JP2023067046A - Immunochromatographic measurement kit for estradiol quantification - Google Patents

Abstract

To provide an immunochromatographic measurement kit which allows for quickly, simply, and inexpensively measuring blood estradiol concentration at horse breeding sites.SOLUTION: An immunochromatographic measurement kit for quantifying estradiol in a biological sample is provided, the measurement kit comprising a sample diluent for diluting a biological sample and an immunochromatographic strip for quantifying estradiol, where the immunochromatographic strip consists of a sample adding member, a membrane carrier, and an absorption member successively and directly arranged in the described order.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to an immunochromatographic measurement kit for quantifying estradiol in biological samples. More specifically, the present invention relates to a measurement kit for quantifying estradiol in a biological sample, comprising a specimen diluent for diluting a biological sample (specimen) and an immunochromatographic strip.

Japan is the fifth largest Thoroughbred (racehorse) producing country in the world (2008). In Thoroughbred production, it is very important to produce healthy and strong foals first. Horses have a gestation period of 11 months, and breeders aim to produce one offspring per year. It has become a big problem that the death rate of 5-15% occurs (Non-Patent Document 1, Non-Patent Document 2). Even in the event of early embryonic death, if treatment for corpus luteum regression by administration of prostaglandin F2α can be taken, it is possible to induce re-emergence and remating within the same season. Therefore, it is extremely important in Thoroughbred production to grasp the gestational status early after mating.

Ultrasound diagnosis is the most common method for diagnosing pregnancy in horses, but it requires a skilled veterinarian to perform the diagnosis, and there are also problems in that an expensive ultrasound diagnostic apparatus is required. On the other hand, it is known to evaluate the pregnancy status (fetal growth) of a pregnant horse by examining the estradiol concentration in the blood of the pregnant horse. However, methods for measuring estradiol include gas chromatography, chemiluminescence immunoassay, and the like, which have the problems of complicated operations and taking a lot of time.

Further, Patent Documents 1 and 2 disclose techniques for measuring urinary estradiol using an immunochromatographic strip.

Journal of Japan Veterinary Medical Association, 2008, 62: 630-635 Hippophile, 2012, 49:34-41

Japanese Patent Publication No. 08-500670 JP-A-10-115614

DISCLOSURE OF THE INVENTION The present invention has been devised to solve the above problems. The purpose is to provide a diagnostic kit.

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 estradiol in a biological sample, comprising a specimen diluent for diluting the biological sample and an immunochromatographic strip, the immunochromatographic strip comprising a sample addition member, a membrane carrier, and an absorbing member are connected in order.
(2) The assay kit according to (1), wherein the sample diluent contains a competitive reagent, an antibody against the competitive reagent, and/or a labeled substance in which a labeling substance is bound to a high-affinity substance.
(3) The assay kit according to (2), wherein the competitive reagent contains a complex of estradiol and a compound.
(4) The measurement kit according to (3), wherein the compound is biotin.
(5) The assay kit according to any one of (2) to (4), wherein the antibody against the competitive reagent is an anti-biotin antibody.
(6) The assay kit according to any one of (2) 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 membrane carrier has a test line on which an anti-estradiol antibody is immobilized.
(8) The measurement kit according to any one of (1) to (7), wherein the biological sample is whole blood, plasma or serum.
(9) The measurement kit according to any one of (1) to (8), which is used to determine the pregnancy status of a horse.

INDUSTRIAL APPLICABILITY According to the present invention, an immunochromatographic measurement kit for estradiol quantification is provided, which enables rapid, simple, and inexpensive measurement of estradiol concentration in a biological sample (blood) at a horse production site.

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 the estradiol 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 electrochemiluminescence immunoassay.

The present invention will be described in detail below.

The present invention is an immunochromatographic measurement kit for quantifying estradiol in a biological sample, comprising a sample diluent for diluting the biological sample and an immunochromatographic strip for quantifying estradiol, wherein the immunochromatographic strip is The measurement kit is characterized in that a sample addition member, a membrane carrier, and an absorption member are arranged in series.

(biological sample)
In the present invention, the biological sample is not particularly limited, but blood (which may be whole blood, serum, or plasma) and the like are suitable. As for animal species, the blood of humans, bovines, dogs, cats, etc. can be measured as well as horses.

(Estradiol)
Estrogen is a representative female sex steroid hormone and acts by binding to intracytoplasmic receptors in target organs. Various types of estrogen have been identified, but the three main types are estrone, estradiol, and estriol. Among these, estradiol has the highest physiological activity and is a particularly important hormone. Estradiol is produced primarily by the ovaries. It is also believed to be secreted in large amounts from the fetus and placenta during pregnancy, and is believed to have an important effect on the maintenance of pregnancy and the development of the fetus.

(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 sample diluent preferably contains a nonionic surfactant that improves the spreadability of the biological sample on the immunochromatographic strip and does not affect the immune reaction. 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 specimen 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 to 4% by mass and 1% by mass to 3% by mass. 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 estradiol in a biological sample and can be detected by a label (substance). A complex of estradiol and a compound is preferably used as the competitive reagent. Forming a complex is preferable because estradiol 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 estradiol-low-molecular-weight biotin complex is more likely to compete with estradiol in the biological sample than the estradiol-high-molecular-weight substance complex such as albumin. are doing. A complex of estradiol and a low-molecular-weight substance also has the advantage of being able to keep production costs lower than a complex of estradiol and a protein (high-molecular-weight substance) such as bovine serum albumin.

Estradiol complexed with biotin is an unstable compound that easily undergoes isomerization of the double bond by light or 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, the sample diluent contains a competitive reagent. Incorporation of a competitive reagent into the specimen diluent is preferable because it simplifies on-site operations. Since the competitive reagent must be stored at a low temperature as described above, the specimen diluent containing the competitive reagent is stored at the above temperature. By adding 0.1 pg or more and 10 ng or less of the competitive reagent to 1 mL of the biological sample, the results of the competitive reaction can be obtained with high accuracy. If the difference between the estradiol 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. In addition, since the competitive reagent is unstable as described above, the sample dilution solution to which the competitive reagent is added is stored at 4 ° C. or less, more preferably -20 ° C. or less, more preferably -80 ° C. or less until use. is preferred.

(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, enzyme labeling substances, and the like. Coloring 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, polyvinyl toluene, and silica. Examples of fluorescent dyes include fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives, and the like. 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 nanobeads (NanoAct (registered trademark)) manufactured by Asahi Kasei Corporation. 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 greater the amount of protein bound to one detection particle, and the higher the performance such as sensitivity.

In the present invention, the antibody against the competitive reagent and/or the labeling substance bound to the high-affinity substance are preferably mixed in advance with the specimen diluent.

(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. On the other hand, if the dilution factor is too high, the amount of estradiol 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 7 shown in FIGS. 1 and 2, 1 is an adhesive sheet, 2 is a membrane carrier, 3 is a test line, 4 is a control line, 5 is a sample addition member, and 6 is an absorption member. The membrane carrier 2 consists of an elongated strip-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 2, a competitive reagent (estradiol and compound A test line 3 is formed (an anti-estradiol antibody is linearly immobilized) for competitively capturing the complex) and estradiol in the biological sample. Further, a control line 4 (an antibody that specifically binds to the compound in the label is linearly immobilized) 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 2. . 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. Control line 4 is for confirming that immunochromatographic development was performed regardless of the presence or absence of estradiol, which is the substance to be analyzed.

(Sample addition member)
In the present invention, the sample application member 5 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.

(membrane carrier)
In the present invention, a nitrocellulose membrane filter is used as the membrane carrier 2 as long as it is capable of chromatographically developing estradiol contained in the biological sample and immobilizing substances such as antibodies forming the test line 3. , 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 3 may be an anti-estradiol antibody that can specifically bind to estradiol, and may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are preferred from the viewpoint of sex.

(anti-estradiol antibody)
Estradiol (molecular weight 272) is a low-molecular-weight compound and lacks sufficient complexity to normally elicit an immune response. Therefore, in order to induce antibody production in immunized animals, it is necessary to use, as an immunogen, a carrier protein such as ovalbumin that is chemically bound to estradiol. Injection of 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 produced by, for example, immunizing an animal such as a mouse with a conjugate of estradiol and ovalbumin together with an appropriate adjuvant, then fusing the spleen cells of the immunized animal with myeloma cells to obtain only the fused cells. It can be obtained by culturing in a selective medium that allows proliferation, and then selecting the proliferated cells using the estradiol conjugate, for example, using an enzyme-labeled immunoassay.

(control line)
In the present invention, the control line 4 is preferably immobilized with an antibody that specifically binds to the compound in the label. An anti-IgG antibody can be used as the antibody, and specifically, it can be formed by immobilizing an anti-rabbit IgG antibody, an anti-mouse 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 6 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 8 or the like for protection and easy handling (Fig. 3). In this case, for example, a sample dropping part 9 is opened above the sample adding member 5 of the immunochromatographic strip, and a determination part (determination window) 10 is preferably opened above the test line 3 and the control line 4 .

(Immunochromatographic development)
A method for quantifying estradiol according to the present invention will be described. First, a biological sample and a specimen diluent are mixed to prepare a biological sample diluent. The obtained biological sample diluted solution is dropped onto the sample dropping part of the immunochromatographic strip, and is spread on the immunochromatographic strip by utilizing capillary action. Estradiol in the developing biological sample diluent is captured by an anti-estradiol antibody that forms a test line competitively with a competing reagent (estradiol-compound complex). In addition, the label in the biological sample diluent binds to the competing reagent (complex of estradiol and compound) captured by the anti-estradiol antibody. Estradiol in the biological sample can be quantified by measuring the signal (coloration) of the obtained 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 estradiol and the competitive reagent (complex of estradiol and the compound) occurs most effectively, and changes in the measured values in response to changes in the concentration of estradiol are obtained at the test line, This is preferable because the difference in estradiol concentration is accurately reflected in the measured value. If it is less than 5 minutes, the reaction between the anti-estradiol antibody in the test line and the estradiol in the biological sample or the competitive reagent (complex of estradiol and the compound) is insufficient, resulting in a low measurement value or a decrease in the estradiol 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-estradiol antibody increases, so that it may not be possible to obtain an accurate measurement value change according to the estradiol concentration in the biological sample.

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

(Immunochromatographic measurement kit)
The immunochromatographic measurement kit of the present invention includes at least a sample diluent for diluting the sample in addition to the immunochromatographic strips described above, and optionally, an estradiol standard solution for preparing a calibration curve, a biological sample dilution Including containers for preparing liquids. 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 (estradiol-biotin complex) was prepared by biotinylating estradiol (Nacalai Tesque, 14541-61) with Biotin-hydrazide (Dojindo Laboratories, B303). After preparation, it was stored at -30°C until use.

(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, after performing a washing operation, the product was suspended in PBS containing 1% by mass of sucrose (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), competitive reagent, label, Triton X-100 (Sigma-Aldrich, 10789704001), polyethylene glycol, and NaCl concentrations of 20 ng/ mL, 0.003% by mass, 0.15% by mass, 2.0% by mass, and 1.5% by mass were added and dissolved to prepare specimen diluents.

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

(Preparation of membrane carrier)
After adjusting the concentration of the anti-estradiol antibody prepared above to 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-rabbit IgG antibody (MyBiosource. Inc., MBS539780) was prepared at a concentration of 1 mg/mL, and then linearly applied to the nitrocellulose membrane filter at an amount of 1.0 μL/cm to form a control line. 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)
The prepared membrane carrier, sample addition member, and absorption member were placed on the adhesive sheet to prepare an immunochromatographic strip.

(Preparation of estradiol standard solution)
The concentration of estradiol in serum obtained by collecting blood from a pregnant horse was measured by a chemiluminescence immunoassay method, and the value obtained was used as an estradiol standard solution. The measurement by the chemiluminescence immunoassay method was performed at the Clinical Laboratory Center (Kinki Preventive Medical Research Institute Co., Ltd.).

(quantitation using a measurement kit)
An estradiol (biological sample) diluent having each concentration shown in Table 1 was prepared by adding the specimen diluent to the above standard solution. 100 μL of the obtained estradiol diluted solution was dropped on the sample dropping part of the immunochromatographic strip and developed. 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 estradiol can be quantified with high accuracy by using the measurement kit of the present invention.

(Comparison experiment with chemiluminescence immunoassay measurement)
Serum estradiol concentrations obtained from 12 female horses were measured using the chemiluminescent immunoassay method and the immunochromatographic assay kit of the present invention, respectively. The measurement by the chemiluminescence immunoassay method was performed at the Clinical Laboratory Center (Kinki Preventive Medical Research Institute Co., Ltd.). In the immunochromatographic method, the estradiol concentration of each serum 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 chemiluminescence immunoassay method was 0.96, indicating a good correlation. It was confirmed that estradiol can be quantified with high accuracy by using the measurement kit of the present invention.

INDUSTRIAL APPLICABILITY According to the present invention, blood estradiol concentration can be measured quickly, easily, and inexpensively at a horse production site, so that the pregnancy status can be grasped and efficient horse production can be achieved. .

1 Adhesive sheet 2 Membrane carrier 3 Test line 4 Control line 5 Sample addition member 6 Absorbing member 7 Immunochromato strip 8 Housing case 9 Sample dropping unit 10 Determination unit (determination window)

Claims (9)

An immunochromatographic measurement kit for quantifying estradiol in a biological sample, comprising a specimen diluent for diluting the biological sample and an immunochromatographic strip, wherein the immunochromatographic strip includes a sample addition member, a membrane carrier, and an absorbent member. A measurement kit characterized by being arranged in sequence. 2. The assay kit according to claim 1, wherein the sample diluent contains a competitive reagent, an antibody against the competitive reagent, and/or a labeled substance in which a labeling substance is bound to a high-affinity substance. 3. The assay kit according to claim 2, wherein the competitive reagent contains a complex of estradiol and a compound. 4. The measurement kit according to claim 3, wherein said compound is biotin. The measurement kit according to any one of claims 2 to 4, wherein the antibody against the competitive reagent is an anti-biotin antibody. The measurement kit according to any one of claims 2 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 membrane carrier comprises a test line on which an anti-estradiol antibody is immobilized. The measurement kit according to any one of claims 1 to 7, wherein the biological sample is whole blood, plasma or serum. 9. The measurement kit according to any one of claims 1 to 8, which is used for determining the pregnancy status of a horse.

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