L-Thyroxine-specific immunoassay

Abstract

To provide a method for specifically measuring L-T4 in an immunoassay method.SOLUTION: An immunoassay method specifically recognizes L-thyroxine using an anti-thyroxine antibody and an anti-immunocomplex antibody.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to an immunoassay which specifically recognizes L-thyroxine. [Background technology]

[0002] L-thyroxine is a thyroid hormone secreted in the body for energy metabolism, such as sugar metabolism and protein synthesis. In addition, chemically synthesized thyroxine preparations are used as hormone replacement agents for hypothyroidism and to inhibit the growth of thyroid tumors.

[0003] L-thyroxine is a hormone synthesized by condensing two molecules of the amino acid tyrosine, and therefore has one asymmetric carbon derived from the amino acid. D-thyroxine is an enantiomer of L-thyroxine, but it is known to lower blood cholesterol without affecting metabolism (Non-Patent Document 1). Thus, D-thyroxine and L-thyroxine are compounds with different physiological activities.

[0004] However, in competitive immunoassays used to measure thyroxine concentrations in samples, it has been difficult to distinguish between D- and L-thyroxine.

[0005] As a method for distinguishing between D- and L-thyroxine, there is an HPLC-based method as described in Non-Patent Document 2. However, due to the characteristics of HPLC, there are problems in terms of achieving high throughput and high sensitivity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of Chromatography A, 1217 (2010) 4255-4266 [Non-Patent Document 2] Pharm Res. 2005 Apr;22(4):667-75.2005 Apr 7. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an immunoassay method which specifically recognizes L-thyroxine. [Means for solving the problem]

[0008] The present invention, made in view of the above problems, includes the following aspects. (1) An immunoassay method that specifically recognizes L-thyroxine using an anti-thyroxine antibody and an anti-immunocomplex antibody. (2) The method according to (1), characterized in that the cross-reaction rate with 3,3',5,5'-tetraiodothyroacetic acid is 5% or less. (3) The method according to (1) or (2), wherein the anti-thyroxine antibody is a rabbit monoclonal antibody. (4) The method according to any one of (1) to (3), wherein the anti-immunocomplex antibody is a mouse monoclonal antibody.

[0009] The present invention will be described in detail below. (1) L-Thyroxine L-thyroxine (hereinafter sometimes referred to as "L-T4") is a type of thyroid hormone, and is sometimes written as thyroxine. As a pharmaceutical drug, levothyroxine is used. In the present invention, the L-T4 to be measured by the immunoassay may be a chloride such as levothyroxine sodium (levothyroxine Na), and is not particularly limited as long as it is a substance that can take the structure of L-T4 in an aqueous solution. (2) Measurement target solution The solution to be measured is not particularly limited as long as it contains L-T4, and examples thereof include human blood (whole blood, plasma, serum, etc.) and body fluids such as urine. (3) Anti-immunocomplex antibodies The anti-immunocomplex antibody is not an antibody against L-T4, nor an antibody against an antibody against L-T4, but an antibody against a complex of L-T4 and an antibody against L-T4. The species of immunized animals used to obtain the anti-immunocomplex antibody is not particularly limited. Examples of animal species include mice, rats, goats, and sheep. In the present invention, it is particularly preferable to use anti-immunocomplex monoclonal antibodies derived from mice. (4) Antithyroxine antibody The anti-thyroxine antibody (hereinafter sometimes referred to as "anti-T4 antibody") preferably has high affinity, and is desirably a monoclonal antibody. Examples of animal species from which monoclonal antibodies with high affinity can be obtained include goats, sheep, and rabbits, and in particular, monoclonal antibodies derived from rabbits are preferred. The affinity is such that the dissociation constant (KD) is 10 -10 (M) or less, and more preferably 10 -11 It is preferable that the thickness is equal to or less than (M). (5) Water-insoluble carrier The water-insoluble carrier is not particularly limited, and examples thereof include fine particles, particles, beads, plates, etc., such as resin, glass, and polystyrene. Anti-T4 antibody or anti-immunocomplex antibody is immobilized on the water-insoluble carrier. There is no particular limit to the immobilization method, and immobilization may be performed directly or indirectly (for example, via avidin-biotin binding, etc.). (6)Signs The label is not particularly limited, but for example, an enzyme, a radioisotope, a dye, etc. can be used. As the enzyme, alkaline phosphatase, etc. can be used. The label is bound to an anti-T4 antibody or an anti-immunocomplex antibody. There is no particular limit to the labeling method, and labeling may be performed directly or indirectly (for example, via avidin-biotin binding, etc.). (7)Measurement method It is preferable to use a fully automated immunoassay device such as that shown in Biological Sample Analysis Vol. 39, No. 4 (2016). For example, it is preferable to perform the following automated measurement using a reagent cup with two cells as shown in Figure 1. 1) A water-insoluble carrier having an anti-T4 antibody immobilized thereon is dispensed into one cell of a two-cell cup, and an enzyme-labeled anti-immunocomplex antibody is dispensed into the other cell to form a reagent cup. 2) Place the reagent cup and the solution to be measured into the automated immunoassay device. 3) In an automated immunoassay device, a solution to be measured containing a hapten is dispensed into a cell on the water-insoluble carrier side of the reagent cup and allowed to react. 4) After the reaction is completed, the components not adsorbed to the water-insoluble carrier on which the anti-T4 antibody is immobilized are washed away. 5) The enzyme-labeled anti-immunocomplex antibody in the reagent cup is transferred to the cell on the water-insoluble carrier side. 6) After the reaction is completed, the components not adsorbed to the water-insoluble carrier on which the anti-T4 antibody is immobilized are washed away. 7) The enzyme substrate is added and the luminescence intensity of the product resulting from the enzyme reaction is measured. (8)specific In the present invention, the term "specific" refers to a low cross-reactivity rate with a cross-reacting substance. A cross-reactivity rate of 5% or less is sufficient, and a cross-reactivity rate of 1% or less is more preferable. Effect of the Invention

[0010] In the measurement method of the present invention, double molecular recognition is performed using an anti-T4 antibody and an anti-immunocomplex antibody, making it possible to distinguish between thyroxine enantiomers and specifically measure the L-isomer. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a diagram showing an example of a reagent cup that can be used in the present invention. [Diagram 2] FIG. 2 is a diagram showing a calibration curve when measuring L-T4 in Example 1. [Diagram 3] FIG. 13 is a diagram showing a reagent cup in Comparative Example 2. EXAMPLES

[0012] Hereinafter, the present invention will be described in detail with reference to an anti-T4 rabbit monoclonal antibody and an anti-immunocomplex mouse monoclonal antibody in the examples, but the present invention is not limited thereto. [Example 1] (1) Anti-T4 rabbit monoclonal antibody The anti-T4 rabbit monoclonal antibody was obtained by the method described in Japanese Patent No. 5663834. (2) Anti-immunocomplex mouse monoclonal antibody The antibody against the immune complex of L-T4 and anti-T4 rabbit monoclonal antibody (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent No. 6,031,944. (3) Measurement The automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method. 1) Preparation of the reagent cup A reagent cup for use in automatic analysis was prepared using a cup with two cells as shown in Figure 1. One cell of the cup is called the particle side cell, and the other cell is called the conjugate side cell.

[0013] A solution containing microparticles immobilized with anti-T4 rabbit monoclonal antibody was dispensed into the cell on the microparticle side. An anti-immunocomplex mouse monoclonal antibody labeled with alkaline phosphatase was dispensed into the cell on the conjugate side. The solution was freeze-dried and sealed with an aluminum seal to prepare a reagent cup for L-T4 measurement. 2) Measurement The L-T4 measurement reagent cup prepared in 1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, Tosoh Corporation), and six samples with known L-T4 concentrations (cal1 to cal6) were measured to create a calibration curve. For the measurement, 5 μL of a sample with known concentration (serum base) and 45 μL of diluent were dispensed into the cell on the particle side of the L-T4 measurement reagent cup, and reacted for 5 minutes (primary reaction), followed by washing with a washing solution (B / F separation), and then the reagent (50 μL) from the conjugate side cell dissolved in the diluent was transferred to the particle side cell and reacted (secondary reaction, 3 minutes). After the secondary reaction was completed, a second wash with a washing solution was performed. Then, the enzyme substrate was added and the luminescence intensity was measured. The measurement results are shown in Table 1 and Figure 2. It was confirmed that the luminescence intensity increased depending on the concentration of L-T4, and it was confirmed that a measurement system for L-T4 using anti-immunocomplex antibodies could be constructed.

[0014] [Table 1]

[0015] (4) Cross-reaction rate evaluation The cross-reaction rate was evaluated by adding the cross-reacting substances listed in Table 2 to the zero concentration sample to a concentration of 100,000ng / dL or 10,000ng / dL, respectively, and measuring. As described in Pharmaceutical Research, vol. 22, No. 4, 2005, 667, each substance used in the cross-reaction rate evaluation binds to albumin or thyroxine-binding globulin in serum, so the added concentration does not match the free substance concentration in the sample solution. For this reason, the reaction rate when L-T4 was added was set to 100%, and the cross-reaction rate of each substance was calculated. Specifically, the cross-reaction rate was calculated using the following formula. Cross-reaction rate (%) = [(calculated concentration from the standard curve when the cross-reacting substance was added) / (molecular weight of the cross-reacting substance)] / [(calculated concentration from the standard curve when the same amount of L-T4 was added) / (molecular weight of L-T4)] x 100 [Comparative Example 1] (1) Measurement reagents AIA-Pack CL FT4 (Tosoh Corporation), a competitive immunoassay reagent, was used. AIA-Pack CL FT4 was prepared by dispensing a solution containing microparticles with anti-T4 rabbit polyclonal antibodies immobilized in the microparticle side cell, dispensing a solution containing alkaline phosphatase-labeled T4 in the conjugate side cell, freeze-drying the cells, and sealing them with an aluminum seal. (2) Measurement AIA-Pack CL FT4 (Tosoh Corporation) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, Tosoh Corporation), and six samples (cal1 to cal6) with known L-T4 concentrations were measured to create a calibration curve. For the measurement, 5 μL of a sample with known concentration (serum base) and 45 μL of diluent were dispensed into the cell on the microparticle side of the AIA-Pack CL FT4 cup, and reacted for 6 minutes (primary reaction), after which the reagent (50 μL) in the cell on the conjugate side dissolved in the diluent was transferred to the cell on the microparticle side and reacted (secondary reaction, 3 minutes). After the secondary reaction was completed, washing was performed with a washing solution. Then, the enzyme substrate was added, and the luminescence intensity was measured. In addition, the cross-reaction rate was calculated using the same sample as that used in the cross-reaction rate evaluation in Example 1 (4).

[0016] The cross-reactivity rates calculated in Example 1 and Comparative Example 1 are shown in Table 2. From these results, it was confirmed that the measurement method of the present invention does not cross-react with D-T4, which shows a cross-reactivity rate of 80% or more in the competitive method (Comparative Example 1), and can specifically measure L-T4. It was also confirmed that no cross-reactivity was observed with 3,3',5,5'-tetraiodothyroacetic acid, which shows a cross-reactivity rate of 20% or more in Comparative Example 1.

[0017] [Table 2]

[0018] [Comparative Example 2] A comparative example in which evaluation was performed by the competitive method using the anti-T4 rabbit monoclonal antibody used in Example 1 is shown below. (1) Anti-T4 rabbit monoclonal antibody The anti-T4 rabbit monoclonal antibody used was the same as that used in Example 1. (2) Measurement The automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

[0019] 1) Preparation of the reagent cup A reagent cup for use in automatic analysis was prepared using a cup with two cells as shown in Figure 3. One cell of the cup is called the particle side cell, and the other cell is called the conjugate side cell.

[0020] A solution containing microparticles immobilized with anti-T4 rabbit monoclonal antibody was dispensed into the cell on the microparticle side. A solution containing alkaline phosphatase-labeled T4 was dispensed into the cell on the conjugate side. The solution was freeze-dried and sealed with an aluminum seal to prepare a measurement reagent cup. 2) Measurement The measurement reagent cup prepared in 1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation), and six samples with known L-T4 concentrations (cal1 to cal6) were measured, and a calibration curve was created. For the measurement, 5 μL of a sample with known concentration (serum base) and 45 μL of diluent were dispensed into the cell on the particle side, and reacted for 6 minutes (primary reaction), and then the reagent (50 μL) in the cell on the conjugate side dissolved in the diluent was transferred to the cell on the particle side and reacted (secondary reaction, 3 minutes). After the secondary reaction was completed, washing was performed with a washing solution. Then, the substrate of the enzyme was added, and the luminescence intensity was measured. In addition, the cross-reaction rate was calculated using the same sample as that used in the cross-reaction rate evaluation in Example 1 (4).

[0021] The cross-reactivity rates calculated in Example 1, Comparative Example 1, and Comparative Example 2 are shown in Table 3. From these results, it was confirmed that the measurement method of the present invention can specifically measure L-T4 without cross-reactivity with D-T4 (Comparative Example 2), which shows a cross-reactivity rate of 100% or more in a competitive method when the same antibody is used. It was also confirmed that no cross-reactivity was shown with 3,3',5,5'-tetraiodothyroacetic acid, which shows a cross-reactivity rate of 25% or more in Comparative Example 2.

[0022] [Table 3]

[0023] From the above results, it was confirmed that L-T4 can be specifically measured using anti-T4 antibody and anti-immunocomplex antibody.

Claims

1. An immunoassay method using an anti-thyroxine antibody and an anti-immunocomplex antibody, which specifically recognizes L-thyroxine and does not cross-react with D-thyroxine.

2. 2. The method according to claim 1, wherein the cross-reaction rate with 3,3',5,5'-tetraiodothyroacetic acid is 5% or less.

3. 3. The method according to claim 1 or 2, wherein the anti-thyroxine antibody is a rabbit monoclonal antibody.

4. The method according to any one of claims 1 to 3, wherein the anti-immunocomplex antibody is a mouse monoclonal antibody.

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