JP7619127B2 - Vitamin A quantification method - Google Patents

Description

The present invention relates to a method for easily measuring vitamin A concentration in a biological sample.

In beef cattle, particularly in the production of Wagyu beef, which produces high-quality 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 has become mainstream in recent years, in which fat marbling is induced by drastically reducing the amount of vitamin A fed, which has the function of promoting the breakdown of fat. However, an extreme deficiency of vitamin A can cause health problems such as impaired liver function, and can also cause muscle edema and myositis, which can lead to a decline in meat quality, which can be a major problem. For this reason, the concentration of vitamin A in the blood is measured to understand the nutritional status of cattle and prevent the occurrence of problems such as those mentioned above.

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 only available several days later, meaning that there is a drawback to this method in that immediate action cannot be taken even if there is a vitamin A deficiency or excess.

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 centrifugal separation of the serum, followed by further steps to remove protein and extract vitamin A components with heptane, which makes the procedure complicated for on-site implementation and takes time and effort.

JP 2010-230447 A JP 2015-169627 A

The present invention aims to provide a method for quickly, easily, and inexpensively measuring vitamin A concentrations in biological samples (blood) at breeding sites for beef cattle, etc.

As a result of extensive research into solving the above problems, the inventors discovered that the above problems could be solved by the following means, and arrived at the present invention.

That is, the present invention comprises the following configuration.
(1) mixing a biological sample, a competitive reagent, and a specimen diluent to prepare a biological sample diluent;
A step of dropping the diluted biological sample onto an immunochromatographic strip and developing the same;
a step of competitively capturing the competitive reagent and vitamin A in the biological sample on a test line of the immunochromatographic strip;
A method for quantifying vitamin A in a biological sample, comprising:
The quantitative determination method is characterized in that 10 ng to 10 μg of the competitive reagent is added per 1 ml of the biological sample.
(2) The method according to (1), wherein the competitive reagent contains a complex of vitamin A and a compound.
(3) The method according to (1) or (2), wherein the compound is biotin.
(4) The quantitative method according to 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 this order, and the membrane carrier has a test line on which anti-vitamin A antibodies are fixed.
(5) The quantitative method according to (4), wherein the immunochromatographic strip comprises an impregnating member disposed between a sample application member and a membrane carrier, and the impregnating member is impregnated with a label.
(6) The method for quantifying a biological sample according to any one of (1) to (5), wherein the biological sample is blood, plasma, or serum.

This invention makes it possible to measure vitamin A concentrations in biological samples (blood) quickly, easily, and inexpensively at beef cattle production sites.

FIG. 1 is a schematic diagram showing an example of an immunochromatographic strip used in the measurement method of the present invention. FIG. 2 is a schematic diagram showing an example of an immunochromatographic strip used in the measurement method of the present invention housed in a housing case.

The present invention is described in detail below.

The present invention is a method for quantifying vitamin A in a biological sample, comprising the steps of mixing a biological sample, a competitive reagent, and a specimen dilution solution to prepare a biological sample dilution solution, dropping and spreading the biological sample dilution solution onto an immunochromatographic strip, and competitively capturing the conjugate and vitamin A in the biological sample on the test line of the immunochromatographic strip, characterized in that 10 ng to 10 μg of the competitive reagent is added per 1 ml of the biological sample.

(Biological samples)
In the present invention, the biological sample is not particularly limited and may be whole blood, serum, or plasma, but whole blood is preferred because it can be easily prepared on-site. Blood from animals such as cattle, humans, horses, dogs, and cats can also be measured.

(Competitive Reagents)
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.

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. For this reason, it is preferable to store the competitive reagent in a cool, dark place until use. Storage temperatures of 4°C or lower are preferable, -20°C or lower are more preferable, and -80°C or lower are even more preferable.

In the present invention, it is preferable to add 10 ng to 10 μg of competitive reagent per 1 ml of biological sample. Since the vitamin A concentration in biological samples, particularly beef cattle plasma, is 200 ng/ml to 1 μg/ml, if the difference between the vitamin A concentration in the biological sample and the amount of competitive reagent added is too large, the competition may not go well and the quantitativeness may decrease. The competitive reagent may be freeze-dried or in solution. The competitive reagent may also be mixed in advance with a specimen diluent and stored at low temperature.

(Specimen diluent)
In the present invention, any type of buffer may be used as the specimen dilution solution so long as it has sufficient buffering capacity within a specified pH range, and examples thereof include Tris, phosphoric acid, phthalic acid, citric acid, maleic acid, succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonate, Good's buffer (MES, ADA, PIPES, ACES, cholamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycineamide, bicine), and the like.

In order to hemolyze red blood cells in the biological sample and to improve the spreading of the biological sample, the specimen dilution solution preferably contains a nonionic surfactant that does not affect the immune reaction. 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 dilution solution is preferably 0.01% to 0.2% by mass, more preferably 0.05% to 0.2% by mass, and even more preferably 0.05% to 0.15% by mass. If the concentration is too low, the biological sample dilution solution is difficult to spread, and if the concentration is too high, the signal of the test line is low.

The specimen dilution solution 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 dilution solution are preferably 1% to 4% by mass and 1% to 3% by mass, respectively, taking into consideration the dilution rate of the biological sample. The number-average molecular weight of the polyethylene glycol used is preferably 2,000 to 16,000, more preferably 5,000 to 10,000. 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 dilution solution may increase, reducing the spread on the immunochromatographic strip.

(Labeled body)
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.

There are no particular limitations on the labeling substance, and examples include color-changing (including fluorescent) labeling substances and enzyme labeling substances, but color-changing labeling substances are preferred because test results can be obtained quickly. Examples of color-changing 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 pigments such as red and blue. 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, and cyanine and its derivatives.

The color of the cellulose-based colored microparticles is not particularly limited, but examples include red, blue, yellow, green, black, white, and fluorescent colors. Among these, blue and black are preferred, as they are less affected by the red color derived from hemoglobin in the background, and blue is more preferred. Examples of such cellulose-based colored microparticles 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.

In addition, it is preferable to treat the surface of the labeled substance with a blocking agent 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.

(Anti-vitamin A antibodies)
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, 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. 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 the cells in a selective medium in which only the fused cells can grow, and selecting the grown cells by, for example, enzyme-labeled immunization using the conjugate of vitamin A.

(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 absorbing member. The membrane carrier 3 is a strip-shaped nitrocellulose membrane with a width of 5 mm and a length of 25 mm, and is attached to the middle of the adhesive sheet 1, which also has a width of 5 mm. On the membrane carrier 3, a test line 4 (an anti-vitamin A antibody is immobilized in a linear shape) for competitively capturing a competitive reagent (a complex of vitamin A and a compound) and vitamin A in a biological sample is formed 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 from the end of the upstream side of the membrane carrier 3 toward the downstream side. 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 vitamin A, which is the substance to be analyzed.

(Test Line)
The test line has immobilized thereon an anti-vitamin A antibody that specifically binds to vitamin A. From the viewpoint of reaction specificity, the anti-vitamin A antibody is preferably a monoclonal antibody.

(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 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).

(Impregnated member)
The impregnated member 2 uses glass fiber, but is not limited to this, and for example, 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 then drying it.

(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 the analyte (vitamin A) contained in the 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.

(Sample Addition Member)
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. The absorbent member 7 can be made of any material that can rapidly 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 optimal.

(Absorption member)
The absorbent member 7 may be made of any material that can rapidly 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.

As shown in FIG. 1, the immunochromatographic strip 8 is prepared by attaching the membrane carrier 3 to the middle of the adhesive sheet 1, superimposing and connecting the downstream end of the impregnated member 2 on the upstream end of the membrane carrier 3, and attaching the upstream portion of the impregnated member 2 to the adhesive sheet 1. Furthermore, the downstream end of the sample addition member 6 is superimposed and connected to the upper surface of the upstream end of the impregnated member 2, and the upstream portion of the sample addition member 6 is attached to the adhesive sheet 1. The upstream end of the absorbent member 7 is superimposed and connected to the upper surface of the downstream end of the membrane carrier 3, and the downstream portion of the absorbent member 5 is attached to the adhesive sheet 1, to prepare the test strip 8.

The immunochromatographic strip may be housed in a plastic housing case 9 for protection and ease of handling (Figure 2). This case is preferably provided with a sample dropping section 10 and a determination section (determination window) 11 open above the sample addition member 6, test line 4, and control line 5 of the immunochromatographic strip.

(Immunochromatography development)
The method for quantifying vitamin A according to 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, it dissolves the label, 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. 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.

(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 (a complex of vitamin A and a compound), and a specimen dilution solution are mixed, and the biological sample dilution solution is dropped onto an immunochromatographic strip and developed, whereby vitamin A in the sample and the competitive reagent are competitively captured by the anti-vitamin A antibody forming a test line. The captured competitive reagent is colored with a label (substance), and quantified by measuring the signal on the test line.

(Preparation of sample dilution solution)
Triton X-100 (Sigma-Aldrich, 10789704001), polyethylene glycol #6000 (Nacalai Tesque, Code: 10200-25), and sodium chloride were added to phosphate buffered saline (pH 7.4, Nacalai Tesque, 27576-21) to give concentrations of 0.102 mass %, 2.04 mass %, and 1.84 mass %, respectively, to prepare a specimen dilution solution.

(Preparation of competitive reagent)
Vitamin A (retinal, MyBiosource.Inc., MBS6023224) was biotinylated using 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.

(Preparation of anti-biotin antibody-bound cellulose particles)
The 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 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 of casein was added, and the mixture was left 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 of sucrose at pH 7.4 to prepare an anti-biotin antibody-bound colored cellulose particle liquid.

(Preparation of immunochromatographic strips)
(1) Preparation of Impregnated Member An 8 mm x 150 mm strip of glass fiber nonwoven fabric was impregnated with 0.5 ml of a suspension of the anti-biotin antibody-bound colored cellulose particles obtained above in a solution of 20 mM Tris-HCl, 0.05% by mass PEG20000, 37.5 mM NaCl, 0.25% by mass BSA, and 3% by mass sucrose. After drying at room temperature, the fabric was cut into a size of 8 mm x 5 mm to prepare an impregnated member impregnated with the anti-biotin antibody-bound colored cellulose particles .

(2) Preparation of test line and control line Anti-vitamin A antibody (Cloud-Clone Corp., PAD051Ge01) was adjusted to a concentration of 1 mg/ml, and then applied in a line shape to a 25 mm x 300 mm nitrocellulose membrane filter in an amount of 1.0 μl/cm to prepare a test line (FIG. 1). 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 nitrocellulose membrane filter in an amount of 1.0 μl/cm to prepare a control line (FIG. 1). 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 x 5 mm to prepare a membrane carrier.

(3) Preparation of Immunochromatographic Strip As shown in FIG. 1, the membrane carrier 3 obtained in (2) above, the impregnated member 2 obtained in (1) above, the sample addition member 6, and the absorption member 7 were arranged on an adhesive sheet 1 to prepare an immunochromatographic strip.

(Signal Strength)
Only the diluted specimen was applied to the immunochromatographic strip, and the absorbance measured at the test line after 10 minutes was regarded as the signal intensity. A test line with sufficient visibility of 300 mAbs or more was rated as ◯ (good), 200 mAbs or more but less than 300 mAbs was rated as △ (average), and less than 200 mAbs was rated as × (bad).

(Absorbance at high concentration of vitamin A)
Bovine blood was diluted with a sample dilution solution so that the vitamin A concentration was 50 ng/ml. The vitamin A concentration in bovine blood was measured by HPLC analysis. This sample was developed on the immunochromatographic strip, and the absorbance at the test line after 10 minutes was measured. If the absorbance is sufficiently reduced even at a high concentration of vitamin A of 50 ng/ml, it can be considered that there is a wide measurement range from 0 ng/ml to about 50 ng/ml. The absorbance at 50 ng/ml was rated as ◯ (good) when it was less than 50 mAbs, △ (average) when it was 50 mAbs or more and less than 100 mAbs, and × (bad) when it was 100 mAbs or more.

Example 1
0.05 ng of competitive reagent was added to a mixture of 10 μL of bovine blood and 490 μL of specimen dilution solution, and dissolved to prepare a biological sample dilution solution. 100 μL of the obtained biological sample dilution solution was dropped into the sample application section of the immunochromatographic strip and developed. 10 minutes after dropping, the color development of the test line was measured using an immunochromatographic reader (Hamamatsu Photonics C10060-10, measurement mode: blue line measurement mode). The results are shown in Table 1.

Example 2
0.5 ng of the competitive reagent was added to a mixture of 10 μL of bovine blood and 490 μL of specimen dilution solution, and dissolved to prepare a biological sample dilution solution. 100 μL of the obtained biological sample dilution solution was dropped into the sample application section of the immunochromatography strip and developed. 10 minutes after dropping, the color development of the test line was measured using an immunochromatography reader. The results are shown in Table 1.

Example 3
5 ng of the competitive reagent was added to a mixture of 10 μL of bovine blood and 490 μL of specimen dilution solution, and dissolved to prepare a biological sample dilution solution. 100 μL of the obtained biological sample dilution solution was dropped into the sample application section of the immunochromatography strip and developed. 10 minutes after dropping, the color development of the test line was measured using an immunochromatography reader. The results are shown in Table 1.

Example 4
50 ng of competitive reagent was added to a mixture of 10 μL of bovine blood and 490 μL of specimen dilution solution, and dissolved to prepare a biological sample dilution solution. 100 μL of the obtained biological sample dilution solution was dropped into the sample application area of the immunochromatography strip and developed. 10 minutes after dropping, the color development of the test line was measured using an immunochromatography reader. The results are shown in Table 1.

(Comparative Example 1)
0.01 ng of competitive reagent was added to a mixture of 10 μL of bovine blood and 490 μL of specimen dilution solution, and dissolved to prepare a biological sample dilution solution. 100 μL of the obtained biological sample dilution solution was dropped into the sample application section of the immunochromatography strip and developed. 10 minutes after dropping, the color development of the test line was measured using an immunochromatography reader. The results are shown in Table 1.

(Comparative Example 2)
100 ng of the competitive reagent was added to a mixture of 10 μL of bovine blood and 490 μL of specimen dilution solution, and dissolved to prepare a biological sample dilution solution. 100 μL of the obtained biological sample dilution solution was dropped into the sample application section of the immunochromatography strip and developed. 10 minutes after dropping, the color development of the test line was measured using an immunochromatography reader. The results are shown in Table 1.

The present invention allows for the rapid and simple measurement of vitamin A concentration in cattle blood at beef cattle breeding sites, making it easy to control fat marbling.

1 Adhesive sheet 2 Impregnated member 3 Membrane carrier 4 Test line 5 Control line 6 Sample addition member 7 Absorption member 8 Immunochromatographic strip

Claims (6)

mixing a biological sample, a competitive reagent, and a specimen diluent to prepare a biological sample diluent;
A step of dropping the diluted biological sample onto an immunochromatographic strip and developing the same;
a step of competitively capturing the competitive reagent and vitamin A in the biological sample on a test line of the immunochromatographic strip;
A step of coloring the competitive reagent captured on the test line with a label and measuring a signal on the test line;
A method for quantifying vitamin A in a biological sample, comprising:
the label is an antibody against the competitive reagent bound to blue or black cellulose particles,
The quantitative determination method is characterized in that 5 ng to 5 μg of the competitive reagent is added per 1 ml of the biological sample. The quantitative method according to claim 1, characterized in that the competitive reagent is a complex of vitamin A and a compound. The method according to claim 2 , wherein the compound is biotin. The quantitative determination method according to any one of claims 1 to 3, characterized in that the sample addition member, the membrane carrier, and the absorption member are arranged in series, and the membrane carrier has a test line to which anti-vitamin A antibodies are immobilized. The quantitative determination method according to claim 4, characterized in that the immunochromatographic strip has an impregnating member disposed between the sample addition member and the membrane carrier, and the label is impregnated into the impregnating member. The quantitative method according to any one of claims 1 to 5, wherein the biological sample is blood, plasma or serum.