JP2023101263A - Immunochromatographic measuring kit for progesterone determination - Google Patents

Abstract

To provide an immunochromatographic measuring kit capable of quickly, easily, and inexpensively measuring a progesterone concentration in a biological specimen such as blood and milk in a dairy field.SOLUTION: An immunochromatographic measuring kit for determining progesterone in a biological specimen contains a specimen diluent liquid for diluting the biological specimen, a competitive reagent, and an immunochromatographic strip. A sample addition member, an impregnation member, a membrane carrier, and an absorption member are sequentially connected and arranged in the immunochromatographic strip. The impregnation member is impregnated with a labeled body obtained by binding a labeling substance to an antibody and/or a high affinity substance for the competitive reagent and an anti-progesterone antibody. The membrane carrier is provided with a test line on which an anti-IgG antibody is immobilized.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to an immunochromatographic measurement kit for quantifying progesterone in biological samples obtained from cattle. More specifically, it relates to a measurement kit for quantifying progesterone in a biological sample, comprising a sample diluent for diluting the biological sample (specimen), a competitive reagent, and an immunochromatographic strip.

Pregnancy of dairy cows is a prerequisite for milk production in dairy farming. Milk production begins only after the cow becomes pregnant and gives birth. Securing more pregnant cows is the most important factor in stabilizing dairy farming. In the event of infertility after fertilization, countermeasures such as remating as early as possible are required in order to suppress economic loss. Therefore, it is extremely important to perform pregnancy diagnosis after insemination and to know the success or failure of pregnancy as early as possible. Pregnancy tests for dairy cows include the rectal examination method, diagnosis using a portable ultrasound imaging device, and the 70-day non-return method (a method that determines that a cow is pregnant if there is no estrus after 70 days from insemination). It has been practiced from the past. On the other hand, as a new pregnancy test method, a method of measuring progesterone in blood or milk has also been proposed. (Non-Patent Document 1, Non-Patent Document 2).

However, conventional hormone measurement by RIA and EIA requires complicated operations and takes a long time to obtain results, and is considered to be of low application value in the field of dairy farming. That is, in the field of dairy farming, there is a demand for a technique for simply measuring the progesterone concentration in biomaterials.

Non-Patent Documents 1 and 2 disclose techniques for measuring progesterone in bovine blood and milk using immunochromatographic strips. Moreover, Patent Document 1 discloses a technique for measuring progesterone using an immunochromatographic method.

Talanta, 132, 685-689, 2015 J. of Dairy Sci. , 103(7), 6600-6611, 2020

Japanese Unexamined Patent Application Publication No. 2002-328127

SUMMARY OF THE INVENTION The present invention was devised to solve the above problems, and an object of the present invention is to provide a measurement kit capable of quickly and simply measuring progesterone concentration present in biological samples such as blood and milk. and

Means for Solving the Problems As a result of intensive studies in order to solve the above problems, the present inventors have found that the above problems can be solved by means shown below, and have completed the present invention.

That is, the present invention consists of the following configurations.
(1) An immunochromatographic measurement kit for quantifying progesterone in a biological sample,
A sample diluent for diluting the biological sample, a competitive reagent, and an immunochromatographic strip,
The immunochromatographic strip comprises a sample addition member, an impregnation member, a membrane carrier, and an absorption member which are arranged in order,
The impregnated member is impregnated with an antibody against a competitive reagent and/or a labeled substance in which a labeling substance is bound to a high affinity substance, and an anti-progesterone antibody,
A measurement kit, wherein the membrane carrier comprises a test line on which an anti-IgG antibody is immobilized.
(2) The assay kit according to (1), wherein the competitive reagent contains a complex of progesterone and a compound.
(3) The measurement kit according to (1) or (2), wherein the compound is biotin.
(4) The anti-IgG antibody is one or more selected from the group consisting of an anti-mouse IgG antibody, an anti-rabbit IgG antibody and an anti-guinea pig IgG antibody, according to any one of (1) to (3). measurement kit.
(5) The assay kit according to any one of (1) to (4), wherein the antibody against the competitive reagent is an anti-biotin antibody.
(6) The assay kit according to any one of (1) to (5), wherein the substance with high affinity for the competitive reagent is avidin and/or streptavidin.
(7) The measurement kit according to any one of (1) to (6), wherein the biological sample is whole blood, plasma, serum, or milk.
(8) The measurement kit according to any one of (1) to (7), which is used to determine the pregnancy status of a bovine.

INDUSTRIAL APPLICABILITY The present invention provides an immunochromatographic measurement kit for quantifying progesterone, which enables rapid, simple, and inexpensive measurement of progesterone concentration in biological samples at dairy farming sites.

Fig. 2 is a schematic diagram (top view) showing an example of an immunochromatographic strip included in the measurement kit of the present invention; 1 is a schematic diagram (side view) showing an example of an immunochromatographic strip included in the measurement kit of the present invention. FIG. FIG. 4 is a schematic diagram showing an example of housing the immunochromatographic strip included in the measurement kit of the present invention in a housing case. FIG. 2 is a diagram (graph) showing an example of progesterone measurement results obtained using the immunochromatographic measurement kit of the present invention. FIG. 2 is a diagram (graph) showing the relationship between the values measured by the immunochromatographic measurement kit of the present invention and the ELISA method.

The present invention will be described in detail below.

The present invention is an immunochromatographic measurement kit for quantifying progesterone in a biological sample, comprising a specimen diluent for diluting the biological sample, a competitive reagent, and an immunochromatographic strip, wherein the immunochromatographic strip is a sample addition member , an impregnated member, a membrane carrier, and an absorbent member are successively arranged, and the impregnated member is impregnated with an antibody against a competitive reagent and/or a labeling substance bound to a high-affinity substance, and an anti-progesterone antibody. The measurement kit is characterized in that the membrane carrier comprises a test line on which an anti-IgG antibody is immobilized.

(biological sample)
In the present invention, the biological sample is not particularly limited, but blood (whole blood, serum, or plasma may be used), milk, and the like are suitable. As for animal species, in addition to bovine, biological samples such as humans, pigs, horses, dogs, and cats can be used as measurement targets.

(progesterone)
Progesterone is a type of steroid hormone synthesized in the corpus luteum of the ovaries. Most of the substances that act as progestins in the body are progesterone. The main function of progesterone is to change the uterus to prepare for pregnancy and, if pregnancy does occur, to maintain the pregnancy until delivery.

(specimen diluent)
In the present invention, the sample diluent may use any type of buffer as long as it has a sufficient buffering capacity in a predetermined pH range. acid, succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, Good's buffer (MES, ADA, PIPES, ACES, colamin hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine) and the like. be done.

In the present invention, the specimen diluent preferably contains a nonionic surfactant that improves the spreadability of the biological sample on the immunochromatographic strip and does not affect immune reactions. 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 and the like can be mentioned. The surfactants may be used alone or in combination of two or more. Further, the concentration of the surfactant in the sample diluent is preferably 0.01% by mass or more and 0.2% by mass or less, and more preferably 0.05% by mass or more and 0.2% by mass or less, although it depends on the dilution ratio of the biological sample. is more preferable, and 0.05% by mass or more and 0.15% by mass or less is even more preferable. If the concentration is too low, the biological sample dilution may be difficult to develop, and if the concentration is too high, the test line signal may be low.

In addition, the specimen diluent preferably contains polyethylene glycol and/or sodium chloride in order to make the competitive reaction more efficient, accelerate, and improve specificity. The concentrations of polyethylene glycol and sodium chloride added to the sample diluent are such that the final concentration in the biological sample diluent is 1% by mass or more and 4% by mass or less, and 1% by mass or more and 3% by mass or less. is preferred. The number average molecular weight of polyethylene glycol to be used is preferably 2000 or more and 16000 or less, more preferably 5000 or more and 10000 or less. If the number-average molecular weight is small, it may not be possible to obtain a sufficient antigen-antibody reaction-promoting action suitable for the present invention. Also, when the number average molecular weight is large, the effect of promoting antigen-antibody reactions may be similarly not obtained, or the viscosity of the sample diluent may increase, resulting in reduced developability on the immunochromatographic strip.

(competitive reagent)
In the present invention, the competitive reagent is not particularly limited as long as it can compete with free or protein-bound progesterone in a biological sample and can be detected by a label (substance). A complex of progesterone and a compound is preferably used as the competitive reagent. A conjugate is preferable because progesterone is stable and an antibody with good performance is readily available. Compounds include bovine serum albumin, ovalbumin and biotin, among which biotin is preferably used. Although the detailed reason is unknown, it is speculated that the complex of progesterone and biotin, which has a low molecular weight, is more likely to compete with the progesterone in the biological sample than the complex of progesterone and a high-molecular-weight substance such as albumin. are doing. In addition, a complex of progesterone and a low-molecular-weight substance has the advantage of being able to keep production costs lower than a complex of progesterone and a protein (high-molecular-weight substance) such as bovine serum albumin.

Progesterone, which forms a complex with biotin, is an unstable compound that easily undergoes isomerization of the double bond due to light and heat, and is easily decomposed because it easily reacts with acid, air, and metal ions. there is fear Therefore, it is preferable to store the competitive reagent at a low temperature in a 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.

In the present invention, it is preferable to add 0.1 ng or more and 10 ng or less of the competitive reagent to 1 mL of the biological sample. If the difference between the progesterone concentration in the biological sample and the amount of the competitive reagent added is too large, the competition may not go well, resulting in a decrease in quantification. The competitive reagent may be freeze-dried or the like, or may be in the form of a solution.

(Label)
In the present invention, a labeled substance can be obtained by binding a labeled substance to an antibody and/or a high-affinity substance against the compound in the competitive reagent. The antibody may be an antibody against the compound in the competitive reagent, and may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred from the viewpoint of reaction specificity. Examples include anti-biotin antibodies. As the high-affinity substance, for example, when the compound is biotin, avidin and streptavidin are preferably used.

The labeling substance is not particularly limited, and examples thereof include color (including fluorescence) labeling substances and enzyme labeling substances. However, coloration labeling substances are preferred because test results can be obtained quickly. Color-changing labeling substances include colloidal metals, colored latex particles, colored cellulose particles, and the like. Representative examples of colloidal metals include platinum colloids, gold colloids, silver colloids, platinum colloids, palladium colloids, gold nanorods, gold nanoplates, silver nanoplates, and the like. The size of colloidal metal particles is usually 3 nm or more and 100 nm or less in diameter. Representative examples of colored latexes include polystyrene latexes colored with respective pigments such as red and blue, polymethyl methacrylate, acrylic acid polymers, and the like. Although the particle size of the latex particles is not particularly limited, those having a particle size of 25 nm or more and 500 nm or less are preferable. In addition, commercially available colored cellulose particles can also be used. Although the particle size of the colored cellulose particles is not particularly limited, a particle size of 100 nm or more and 500 nm or less is preferable. Examples of fluorescent labeling substances include materials such as polystyrene, polymethyl methacrylate, polyvinyltoluene, and silica. Examples of fluorescent dyes include fluorescein and its derivatives, rhodamine and its derivatives, and cyanine and its derivatives. can do.

The color of the colored cellulose particles is not particularly limited, but examples thereof include red, blue, yellow, green, black, white, and fluorescent colors. Among these, blue and black are preferred, and blue is more preferred because they are less susceptible to the red color derived from hemoglobin in the background. Examples of such colored cellulose particles include colored cellulose nanoparticles (NanoAct (registered trademark)) manufactured by Asahi Kasei Corporation. Among them, Navy (BL1), Dark Navy (BL2), and Black (KR1) are preferable, and BL1) and Dark Navy (BL2) are more preferred.

In the present invention, in order to suppress non-specific binding to the surface of the labeling substance, it may be treated with a blocking agent in advance. It is preferable to use polyethylene glycol or protein as the blocking agent. Preferred proteins include Blocking Peptide Fragment (BPF), bovine serum albumin (BSA), casein and the like. These blocking agents may be used if they are commercially available, or they may be separately produced by a known method. The molecular size is also not particularly limited, but an average molecular weight of 100 kDa or less is preferable. In general, the smaller the molecular size of the blocking agent, the higher the amount of protein bound to one detection particle and the higher performance such as sensitivity.

(Dilution ratio)
In the present invention, the dilution ratio of the biological sample is preferably 30 times or more and 300 times or less. If the dilution ratio is too low, contaminants in the biological sample may affect the quantitative value. In addition, if the dilution rate is too high, the amount of estrone in the biological sample will decrease, which may lower the accuracy of measurement.

(Immunochromato strip)
A specific example of the immunochromatographic strip is the immunochromatographic strip 8 shown in FIGS. 1 and 2, 1 is an adhesive sheet, 2 is an impregnation member, 3 is a membrane carrier, 4 is a test line, 5 is a control line, 6 is a sample addition member, and 7 is an absorption member. The membrane carrier 3 consists of an elongated belt-shaped nitrocellulose membrane having a width of 5 mm and a length of 25 mm, and is attached to the middle of the pressure-sensitive adhesive sheet 1 having a width of 5 mm. On the membrane carrier 3, an anti-progesterone antibody for capturing an anti-progesterone antibody is provided at a position of 3 mm or more and 15 mm or less toward the right side (downstream side) from the starting point side of chromatographic development, that is, the left side (upstream side) end in FIGS. A test line 4 is formed (an anti-IgG antibody is linearly immobilized). Furthermore, a control line 5 is formed at a position of 8 mm or more and 25 mm or less toward the downstream side from the upstream end of the membrane carrier 3 (an antibody that specifically binds to the antibody in the label is linearly immobilized). . In addition, it is preferable that the test line is arranged on the upstream side of the control line, and the distance between the test line and the control line is 3 mm or more and less than 10 mm. The control line 5 is for confirming that the immunochromatographic development was performed regardless of the presence or absence of progesterone, which is the substance to be analyzed.

(Sample addition member)
In the present invention, the sample addition member 6 is, for example, a porous synthetic resin sheet or film such as porous polyethylene or porous polypropylene, or cellulose paper or non-woven fabric such as filter paper or cotton cloth. be able to.

(impregnated member)
In the present invention, the impregnated member 2 uses a belt-shaped glass fiber of 5 mm x 15 mm, but is not limited to this. For example, filter paper, nitrocellulose membrane, porous plastic nonwoven fabric such as polyethylene, polypropylene, etc. can. The impregnated member 2 can be produced by impregnating a member such as the glass fiber with a suspension containing the marker and drying it. The impregnation member is pre-impregnated with an antibody against a competitive reagent and/or a labeled substance in which a labeling substance is bound to a high-affinity substance, and an anti-progesterone antibody, and is placed between the sample addition member and the membrane carrier. Let it be used.

(membrane carrier)
In the present invention, a nitrocellulose membrane filter is used as the membrane carrier 3 as long as the progesterone contained in the biological sample can be chromatographically developed and a substance such as an antibody that forms the test line 4 can be immobilized. , and other cellulose membranes, nylon membranes, glass fiber membranes, etc. can also be used.

(test line)
In the present invention, the antibody immobilized on the test line 4 is an antibody capable of specifically binding to mouse IgG, rabbit IgG or guinea pig IgG, that is, anti-mouse IgG antibody, anti-rabbit IgG antibody or anti-guinea pig IgG antibody. Any antibody may be used, and it may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable from the viewpoint of reaction specificity.

(anti-progesterone antibody)
Progesterone (molecular weight 314) is a small molecule and lacks sufficient complexity to normally provoke an immune response. Therefore, in order to induce antibody production in an immunized animal, it is necessary to use, as an immunogen, a carrier protein such as ovalbumin that is chemically bound to progesterone. Injecting an immunogen mixed with an adjuvant increases the strength of the immune response and increases the possibility of obtaining good antibodies. Polyclonal antibodies can be obtained by purifying antisera obtained by immunizing rabbits, mice and the like. Monoclonal antibody-producing cells are obtained by, for example, immunizing an animal such as a mouse with a conjugate of progesterone and ovalbumin together with an appropriate adjuvant, fusing the spleen cells of the immunized animal with myeloma cells, and obtaining only the fused cells. can be obtained by culturing in a selective medium capable of proliferating, and then selecting the proliferated cells using the estrone-bound compound, for example, using an enzyme-labeled immunoassay.

(control line)
In the present invention, the control line 5 is preferably immobilized with an antibody that specifically binds to the label. An anti-IgG antibody can be used as the antibody, and specifically, it can be formed by immobilizing an anti-goat IgG antibody or the like on a 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, that is, that immunochromatographic development has been performed (normally).

(absorbing member)
In the present invention, the absorbent member 7 may be made of a material that can quickly absorb and retain liquid, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabrics made of polyethylene, polypropylene, etc., particularly filter paper. is optimal.

The immunochromato strip is preferably housed in a plastic housing case 9 or the like in order to protect it and facilitate handling (Fig. 3). In this case, for example, a sample dropping portion 10 is opened above the sample addition member 6 of the immunochromatographic strip, and a determination portion (determination window) 11 is preferably opened above the test line 4 and the control line 5 .

(Immunochromatographic development)
A method for quantifying progesterone according to the present invention will be described. First, a biological sample diluent is prepared by mixing a biological sample, a competitive reagent, and a specimen diluent. The obtained biological sample diluted solution is dropped onto the sample drop site of the immunochromatographic strip, and is spread on the immunochromatographic strip using capillary action. The developed biological sample diluent elutes the labeled substance and the anti-progesterone antibody when passing through the impregnated member. Progesterone in the biological sample diluent is captured by an anti-progesterone antibody competitively with a competing reagent (a complex of progesterone and a compound) during development. In addition, the labeled substance binds to a competitive reagent (a complex of progesterone and a compound) captured by an anti-progesterone antibody, and furthermore, this conjugate is captured by an anti-IgG antibody of the test line and exhibits a signal (coloration ). It can be quantified by measuring the signal (coloration) of the test line. It is desirable 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 progesterone and the competitive reagent (complex of progesterone and the compound) occurs most effectively, and changes in the measured values in response to changes in the concentration of progesterone can be obtained at the test line. This is preferable because the difference in concentration is accurately reflected in the measured value. That is, it is possible to accurately measure the progesterone concentration in the biological sample. In less than 5 minutes, the reaction between the anti-progesterone antibody in the test line and the progesterone in the biological sample or the competitive reagent (complex of progesterone and the compound) is not sufficient, resulting in low measured values or low progesterone concentration in the biological sample. It may not be possible to obtain measurements that accurately reflect the differences. Moreover, if the time exceeds 12 minutes, non-specific reaction to the anti-progesterone antibody increases, so that it may not be possible to obtain an accurate measurement value change according to the progesterone concentration in the biological sample.

(competition law)
In the present invention, progesterone in a biological sample is preferably quantified by a competitive method. Since it is difficult to sandwich a low-molecular-weight compound such as progesterone between two kinds of antibodies, it is preferable to employ a competitive method.

(Immunochromatographic measurement kit)
The immunochromatographic measurement kit of the present invention includes, in addition to the immunochromatographic strips described above, at least a specimen diluent for diluting the specimen and a competitive reagent, and, if necessary, a progesterone standard solution for preparing a calibration curve, Including a container for preparing a biological sample diluent. It may also include a measurement device (chromato reader, etc.) for measuring immunochromatography results.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples. The characteristic values measured in the examples were measured according to the following methods.

(Preparation of competitive reagent)
A competitive reagent (progesterone-biotin complex) was prepared by biotinylating progesterone (Sigma-Aldrich, P0130) with Biotin-hydrazide (Dojindo, B303). After preparation, it was stored at -30°C until use.

(Preparation of anti-progesterone antibody)
An anti-progesterone antibody was obtained by purifying IgG by affinity chromatography from serum obtained by immunizing a mouse with a conjugate of progesterone and BSA (bovine serum albumin).

(Anti-mouse IgG antibody)
An anti-mouse IgG antibody (Goat Anti-Mouse IgG, Abcam, ab6708) was used as the anti-mouse IgG antibody.

(Preparation of label solution)
As a labeling substance, a colored cellulose fine particle solution (Asahi Kasei, BL1, 1% by mass) was suspended in 10 mM Tris Buffer (PBS) of pH 7.0, and an anti-biotin antibody (abcam, ab53494) was added and mixed. for 120 minutes to allow the antibody to bind to the surface of the colored cellulose microparticles. Furthermore, in order to suppress non-specific binding to the surface of the colored cellulose fine particles, 1% by mass of casein was added, and the mixture was allowed to stand at 37° C. for 60 minutes for blocking treatment. Thereafter, a washing operation was performed and suspended in 1 mass % sucrose-containing PBS (pH 7.4) to prepare a label solution (anti-biotin antibody-bound cellulose fine particle solution).

(Preparation of specimen diluent)
Phosphate-buffered saline (pH 7.4, Nacalai Tesque, 27576-21) was added with Triton X-100 (Sigma-Aldrich, 10789704001), polyethylene glycol, and NaCl at a final concentration of 0.15% by mass, respectively. 0% by mass and 1.5% by mass were added and dissolved to prepare specimen diluents.

(Production of impregnated member)
An 8 mm × 150 mm band-shaped glass fiber nonwoven fabric was impregnated with a mixture of 0.5 mL of the labeled solution (anti-biotin antibody-bound cellulose fine particle solution) obtained above and 0.5 mL of anti-progesterone antibody (1 mg/ml). let me After drying at room temperature, it was cut into a size of 8 mm×5 mm to obtain an impregnated member.

(Preparation of membrane carrier)
After adjusting the anti-mouse IgG antibody to a concentration of 1 mg/mL, it was linearly applied to a 25 mm×300 mm nitrocellulose membrane filter at an amount of 1.0 μL/cm to prepare a test line.
Next, an anti-goat IgG antibody (Donkey Anti-Goat IgG, Abcam, ab182021) was adjusted to a concentration of 1 mg/mL, and then linearly applied to the nitrocellulose membrane filter at an amount of 1.0 μL/cm. A control line was made.
After the test line and control line were prepared, they were dried at 50° C. for 30 minutes and cut into a size of 25 mm×5 mm to obtain a membrane carrier.

(Preparation of immunochromatographic strip)
An immunochromatographic strip was prepared by arranging a sample addition member and an absorption member in addition to the prepared membrane carrier and impregnated member on an adhesive sheet.

(Preparation of progesterone standard solution)
Progesterone (Sigma-Aldrich, P0130) was added to PBS (phosphate buffered saline) to prepare a progesterone standard solution.

(quantitation using a measurement kit)
A sample diluent was added to the above standard solution to prepare a progesterone diluent having each concentration shown in Table 1. After adding a competitive reagent to the progesterone diluent so that the concentration of the progesterone-biotin complex was 0.3 ng/mL, 100 μL was dropped onto the sample drop portion of the immunochromatographic strip for development. Ten minutes after the dropping, the color development (absorbance) of the test line was measured using an immunochromatographic reader (C10060-10 manufactured by Hamamatsu Photonics, measurement mode: blue line measurement mode). Results are shown in Table 1 and FIG. It was confirmed that progesterone can be quantified with high accuracy by using the measurement kit of the present invention.

(Comparison experiment with ELISA method measurement)
Serum progesterone concentrations obtained from 12 dairy cows were measured using the ELISA method and the immunochromatographic measurement kit of the present invention, respectively. The ELISA method was measured using a Progesterone ELISA kit (Enzo Life Sciences. Inc.). In the immunochromatographic method, the progesterone concentration in each sample was calculated using a standard curve obtained by simultaneously measuring standard solutions, and the measurement results are shown in Table 2 and FIG. The correlation coefficient between the values measured by the immunochromatography method and the values measured by the ELISA method was 0.95, indicating a good correlation. It was confirmed that progesterone can be quantified with high accuracy by using the measurement kit of the present invention.

INDUSTRIAL APPLICABILITY According to the present invention, the progesterone concentration in biological samples such as blood and milk can be measured quickly, easily, and inexpensively at the site of dairy farming, so that the pregnancy status can be grasped, and dairy cattle can be produced efficiently. It is possible to do

1 adhesive sheet 2 impregnation member 3 membrane carrier 4 test line 5 control line 6 sample addition member 7 absorption member 8 immunochromatographic strip 9 housing case 10 sample drop portion 11 determination portion (determination window)

Claims (8)

An immunochromatographic measurement kit for quantifying progesterone in a biological sample,
A sample diluent for diluting the biological sample, a competitive reagent, and an immunochromatographic strip,
The immunochromatographic strip comprises a sample addition member, an impregnation member, a membrane carrier, and an absorption member which are arranged in order,
The impregnated member is impregnated with an antibody against a competitive reagent and/or a labeled substance in which a labeling substance is bound to a high affinity substance, and an anti-progesterone antibody,
A measurement kit, wherein the membrane carrier comprises a test line on which an anti-IgG antibody is immobilized. 2. The assay kit according to claim 1, wherein the competitive reagent contains a complex of progesterone and a compound. 3. The assay kit according to claim 1, wherein said compound is biotin. The measurement kit according to any one of claims 1 to 3, wherein the anti-IgG antibody is one or more selected from the group consisting of anti-mouse IgG antibody, anti-rabbit IgG antibody and anti-guinea pig IgG antibody. The measurement kit according to any one of claims 1 to 4, wherein the antibody against the competitive reagent is an anti-biotin antibody. The measurement kit according to any one of claims 1 to 5, wherein the substance with high affinity for the competitive reagent is avidin and/or streptavidin. The measurement kit according to any one of claims 1 to 6, wherein the biological sample is whole blood, plasma, serum, or milk. 8. The measurement kit according to any one of claims 1 to 7, which is used for determining the pregnancy state of a bovine.