antibody against the competitive reagent is used, the impregnated member is not necessary.

[0032] (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.

[0033] (Absorption material) The absorbent member 7 may be made of any material that can quickly absorb and retain liquid, and examples of such materials include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., with filter paper being particularly suitable.

[0034] The immunochromatographic strip may be housed in a plastic housing case 9 or the like for protection and ease of handling (FIG. 2). This case is preferably provided with 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.

[0035] (Immunochromatography development) The method for quantifying vitamin A of the present invention will be described. First, a biological sample, a competitive reagent, and a specimen dilution solution are mixed to prepare a biological sample dilution solution. The obtained biological sample dilution solution is dropped onto the sample dropping site of the immunochromatographic strip, and developed on the immunochromatographic strip by utilizing the capillary phenomenon. Vitamin A in the biological sample dilution solution during development is captured by the anti-vitamin A antibody that forms a test line competitively with the competitive reagent (complex of vitamin A and a compound). Furthermore, when the biological sample dilution solution passes through the impregnated member, the label is eluted, and the label binds to the competitive reagent (complex of vitamin A and a compound) captured by the anti-vitamin A antibody. Quantification can be performed by measuring the signal (colorization) of the test line. Note that it is preferable to measure the coloration of the test line 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 a compound) occurs most effectively, and a change in the measurement value corresponding 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 measurement value, which is preferable. EXAMPLES

[0036] (Preparation of competitive reagent) Vitamin A (retinal, MyBiosource.Inc., MBS6023224) was biotinylated with Biotin-hydrazide (Dojindo Chemical, B303) to prepare a competitive reagent. After preparation, the reagent was stored at -30°C until use.

[0037] (Preparation of diluent) A 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).

[0038] (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.

[0039] (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.

[0040] (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.

[0041] (3) Preparation of immunochromatographic strips As shown in FIG. 1, a sample application 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.

[0042] Example 1 10 μl of blood was diluted with 490 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 50 times. The final concentration of the surfactant was 0.05% by mass. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the prepared immunochromatographic test piece 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).

[0043] Example 2 10 μl of blood was diluted with 490 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 50 times. The final concentration of the surfactant was 0.1% by mass. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the prepared immunochromatography test strip and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatography reader.

[0044] Example 3 10 μl of blood was diluted with 490 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 50 times. The final concentration of the surfactant was 0.2 mass%. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the immunochromatography test strip prepared above, and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatography reader.

[0045] Example 4 290 μl of specimen dilution solution was added to 10 μl of blood to dilute it, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 30 times. The final concentration of the surfactant was 0.1% by mass. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the prepared immunochromatography test strip and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatography reader.

[0046] Example 5 10 μl of blood was diluted with 990 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 100 times. The final concentration of the surfactant was 0.1% by mass. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the prepared immunochromatography test strip and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatography reader.

[0047] Example 6 10 μl of blood was diluted with 2990 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 300 times. The final concentration of the surfactant was 0.1% by mass. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the prepared immunochromatography test strip and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatography reader.

[0048] Example 7 10 μl of blood was diluted with 4990 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 500 times. The final concentration of the surfactant was 0.1% by mass. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the prepared immunochromatography test strip and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatography reader.

[0049] Example 8 10 μl of blood was diluted with 190 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 20 times. The final concentration of the surfactant was 0.1% by mass. 100 μL of the obtained specimen 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.

[0050] Comparative Example 1 10 μl of blood was diluted with 490 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 50 times. The final concentration of the surfactant was 0.01% by mass. 100 μL of the obtained specimen 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.

[0051] Comparative Example 2 10 μl of blood was diluted with 490 μl of specimen dilution solution, and 0.5 ng of a competitive reagent (vitamin A-biotin complex) was further added to prepare a biological sample dilution solution. The blood was diluted 50 times. The final concentration of the surfactant was 0.3 mass%. 100 μL of the obtained specimen dilution solution was dropped onto the sample drop section of the prepared immunochromatography test strip and allowed to develop. 10 minutes after dropping, the color of the test line was measured using an immunochromatography reader.

[0052] The above-mentioned Examples and Comparative Examples were evaluated as follows using whole blood samples obtained by collecting blood from 20 cows.

[0053] (Variation in measurements) In each Example and Comparative Example, all whole blood samples were measured five times, and the average CV (coefficient of variation) was calculated. The average CV was rated as ◯ (good) when it was less than 5%, △ (average) when it was 5% or more and less than 7.5%, and × (bad) when it was 7.5% or more. The results are shown in Tables 1 and 2.

[0054] (Correlation with HPLC measurements) In each Example and Comparative Example, all whole blood samples were measured five times, and the average values ​​were compared with the values ​​measured by HPLC to determine the correlation coefficient. The HPLC measurements were performed at a clinical testing center (Kinki Preventive Medical Research Institute, Inc.). A correlation coefficient of 0.9 or more was rated as ◯ (good), 0.85 or more but less than 0.9 was rated as △ (average), and less than 0.85 was rated × (bad). The results are shown in Tables 1 and 2.

[0055] [Table 1]

[0056] [Table 2] [Industrial Applicability]

[0057] INDUSTRIAL APPLICABILITY The present invention makes it possible to provide a vitamin A immunochromatography kit that enables rapid and simple measurement of vitamin A concentration in bovine blood at beef cattle production sites. [Explanation of symbols]

[0058] 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

Claims

1. A step of mixing whole blood, a diluent containing a surfactant, and a competitive reagent to obtain a specimen diluent; a step of spreading the sample dilution solution on an immunochromatographic strip on which an anti-vitamin A antibody has been immobilized in advance, and competitively binding the vitamin A in the whole blood and a competitive reagent to the anti-vitamin A antibody; A step of coloring with a label that specifically binds to the competitive reagent bound to the anti-vitamin A antibody; Including, The competitive reagent is a complex of vitamin A and biotin, The label is an antibody against biotin bound to blue or black cellulose particles, A method for quantifying vitamin A in whole blood, characterized in that the concentration of the nonionic surfactant in the specimen dilution solution is 0.05% by mass to 0.2% by mass.

2. 2. The method according to claim 1, wherein the dilution rate of the whole blood is 30 times or more and 300 times or less.

3. The quantitative method according to claim 1 or 2, 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.

4. 4. The method according to claim 3, 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.

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