JP2024031479A - Immunochromatography kit for thyroxine determination - Google Patents

Abstract

[Problem] The present invention provides an immunochromatographic measurement kit that can quickly, easily, and inexpensively measure the concentration of thyroxine in biological samples such as blood at breeding sites for breeding pigs. SOLUTION: The present invention is an immunochromatographic measurement kit for quantifying thyroxine in a biological sample, which includes a specimen diluent for diluting the biological sample, a competitive reagent, and an immunochromatographic strip, the immunochromatographic strip being , a measurement kit characterized in that a sample addition member, an impregnating member, a membrane carrier, and an absorption member are arranged in series, and the membrane carrier is provided with a test line on which an anti-IgG antibody is immobilized. [Selection diagram] None

Description

The present invention relates to an immunochromatography kit for quantifying thyroxine in biological samples. More specifically, the present invention relates to a measurement kit for quantifying thyroxine in a biological sample, which includes a specimen diluent for diluting a biological sample (specimen), a competitive reagent, and an immunochromatographic strip.

In recent years, pig farming operations have become larger and more corporate, and artificial insemination has become quite popular. Under these circumstances, it is becoming increasingly important to diagnose the pregnancy/non-pregnancy of breeding sows early after mating or artificial insemination, and to manage the health of breeding pigs based on an understanding of their farrowing cycles. In pigs, the concentration of thyroxine in the blood is closely related to pregnancy and parturition, and it has been reported that the concentration decreases significantly from late pregnancy to parturition, and then increases sharply from the late lactation period to the end of the weaning period (non-swine). Patent Document 1). For this reason, measuring the thyroxine concentration in the blood is used as an indicator to understand the ovarian activity of breeding pigs.

However, the conventional ELISA method is complicated to operate and takes a long time to produce results, so there is a need for a technology that can easily and quickly measure blood thyroxine concentration in breeding pig breeding sites. ing.

Patent Document 1 discloses a technique related to a non-competitive immunoassay using a novel anti-thyroxine antibody.

Acta. Vet. Scand. 22:369-381, 1981

Japanese Patent Application Publication No. 2010-178649

The present invention was devised to solve the above problems, and an object of the present invention is to provide a measurement kit that can quickly measure the concentration of thyroxine present in a biological sample such as blood.

As a result of intensive studies to solve the above problems, the inventors of the present invention discovered that the above problems could be solved by the means shown below, and arrived at the present invention.

That is, the present invention consists of the following configuration.
(1) An immunochromatographic measurement kit for quantifying thyroxine in a biological sample, comprising a specimen diluent for diluting the biological sample, a competitive reagent, and an immunochromatographic strip,
The immunochromatographic strip comprises a sample addition member, an impregnating member, a membrane carrier, and an absorbing member arranged in sequence,
The membrane carrier is equipped with a test line on which an anti-IgG antibody is immobilized.
A measurement kit characterized by:
(2) The measurement kit according to (1), wherein 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.
(3) The measurement kit according to (1) or (2), wherein the competitive reagent is a complex of thyroxine and a compound.
(4) The measurement kit according to (3), wherein the compound is biotin.
(5) The impregnated member according to (1) or (2) is characterized in that the impregnated member is impregnated with an antibody against a competitive reagent and/or a label in which a labeling substance is bound to a high affinity substance, and an anti-thyroxine antibody. Measurement kit as described.
(6) The measurement kit according to (5), wherein the antibody against the competitive reagent is an anti-biotin antibody.
(7) The measurement kit according to (5), wherein the substance with high affinity for the competitive reagent is avidin and/or streptavidin.
(8) The measurement kit according to (1) or (2), wherein the biological sample is blood, plasma, serum, saliva, or urine.

The present invention provides an immunochromatographic measurement kit for quantifying thyroxine that can quickly, easily, and inexpensively measure the concentration of thyroxine in biological samples at breeding sites for breeding pigs.

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

The present invention will be explained in detail below.

The present invention is an immunochromatography kit for quantifying thyroxine in a biological sample, which includes a specimen diluent for diluting the biological sample, a competitive reagent, and an immunochromatography strip, the immunochromatography strip comprising a sample addition member. , a measurement kit characterized in that an impregnating member, a membrane carrier, and an absorbing member are arranged in series.

(biological sample)
In the present invention, biological samples are not particularly limited, but blood (whole blood, serum, or plasma may be used), saliva, urine, etc. are suitable. In addition to pigs, animal species such as humans, cows, horses, dogs, and cats can also be measured.

(thyroxine)
Thyroxine is a type of thyroid hormone secreted from the follicles of the thyroid gland, and is a modified amino acid that is also a precursor of triiodothyronine, which is also a thyroid hormone. Most of thyroxine is transported in the blood in a state bound to proteins such as thyroxine-binding protein and albumin. Its lifespan in the blood is approximately one week. In addition, thyroxine has been shown to be involved in the control of metabolic rate and to affect growth.

(Sample diluent)
In the present invention, any kind of buffer may be used as the sample diluent as long as it has sufficient buffering capacity in a predetermined pH range, such as Tris, phosphoric acid, phthalic acid, citric acid, maleic acid, etc. Acid, succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, Good buffer (MES, ADA, PIPES, ACES, choramine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine), etc. It will be done.

In the present invention, the specimen diluent preferably contains a nonionic surfactant that improves the spreadability of the biological sample on the immunochromato strip and does not affect the immune reaction. Examples of nonionic surfactants include polyoxyethylene alkyl phenyl ethers (Triton (registered trademark) surfactants, etc.), polyoxyethylene alkyl ethers (Brij (registered trademark) surfactants, etc.), and polyoxyethylene sorbitan. Examples include 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. Further, the surfactants may be used alone or in combination of two or more. Furthermore, the concentration of the surfactant in the specimen diluent is preferably 0.01 mass% or more and 0.2 mass% or less, and 0.05 mass% or more and 0.2 mass% or less, although it depends on the dilution ratio of the biological sample. is more preferable, and even more preferably 0.05% by mass or more and 0.15% by mass or less. If the concentration is too low, the biological sample diluent may be difficult to develop, and if the concentration is too high, the signal on the test line may be low.

Further, the specimen diluent preferably contains polyethylene glycol and/or sodium chloride in order to improve efficiency, promotion, and specificity of competitive reaction. The concentration of polyethylene glycol and sodium chloride to be added to the specimen dilution solution should be such that the final concentration in the biological sample dilution solution is 1% by mass or more and 4% by mass or less, and 1% by mass or more and 3% by mass or less, respectively. preferable. The number average molecular weight of the polyethylene glycol used is preferably 2,000 or more and 16,000 or less, more preferably 5,000 or more and 10,000 or less. If the number average molecular weight is small, a sufficient antigen-antibody reaction promoting effect suitable for the present invention may not be obtained. Furthermore, if the number average molecular weight is large, the promoting effect of the antigen-antibody reaction may not be obtained, or the viscosity of the biological sample dilution solution may become high, resulting in a decrease in spreadability 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 thyroxine in a biological sample and can bind to an anti-thyroxine antibody. As the competitive reagent, it is preferable to use a complex of thyroxine and a compound. Formation into a complex is preferable because thyroxine is stabilized and antibodies with good performance are easily available. Examples of the compound include bovine serum albumin, ovalbumin, and biotin, among which biotin is preferably used. Although the detailed reason is unknown, according to the inventor's study, the complex of thyroxine and biotin, which has a low molecular weight, is more effective against thyroxine in biological samples than the complex of thyroxine and a high molecular weight substance such as albumin. There is a tendency for the competition principle to work more easily. Further, a complex of thyroxine and a low molecular weight substance has the advantage that manufacturing costs can be kept lower than a complex of thyroxine and a protein (high molecular weight substance) such as bovine serum albumin.

In addition, thyroxine that forms a complex with biotin is an unstable compound, and its double bonds are easily isomerized by light and heat, and it also easily reacts with acids, air, and metal ions, so it is easily decomposed. There is a fear that it will end. 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 that 0.01 ng or more and 10 ng or less of the competitive reagent be added to 1 mL of the biological sample. If the difference between the thyroxine concentration in the biological sample and the amount of competitive reagent added is too large, competition may not be successful and quantitative performance may deteriorate. The competitive reagent may be freeze-dried or in the form of a solution.

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

The labeling substance is not particularly limited, and examples thereof include coloring (including fluorescence) labeling substances, enzyme labeling substances, etc., but coloring labeling substances are preferable because test results can be obtained quickly. Examples of the coloring labeling substance include colloidal metals, colored latex particles, and colored cellulose fine particles. Typical examples of colloidal metals include platinum colloid, gold colloid, silver colloid, palladium colloid, gold nanorods, gold nanoplates, silver nanoplates, and the like. The size of the colloidal metal particles is usually 3 nm or more and 100 nm or less in diameter. Typical examples of colored latex include polystyrene latex colored with red and blue pigments, polymethyl methacrylate, acrylic acid polymer, and the like. The particle size of the latex particles is not particularly limited, but preferably has a particle size of 25 nm or more and 500 nm or less. In addition, commercially available colored cellulose fine particles can also be used. Although the particle size of the colored cellulose fine 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 those made of materials such as polystyrene, polymethyl methacrylate, polyvinyltoluene, and silica, and examples of fluorescent dyes include fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives, etc. can do.

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

In the present invention, the surface of the labeling substance may be treated with a blocking agent in advance in order to suppress non-specific binding to the surface. It is preferable to use polyethylene glycol or protein as the blocking agent. Preferred proteins include Blocking Peptide Fragment (BPF, manufactured by Toyobo Co., Ltd.), bovine serum albumin (BSA), casein, and the like. If these blocking agents are commercially available, they may be used, or they may be separately produced by a known method. The molecular size is also not particularly limited, but the average molecular weight is preferably 100 kDa or less. Generally, 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.

(Immunochromato strip)
A specific example of the immunochromato strip is an immunochromato strip 8 as shown in FIGS. 1 and 2. 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 is made of a long and narrow 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. The membrane carrier 3 has a film for capturing an anti-thyroxine antibody located at a distance of 3 mm or more and 15 mm or less from the end of the left side (upstream side) in FIGS. A test line 4 is formed (an anti-IgG antibody is fixed in a linear manner). Furthermore, a control line 5 is formed at a position of 8 mm or more and 25 mm or less from the upstream end of the membrane carrier 3 toward the downstream side (an antibody that specifically binds the compound in the label is immobilized in a linear manner). . Note that the test line is preferably placed 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. Control line 5 is for confirming that immunochromatography was performed regardless of the presence or absence of thyroxine, which is the substance to be analyzed.

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

(Impregnated member)
In the present invention, the impregnated member 2 is made of glass fiber in the form of a band 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 also be used. can. The impregnated member 2 is obtained by impregnating a member such as glass fiber with a suspension containing an antibody against a competitive reagent and/or a labeled substance bound to a high affinity substance, and an anti-thyroxine antibody, and drying the suspension. It can be made.

(membrane carrier)
In the present invention, a nitrocellulose membrane filter is used as the membrane carrier 3, but as long as it is capable of chromatographic development of thyroxine contained in a biological sample and capable of immobilizing a substance such as an antibody forming the test line 4. , any other material may be used, and other cellulose films, nylon films, glass fiber films, etc. can also be used.

(test line)
In the present invention, the antibody immobilized on the test line may be an antibody that can specifically bind to mouse IgG, rabbit IgG, or guinea pig IgG, that is, an anti-mouse IgG antibody, an anti-rabbit IgG antibody, or an anti-guinea pig IgG antibody. The antibody may be a polyclonal antibody or a monoclonal antibody, but from the viewpoint of reaction specificity, a monoclonal antibody is preferable. It is the anti-thyroxine antibody (mouse IgG) added to the biological sample diluent that directly binds to the antibody immobilized on the test line, and the amount of this binding reflects the amount of thyroxine present in the sample. Therefore, the reaction specificity of the immobilized antibody is very important.

(Anti-thyroxine antibody)
Thyroxine (molecular weight 777) is a low-molecular compound and does not have sufficient complexity to normally elicit an immune response. Therefore, in order to cause an immunized animal to produce antibodies, it is necessary to use as an immunogen a chemically bound thyroxine to a carrier protein such as ovalbumin. In addition, injecting the 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 antiserum obtained by immunizing rabbits, mice, etc. Monoclonal antibody producing cells can be obtained by, for example, immunizing an animal such as a mouse with a conjugate of thyroxine and ovalbumin together with an appropriate adjuvant, then fusing the immunized animal's splenocytes and myeloma cells, and producing only the fused cells. It can be obtained by culturing in a selective medium that allows proliferation, and selecting the proliferated cells using a conjugate with thyroxine, for example, by enzyme-labeled immunoassay.

(control line)
In the present invention, it is preferable that an antibody that specifically binds to the anti-biotin antibody in the label is immobilized on the control line. As the antibody, an anti-IgG antibody can be used. For example, if the anti-biotin antibody is derived from goat, it can be formed by immobilizing the anti-goat IgG antibody on a membrane carrier. In this case, it is only necessary that the anti-goat antibody and the anti-biotin antibody (goat IgG) specifically bind to each other and the coloration of the line can be confirmed. By using the control line, it can be confirmed that the labeled substance has migrated to the most downstream part of the membrane carrier, that is, that the immunochromatographic development has been carried out (normally).

(absorbing member)
In the present invention, the absorbent member 7 may be made of any material as long as it can absorb and retain liquid quickly, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., but especially filter paper. is optimal.

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

(Immunochromatographic development)
The method for quantifying thyroxine of the present invention will be explained. First, a biological sample diluted solution is prepared by mixing a biological sample, a competitive reagent, and a sample diluted solution. The obtained biological sample diluted solution is dropped onto the sample dropping site of the immunochromato strip and spread on the immunochromato strip using capillary action. The developed biological sample diluted solution elutes the label and the anti-thyroxine antibody when passing through the impregnation member. Thyroxine in the biological sample diluted solution is competitively captured by the anti-thyroxine antibody with a competitive reagent (a complex of thyroxine and a compound) during development. The labeled substance binds to the competitive reagent (complex of thyroxine and compound) captured by the anti-thyroxine antibody, and this conjugate is further captured by the test line anti-mouse IgG antibody and exhibits a signal (coloration). . Then, by measuring the signal (coloration) of this test line, thyroxine in the biological sample can be quantified. Note that 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 time, the competitive reaction between thyroxine and the competitive reagent (complex of thyroxine and the compound) will occur most effectively, and a measurement value corresponding to the concentration of thyroxine will be obtained at the test line, and the thyroxine concentration will be measured. This is preferable because it is accurately reflected in the value. That is, the thyroxine concentration in a biological sample can be accurately measured. If the time is less than 5 minutes, the reaction between the anti-thyroxine antibody in the test line and the thyroxine in the biological sample or the competitive reagent (complex of thyroxine and a compound) may not be sufficient, resulting in a low measured value or the concentration of thyroxine in the biological sample. It may not be possible to obtain accurate measurements. Furthermore, if the time exceeds 12 minutes, non-specific reactions to anti-thyroxine antibodies increase, so that accurate measurement values depending on the thyroxine concentration in the biological sample may not be obtained.

(Immunochromatographic measurement kit)
In addition to the immunochromatographic strip described above, the immunochromatographic measurement kit of the present invention includes at least a specimen diluent for diluting the specimen and a competitive reagent, and if necessary, a thyroxine standard solution for creating a calibration curve, Including containers for preparing sample dilution solution. It may also include a measuring device (such as a chromatoreader) for measuring immunochromatography results.

(dilution ratio)
In the present invention, it is appropriate that the dilution ratio of the biological sample is 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. Furthermore, if the dilution ratio is too high, the amount of thyroxine in the biological sample will decrease, which may reduce measurement accuracy.

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

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. Note that the characteristic values obtained in the examples were measured according to the following method.

(Preparation of competitive reagent)
A competitive reagent (thyroxine-biotin complex) was prepared by biotinylating thyroxine (Nacalai Tesque, 33917-74) using Biotin-hydrazide (Dojindo Chemical, B303). After preparation, it was stored at -30°C until use.

(Preparation of sample dilution solution)
Dissolve Triton pH 7.4) was prepared.

(Preparation of impregnated member)
A band-shaped glass fiber nonwoven fabric measuring 8 mm x 150 mm was impregnated with 0.5 mL of the control reagent (anti-biotin antibody-bound cellulose particle liquid) obtained above. After drying at room temperature, it was cut into a size of 8 mm x 5 mm to obtain an impregnated member.

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

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

(Preparation of test line and control line)
After adjusting the anti-mouse IgG antibody to a concentration of 1 mg/mL, it was linearly applied to a 25 mm x 300 mm nitrocellulose membrane filter in an amount of 1.0 μL/cm to create a test line.
Next, an anti-goat IgG antibody (Donkey Anti-Goat IgG, Abcam, AB182021) was prepared at a concentration of 1 mg/mL, and then linearly applied to the nitrocellulose membrane filter in an amount of 1.0 μL/cm. A control line was created.
After preparing a test line and a control line, they were dried at 50° C. for 30 minutes and cut into a size of 25 mm×5 mm to form a membrane carrier.

(Preparation of immunochromato strip)
In addition to the prepared membrane carrier and impregnation member, a sample addition member and an absorption member were placed on the adhesive sheet to prepare an immunochromatography strip.

(Preparation of thyroxine standard solution)
Thyroxine (Nacalai Tesque, 33917-74) was added to PBS (phosphate buffered saline) to prepare an estrone standard solution.

(Quantification using immunochromatography kit)
A specimen diluent was added to the above standard solution to prepare thyroxine diluted solutions (biological sample diluted solutions) having various concentrations shown in Table 1. A competitive reagent was added to each of the obtained thyroxine dilutions so that the amount of thyroxine-biotin complex was 300 pg, and then 100 μL was dropped onto the sample dropping portion of the immunochromato strip and allowed to develop. Ten minutes after dropping, the color development (absorbance) of the test line was measured using an immunochromatoreader (C10060-10 manufactured by Hamamatsu Photonics, measurement mode: blue line measurement mode). The results are shown in Table 1 and FIG. 4. It was confirmed that thyroxine can be quantified with high accuracy by using the measurement kit of the present invention.

(Comparative experiment with measurement by ELISA method The thyroxine concentration in the blood collected from 12 sows was measured using the ELISA method and the immunochromatography measurement kit of the present invention, respectively. For the measurement by the ELISA method, DetectX Thyroxine (T4 ) Measured using the Enzyme Immunoassay Kit (Arbor Assays LLC). In the immunochromatography method, the thyroxine concentration in each blood sample was calculated using the standard curve obtained by simultaneously measuring the standard solution, and the measurement results are shown in Table 2. and shown in Figure 5.The correlation coefficient between the measured values of the immunochromatography method and the measured values of the ELISA method was 0.96, indicating a good correlation.By using the measurement kit of the present invention, thyroxine can be accurately measured. It was confirmed that it could be quantified well.

According to the present invention, the concentration of thyroxine in biological samples such as blood can be measured quickly, easily, and inexpensively at the site where breeding pigs are raised, so pregnancy status can be determined and pig production can be carried out efficiently. becomes possible.

1 Adhesive sheet 2 Impregnation member 3 Membrane carrier 4 Test line 5 Control line 6 Sample addition member 7 Absorption member 8 Immunochromatography strip 9 Housing case 10 Sample dropping part 11 Judgment part (judgment window)

Claims (8)

An immunochromatographic measurement kit for quantifying thyroxine in a biological sample, comprising a specimen diluent for diluting the biological sample, a competitive reagent, and an immunochromatographic strip,
The immunochromatographic strip comprises a sample addition member, an impregnating member, a membrane carrier, and an absorbing member arranged in sequence,
The membrane carrier is equipped with a test line on which an anti-IgG antibody is immobilized.
A measurement kit characterized by: The measurement kit according to claim 1, wherein 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. The measurement kit according to claim 1 or 2, wherein the competitive reagent is a complex of thyroxine and a compound. The measurement kit according to claim 3, wherein the compound is biotin. 3. The measurement kit according to claim 1, wherein the impregnated member is impregnated with an antibody against a competitive reagent and/or a label in which a labeling substance is bound to a high affinity substance, and an anti-thyroxine antibody. The measurement kit according to claim 5, wherein the antibody against the competitive reagent is an anti-biotin antibody. The measurement kit according to claim 5, wherein the substance with high affinity for the competitive reagent is avidin and/or streptavidin. The measurement kit according to claim 1 or 2, wherein the biological sample is blood, plasma, serum, saliva, or urine.