D-thyroxine specific measurement method

Abstract

To provide a method that specifically and highly sensitively measures D-T4 concentration in a short time in an immunoassay.SOLUTION: A D-T4 measurement method is configured to: react D-T4 in a sample with a molecule (T4 conjugate molecule) capable of conjugating to the T4; generate a complex of the D-T4 and T4 conjugate molecule; then, react a molecule (a capture molecule) capable of conjugating to the complex; and detect a conjugate of the complex and the capture molecule, in which the T4 conjugate molecule is an anti T4 antibody capable of conjugating to any of the D-T4 and an L-thyroxine (L-T4), and the capture molecule is an anti-immunocomplex antibody specifically conjugating to the complex.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to a method for specifically measuring D-thyroxine (hereinafter referred to as "D-T4"). [Background technology]

[0002] Methods for detecting specific substances using antibodies (hereafter referred to as "immunoassays") have been well studied, and many methods have been reported. However, if we focus on the core of the technology, we can classify them into several types. Among them, the most representative methods are the sandwich method and the competitive method. The sandwich method is a method in which an antigen is captured and detected by sandwiching it between two types of antibodies. In this method, first, one antibody (primary antibody) is immobilized on a carrier, and the antigen is immobilized on the carrier by the action of this primary antibody. Next, the other antibody (secondary antibody) labeled with an enzyme or fluorescent dye is added. This labeled secondary antibody captures and binds to the antigen immobilized on the carrier. The antigen is then quantified by detecting the signal emitted by this labeled secondary antibody (Figure 1). On the other hand, the competitive method is a method for detecting antigens using one type of antibody. In this method, the antibody is immobilized on a carrier, and a sample containing the antigen and an antigen labeled with an enzyme or fluorescent dye (labeled antigen) are added. If the amount of antigen in the sample increases, the amount of labeled antigen that binds to the antibody decreases, and the signal becomes weaker. The intensity of the signal emitted from the labeled antigen when a sample containing no antigen is added is set to 100%, and the intensity of the signal emitted from the labeled antigen when a sample containing the antigen is added is calculated as a relative value to this, and the amount of antigen in the sample is calculated (Figure 2).

[0003] Both the sandwich method and the competitive method can quantitatively detect antigens. However, due to the difference in the measurement principle, each method has advantages and disadvantages. In the sandwich method, an excess amount of primary and secondary antibodies can be used, so antigens can be detected with high sensitivity. In addition, in this method, antigens are recognized by two types of antibodies, so very high reaction specificity can be achieved. The advantage of the sandwich method is that it exhibits excellent performance in both sensitivity and reaction specificity. On the other hand, this method is a measurement method that is difficult to apply to all antigens. In the sandwich method, two types of antibodies need to bind to the antigen simultaneously. Therefore, antigens that can be applied to this method are limited to those with relatively large molecular weights that have two or more interaction sites with the antibody. This restriction is a disadvantage of the sandwich method. In contrast, the competitive method requires only one type of antibody. Therefore, even low molecular weight compounds with few sites that interact with the antibody (Non-Patent Document 1) can be detected. In addition, since the experimental operation is one step less than the sandwich method, it is also possible to shorten the measurement time. However, the competitive method cannot use an excess amount of antibody like the sandwich method. Therefore, when comparing using antibodies with the same performance, the sensitivity is inferior to the sandwich method. Also, since only one type of antibody is involved in the reaction, the specificity is also inferior to the sandwich method.

[0004] Thyroxine (hereinafter referred to as "T4") is a type of hormone secreted by the thyroid gland. In the body, T4 plays an important role in sugar metabolism and protein synthesis (Non-Patent Document 2). It is also known that when abnormalities occur in the thyroid gland due to Graves' disease, thyroiditis, Hashimoto's disease, etc., the hormone balance is disrupted, resulting in various systemic symptoms such as palpitations, weight loss, finger tremors, sweating, loose stools, and muscle weakness. For this reason, T4 is widely used as an essential diagnostic marker for checking thyroid function.

[0005] Although the blood concentration of T4 is very important clinically, it is not easy to construct a measurement system for it. In particular, the blood concentration of T4 is very low, so it is necessary to construct a sensitive measurement system to detect it. Furthermore, T4 has an asymmetric center inside, so there are optical isomers of L-thyroxine (hereinafter referred to as "L-T4") and D-T4. The structure of D-T4 is shown in the following formula (I).

[0006] [ka]

[0007] It is known that these optical isomers have different functions in the body (Non-Patent Document 2). Therefore, in order to perform accurate clinical diagnosis, it is desirable to distinguish between L-T4 and D-T4. However, since T4 has a small molecular weight, it is measured by a competitive method in conventional immunoassays. However, as mentioned above, the competitive method does not provide high reaction specificity, making it difficult to distinguish between L-T4 and D-T4.

[0008] Methods such as HPLC have been reported as methods for distinguishing and measuring L-T4 and D-T4, but these methods are far inferior to immunoassays in terms of sensitivity and measurement speed, which has been a major issue until now (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Biochemistry, 82(8), 710:2010 [Non-Patent Document 2] Journal of Chromatography A, 1217 (2010) 4255-4266 [Non-Patent Document 3] Pharm Res. 2005 Apr;22(4):667-75.2005 Apr 7. Summary of the Invention [Problem to be solved by the invention]

[0010] The problem to be solved by the present invention is to measure the concentration of D-T4 specifically, sensitively and in a short period of time in an immunoassay. [Means for solving the problem]

[0011] The present invention, made in view of the above problems, includes the following aspects. (1) D-thyroxine (D-T4) in the sample and - Molecules that can bind to thyroxine (T4) (T4-binding molecules) to generate a "complex of D-T4 and T4 binding molecule", Next, a molecule capable of binding to the complex (a capture molecule) is reacted with the complex, A method for detecting binding between the complex and a capture molecule, comprising the steps of: -T4 binding molecules specifically bind to D-T4 and / or The capture molecule specifically binds to the complex. A method for measuring D-T4, comprising: (2) The measurement method described in (1), wherein the T4 binding molecule is a molecule capable of binding to both D-T4 and L-thyroxine (L-T4), and the capture molecule is a molecule that specifically binds to the complex. (3) The method according to (1) or (2), wherein the T4 binding molecule is an anti-T4 antibody. (4) The method according to any one of (1) to (3), wherein the capturing molecule is an anti-immunocomplex antibody. (5) The method for measuring D-T4 according to any one of (1) to (4), wherein D-T4 is measured by ELISA. (6) The method for measuring D-T4 according to any one of (1) to (5), wherein the cross-reactivity rate with compounds other than D-T4 is 5% or less. (7) The method for measuring the amount of the compound other than D-T4 described in (6), wherein the compound other than D-T4 is L-T4.

[0012] The present invention will be described in detail below.

[0013] The D-T4 to be measured in the present invention has various names such as D-thyroxine, D-thyroxine, and dextrothyroxine, but the name is not particularly limited as long as it has the structure shown in chemical formula (I), and any compound may have any name. In addition, the compound to be measured in the present invention is not particularly limited in type or structure as long as it can have the structure of D-T4 in an aqueous solution, and may be a chloride of these compounds.

[0014] Furthermore, the sample of the present invention is not particularly limited in type as long as it contains D-T4, and can be, for example, human blood (whole blood, plasma, serum, etc.), urine, sweat, saliva, tears, and other bodily fluids, or these bodily fluids diluted with water or a buffer solution.

[0015] The T4 binding molecule used in the present invention may be capable of binding to D-T4, and may be capable of specifically binding to D-T4, or may be capable of binding to both D-T4 and L-T4. The type of the T4 binding molecule is not particularly limited, and aptamers, peptides, receptors, antibodies, etc. can be used, but antibodies are preferred. When the D-T4 binding molecule is an antibody, the subclass is not particularly limited, and IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgD, IgE, etc. can be used. The origin and format of the antibody are not particularly limited, and antibodies derived from mice, rats, rabbits, goats, sheep, camels, fish, birds, etc., and antibodies in various formats such as single-chain antibodies, bispecific antibodies, F(ab), F(ab'), F(ab')2, scF(ab), Fv, scFv, sdAb, etc. can be used.

[0016] Furthermore, the type of the capture molecule used in the present invention is not particularly limited as long as it can bind to the "complex of D-T4 and T4 binding molecule", and aptamers, peptides, antibodies, etc. can be used. When the capture molecule is an antibody, it is preferably an anti-immunocomplex antibody, specifically an antibody against the "complex of D-T4 and anti-T4 antibody". The subclass of the antibody is not particularly limited, and IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgD, IgE, etc. can be used. The origin and format of the antibody are not particularly limited, and antibodies derived from mice, rats, rabbits, goats, sheep, camels, fish, birds, etc., and antibodies in various formats such as single chain antibodies, bispecific antibodies, F(ab), F(ab'), F(ab')2, scF(ab), Fv, scFv, sdAb, etc. can be used.

[0017] In addition, a water-insoluble carrier can be used in the present invention. The type of water-insoluble carrier is not particularly limited as long as it can immobilize D-T4 binding molecules or capture molecules, and resin, glass, polystyrene particles, magnetic particles, beads, plates, etc. can be used. In addition, the method of immobilization to this water-insoluble carrier is not particularly limited as long as it can immobilize T4 binding molecules or capture molecules, and immobilization can be performed by a direct or indirect method (for example, via avidin-biotin binding, etc.).

[0018] Furthermore, the present invention allows the use of a label. The type of label is not limited as long as it can be bound to the T4 binding molecule or the capture molecule, and an enzyme, a radioisotope, a dye, or the like can be used. This label is preferably an enzyme, but the type is not particularly limited, and alkaline phosphatase, horseradish peroxidase, or the like can be used. The method of binding this label to the T4 binding molecule or the capture molecule is not particularly limited, and labeling can be performed by a direct or indirect method (for example, via an avidin-biotin bond, etc.).

[0019] In the present invention, the T4 binding molecule specifically binds to D-T4, and / or The capture molecule specifically binds to the "complex of D-T4 and T4-binding molecule" It is essential that: In particular, it is preferable that the T4-binding molecule is capable of binding to both D-T4 and L-T4, and the capture molecule is one that specifically binds to the complex.

[0020] In the present invention, it is also preferred that the T4 binding molecule is immobilized on a water-insoluble carrier, and the capture molecule is bound to a label.

[0021] In addition, the measurement method used in the present invention is not particularly limited as long as it can quantify D-T4 bound to T4-binding molecules, and manual or fully automated measurement can be used. In addition, even if this measurement method is fully automated, the type of device is not particularly limited, and for example, a fully automated immunoassay device such as that shown in Biological Sample Analysis Vol. 39, No. 4 (2016) can be used, and the following automatic measurement can be performed using a reagent cup as shown in Figure 3.

[0022] A) Prepare a two-cell reagent cup in which one cell contains a water-insoluble carrier on which a T4-binding molecule (anti-T4 antibody) is immobilized, and the other cell contains an enzyme-labeled capture molecule (anti-immunocomplex antibody). B) Place the reagent cup and the test solution in the automated immunoassay device. C) In an automated immunoassay device, the test solution containing D-T4 is added to a cell in the reagent cup containing a water-insoluble carrier, and allowed to react. D) After the reaction is completed, the components that are not adsorbed onto the water-insoluble carrier are washed / removed. E) The enzyme-labeled capture molecules in the reagent cup are transferred to a cell containing a water-insoluble carrier and reacted. F) After the reaction is completed, the components that are not adsorbed onto the water-insoluble carrier are washed / removed. G) A substrate for the enzyme is added and a signal resulting from the product produced by the enzyme reaction is measured.

[0023] According to the present invention, D-T4 can be specifically measured, and cross-reactivity with compounds other than D-T4, such as L-T4, can be measured at 5% or less.

[0024] Furthermore, according to the present invention, D-T4 can be measured with high sensitivity, and can be measured at a D-T4 concentration of 10 pg / ml to 10 ng / ml, preferably 20 pg / ml to 3 ng / ml, and more preferably 40 pg / ml to 1 ng / ml.

[0025] Furthermore, according to the present invention, the process from placing the sample in the device to detecting the complex between the "complex of D-T4 and the T4 binding molecule" and the capture molecule can be completed in a short time, for example, within 15 minutes. If it is necessary to further increase the detection sensitivity, the reaction time can be extended, and for example, the complex between the "complex of D-T4 and the T4 binding molecule" and the capture molecule can be detected within 25 minutes after placing the sample in the device. Effect of the Invention

[0026] According to the present invention, it is possible to quantitatively determine the concentration of D-T4 specifically, sensitively, and in a short period of time in an immunoassay. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is a diagram illustrating the principle of measurement by the sandwich method. [Diagram 2] FIG. 1 is a diagram illustrating the principle of measurement by a competitive method. [Diagram 3] FIG. 2 is a diagram showing an example of a reagent cup that can be used in the present invention. [Figure 4] This is a graph showing the results of measuring D-T4 and L-T4 by the competitive method. [Diagram 5] This is a graph showing the results of measuring D-T4 and L-T4 using the sandwich method. EXAMPLES

[0028] The following examples of the present invention will be described in detail with respect to anti-T4 rabbit monoclonal antibody and anti-immunocomplex mouse monoclonal antibody, but the present invention is not limited thereto. The compositions and pH values ​​of the chemicals used in the examples are as follows: Carbonate buffer (pH 9.6) 12mM Sodium Carbonate 38mM Sodium Bicarbonate Washing buffer (pH 7.4) 1mM Tris(hydroxymethyl)-aminomethane 7.5mM Sodium Chloride 0.05% tween20 Blocking buffer (pH 7.5) 8.1mM disodium hydrogen phosphate dodecahydrate 1.5mM Potassium hydrogen phosphate 137mM Sodium Chloride 2.7mM Potassium Chloride 1% skim milk Incubation buffer (pH 7.5) 8.1mM disodium hydrogen phosphate dodecahydrate 1.5mM Potassium hydrogen phosphate 137mM Sodium Chloride 2.7mM Potassium Chloride 0.1% skim milk ALP buffer (pH9.8) 1M Diethanolamine 0.5mM Magnesium chloride 10mM 4-Methylumbelliferyl phosphate PBS buffer (pH7.4) 137mM Sodium Chloride 8.1mM Disodium hydrogen phosphate 2.68mM Potassium Chloride 1.47mM Potassium dihydrogen phosphate (1) Preparation of immunogen for isolating T4-binding antibodies Bovine serum albumin (BSA) was dissolved in 50 mM borate buffer (pH 8.5). Next, T4 with a linker was dissolved in dimethyl sulfoxide and added to the BSA solution, and reacted at room temperature for 30 minutes. The reaction solution was then placed in a dialysis tube and dialyzed against a PBS solution to remove unreacted T4. After dialysis, the T4-labeled BSA was collected from the dialysis tube and used as an immunogen for isolating T4-binding antibodies.

[0029] (2) Immunization of laboratory animals to isolate T4-binding antibodies Rabbits (female, Japanese white, 12 weeks old) were prepared as immunized animals. A 1:1 mixture of 2mg / ml of immunizing antigen solution for isolating T4-binding antibodies and Freund's complete adjuvant was prepared. This mixture was then sonicated on ice to emulsify. 1ml of this solution was then injected subcutaneously into the back of each rabbit to immunize them.

[0030] One week after the first immunization, the rabbits were immunized again as follows. First, a 1:1 mixture of 2 mg / ml of the immunizing antigen solution for isolating T4-binding antibodies and Freund's incomplete adjuvant was prepared. This mixture was then subjected to ultrasonic waves on ice to emulsify. Then, 1 ml of this solution was injected subcutaneously into the back of each of the rabbits for immunization. After the second immunization, this immunization procedure using Freund's incomplete adjuvant was repeated once a week.

[0031] (3) Cell fusion to isolate T4-binding antibodies After the immunization procedure was completed, the immunized rabbits were euthanized and their spleens were removed. B cells were isolated from the spleens and mixed with myeloma cells, and fused using the PEG method. The fused cells were then collected and suspended in fusion cell medium (E-RDF + 10% bovine serum + 5% BM conditioned + HAT). This cell suspension was plated on a 384-well plate and cultured at 37°C in an environment of 5% CO2.

[0032] (4) Screening of T4-binding antibodies After culturing the fused cells, the reactivity of the antibodies secreted into the culture supernatant with the antigen was examined by ELISA as described below. (A) Anti-rabbit antibody was dissolved in carbonate buffer to a concentration of 0.5 μg / ml, and 50 μl of this solution was dispensed into each well of an ELISA plate (384 wells). The plate was then left to stand at room temperature for 1 hour, and each well was washed three times with washing buffer. (B) 100 μl of blocking buffer was added to each well and incubated at room temperature for 1 hour, after which each well was washed once with washing buffer. (C) 50 μl of incubation buffer was added to each well. (D) 2 μl of culture supernatant was added to each well and then allowed to stand at room temperature for 1 hour. At this time, two sets of identical plates containing culture supernatant were prepared in order to compare the reactivity with T4 by adding two types of solutions in the next step. After that, each well of these plates was washed three times with washing buffer.

[0033] (E) 50 μl of the incubation buffer containing alkaline phosphatase-labeled T4 was added to each well of one plate and incubated at room temperature for 1 h. 50 μl of the incubation buffer containing alkaline phosphatase-labeled T4 and unlabeled T4 was added to each well of the other plate and incubated at room temperature for 1 h. Then, each well of these plates was washed three times with washing buffer. (F) 50 μl of ALP buffer was added to each well and allowed to stand for 30 minutes. (G) Fluorescence intensity was measured (Excitation 360nm / Emission 465nm) to examine wells containing antibodies reactive with the antigen. Through such screening, wells containing antibodies that strongly bind to the antigen were identified. Cells were then collected from these wells.

[0034] (5) Obtaining the gene sequence of T4-binding antibody Using RNeasy Plus Micro kit (QIAGEN), mRNA was purified from the collected cells. Then, using this mRNA as a template, cDNA was synthesized using Sensiscript RT kit (QIAGEN). Next, using this cDNA as a template, the heavy and light chain genes of the antibody were amplified by PCR. The gene sequences of the PCR products were then analyzed, and the full-length sequences of the heavy and light chain genes of the antibody reactive with T4 were determined.

[0035] (6) Expression and purification of T4-binding antibodies The cloned heavy and light chain genes of the T4-binding antibody were transfected into animal cells using Neon (Invitrogen). The cells were then suspended in animal cell culture medium (E-RDF+10% FCS) and cultured at 37°C in an environment of 5% CO2. Three days after transfection, G-418 sulfate was added to the culture medium to a final concentration of 500 μg / ml. Four days after the addition of G-418 sulfate, the cells were suspended in selection medium (E-RDF+10% FCS+500 μg / ml G418 sulfate) and plated on a 384-well plate for limiting dilution. Five days after limiting dilution, wells containing cell lines expressing the antibody were examined by the ELISA method described below.

[0036] (A) Anti-rabbit antibody was dissolved in carbonate buffer to a concentration of 0.5 μg / ml, and 50 μl of this solution was dispensed into each well of an ELISA plate (384 wells). The plate was then left to stand at room temperature for 1 hour, and each well was washed three times with washing buffer. (B) 100 μl of blocking buffer was added to each well and incubated at room temperature for 1 hour, after which each well was washed once with washing buffer. (C) 50 μl of incubation buffer was added to each well. (D) 2 μl of culture supernatant was added to each well and allowed to stand at room temperature for 1 hour, after which each well of the plate was washed three times with washing buffer. (E) 50 μl of an incubation buffer containing an anti-rabbit antibody labeled with alkaline phosphatase was added to each well, and the wells were left to stand at room temperature for 1 hour. Then, each well of the plate was washed three times with a washing buffer. (F) 50 μl of ALP buffer was added to each well and allowed to stand for 30 minutes. (G) Fluorescence intensity was measured (Excitation 360 nm / Emission 465 nm) and wells containing cells expressing rabbit antibodies were examined.

[0037] The wells in which rabbit antibodies were strongly expressed were observed under a microscope to determine where the cells in the wells were monocloned. The cells were then collected from the wells and suspended in animal cell culture medium. These cells were then cultured again at 37°C in 5% CO2. The above-mentioned limiting dilution and ELISA were then performed several times using these cells to obtain monoclonal antibody-expressing cells.

[0038] The monoclonal T4-binding antibody expressing cells were suspended in an animal cell culture medium and cultured at 37°C in an environment of 5% CO2. The culture supernatant was then collected and the T4-binding antibody was purified.

[0039] (7) Isolation of anti-immunocomplex mouse monoclonal antibody The purified T4-binding antibody was digested with papain to prepare F(ab)'2. This F(ab)'2 was then mixed with D-T4 to prepare the immunization antigen. This immunization antigen was mixed with equal amounts of Freund's complete adjuvant and emulsified by sonication on ice. This was injected into the abdominal cavity of a mouse prepared as an immunization animal, and immunization was performed.

[0040] One week after the first immunization, the mice were immunized again as follows. First, equal amounts of the immunization antigen and Freund's complete adjuvant were mixed. The mixture was then emulsified by ultrasonication on ice and injected into the abdominal cavity of the mice for immunization. After the second immunization, this immunization procedure using Freund's incomplete adjuvant was repeated once a week.

[0041] After the immunization procedure was completed, the immunized mice were euthanized and their spleens were removed. B cells were isolated from the spleens and mixed with myeloma cells to cause cell fusion by electric fusion. The fused cells were then collected and suspended in a fusion cell medium (E-RDF + 10% bovine serum + 5% BM-conditioned + HAT) at 2 × 10 5 The cell density was adjusted to 100 cells / ml. The cell suspension was then plated onto a 384-well plate and cultured at 37°C in an environment of 5% CO2.

[0042] After culturing the fused cells, the reactivity of the antibody secreted into the culture supernatant with the "complex of D-T4 and T4 binding molecule" was examined according to a method already reported (see Patent No. 6031944). Then, cells were collected from the wells containing the antibody that specifically binds to the complex, suspended in animal cell culture medium, and cultured at 37°C and 5% CO2. Next, the cells were subjected to limiting dilution to monoclonate the capture antibody-expressing cell line. The monoclonal capture antibody-expressing cells were suspended in animal cell culture medium and cultured at 37°C and 5% CO2. The capture antibody was then purified from the culture medium. The purified capture antibody was labeled with Alkaline Phosphatase Labeling Kit-NH2 (manufactured by Dojindo Laboratories). The capture antibody labeled with alkaline phosphatase was used to measure the D-T4 concentration by the sandwich method.

[0043] (8) Measurement of T4 concentration by competitive method (Comparative Example 1) First, a calibrator solution with a known T4 (D-T4 or L-T4) concentration was prepared. Next, a certain amount of each calibrator solution was added to the magnetic particles on which the T4-binding antibody was immobilized, and incubated for 10 minutes. A certain amount of T4 labeled with ALP was then added to this, and reacted for another 3 minutes. After the reaction, B / F separation was performed, and the ALP substrate was added, and the signal emitted from CJ bound to the solid phase was detected. The signal (B0) when the calibrator solution containing no T4 was added was set to 100%, and the signal (B) when each calibrator solution containing a known concentration of T4 was added was calculated as a relative value [(B / B0) x 100]. The horizontal axis was the T4 concentration (logarithm) and the vertical axis was the B / B0 (%), and the measurement results when each calibrator solution was added were plotted to create a calibration curve (Figure 4). This series of operations was performed fully automatically at 37°C using a Tosoh AIA-CL2400. The results of the experiment showed that the competitive method could detect D-T4 if the concentration was 0.25ng / ml or higher. In addition, the T4 concentrations at which the B / B0 of the standard curves made with L-T4 and D-T4 was 40% were calculated and compared, resulting in 2.8ng / ml and 0.98ng / ml, respectively. From these results, the cross-reaction rate with L-T4 in the competitive method was calculated to be 0.98÷2.8×100=35%.

[0044] (9) Measurement of D-T4 concentration by sandwich method (Example 1) First, a calibrator solution (with a known T4 (D-T4 or L-T4) concentration) was prepared. Next, a fixed amount of each calibrator solution was added to the magnetic particles on which the T4-binding antibody was immobilized, and incubated for 5 minutes. After the reaction, B / F separation was performed, and a fixed amount of the capture antibody labeled with ALP was added to the particles, and incubated for another 3 minutes. After that, B / F separation was performed again, and the ALP substrate was added to detect the signal emitted from CJ bound to the solid phase. Then, T4 The signal (N) obtained when a calibrator solution containing no T4 was added was used as the standard, and the signal (S) obtained when each calibrator solution containing a known concentration of T4 was added was calculated as a relative value (S / N). The horizontal axis represents the T4 concentration (logarithm) and the vertical axis represents the S / N (logarithm), and the measurement results obtained when each calibrator solution was added were plotted to create a calibration curve (Figure 5). This series of operations was performed fully automatically at 37°C using a Tosoh AIA-CL2400. The experimental results showed that when the D-T4 concentration was 0.25 It was revealed that it can be detected even at ng / ml or less. In addition, the S / N ratio when 10ng / ml of L-T4 was added was applied to the calibration curve created when D-T4 was added to calculate the concentration (D-T4 equivalent concentration), which was 0.08ng / ml. From this result, the cross-reaction rate with L-T4 in the sandwich method was calculated to be 0.08÷10×100=0.8%. Furthermore, the S / N ratio when 1000ng / ml of T3 was added was applied to the calibration curve created when D-T4 was added to calculate the concentration (D-T4 equivalent concentration), which was 0.05ng / ml. From this result, the cross-reaction rate with T3 in the sandwich method was calculated to be 0.05÷1000×100=0.005%.

[0045] These results demonstrated that the sandwich method using anti-T4 antibody and anti-immunocomplex antibody can measure only D-T4 with high sensitivity in a short time.

Claims

1. D-thyroxine (D-T4) in the sample; - a molecule capable of binding to thyroxine (T4) (a T4-binding molecule); to produce a "complex of D-T4 and a T4 binding molecule"; Next, a molecule capable of binding to the complex (a capture molecule) is reacted with the complex, A method for detecting binding between the complex and a capture molecule, comprising the steps of: The T4 binding molecule specifically binds to D-T4. and / or The capture molecule specifically binds to the complex. A method for measuring D-T4,

2. The method according to claim 1, wherein the T4-binding molecule is a molecule capable of binding to both D-T4 and L-thyroxine (L-T4), and the capture molecule is a molecule that specifically binds to the complex.

3. The method of claim 1 , wherein the T4 binding molecule is an anti-T4 antibody.

4. The method according to claim 3, wherein the capture molecule is an anti-immunocomplex antibody.

5. The method according to claim 3, wherein D-T4 is measured by an ELISA method.

6. The method according to claim 1, wherein the cross-reactivity rate with compounds other than D-T4 is 5% or less.

7. The method according to claim 6, wherein the compound other than D-T4 is L-T4.

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