KR100616438B1 - Analyzing Method for Activity of Thyroid Autoantibody - Google Patents

Abstract

The present invention relates to a method for measuring the activity of a stimulating thyroid stimulating hormone receptor (TSAB), a method for measuring the activity of a blocking thyroid stimulating hormone receptor (TSBAB) and a kit for measuring the activity of TSAB or TSBAB, in more detail ( a) obtaining a testing fluid from the subject under test; (b) subjecting the test solution to a culture of thyroid cells; And (c) measuring the phosphorylation of S6 kinase or ribosomal S6 protein in the treated thyroid cells, a method for measuring activity of TSAB, a method for measuring activity of TSBAB and a kit for measuring activity of TSAB or TSBAB.

Hyperthyroidism, Hypothyroidism, Thyroid Stimulating Hormone Receptor, Stimulated Thyroid Stimulating Hormone Receptor, Blocked Thyroid Stimulating Hormone Receptor, S6 Kinase, Ribosome S6 Protein

Description

Analytical Method for Activity of Thyroid Autoantibody             

1 is a photograph showing phosphorylation of p70 and p85 type S6 kinase of FRTL-5 thyroid cells by thyroid stimulating hormone (TSH) and stimulated thyroid stimulating hormone receptor (TSAB);

Figure 2 is a photograph showing the phosphorylation of S6 kinase and S6 protein of immunoglobulin, ie, FRTL-5 thyroid cells by TSAB in patients with hyperthyroidism; And

Figure 3 is a photograph showing that TSBAB of hypothyroidism specifically inhibits phosphorylation of ribosomal S6 protein by TSH and TSAB.

The present invention relates to a method for measuring the activity of a stimulating thyroid stimulating hormone receptor (TSAB), a method for measuring the activity of a blocking thyroid stimulating hormone receptor (TSBAB) and a kit for measuring the activity of TSAB or TSBAB.

Hyperthyroidism and hypothyroidism are caused by the production of autoantibodies against thyroid stimulating hormone receptors ("TSHR"). Autoantibodies to TSHR correspond to immunoglobulin G (IgG) and are classified as functionally stimulating and blocking thyroid stimulating hormone receptor antibodies depending on whether thyroid stimulating hormone receptors are activated.

A stimulating type TSHR antibody (hereinafter referred to as "TSAB" (thyroid stimulating antibody)) activates TSHR to cause hyperthyroidism, and a blocking type TSHR antibody , Hereinafter referred to as "TSBAB" (thyroid stimulation blocking antibody), inhibits the action of TSH on TSHR, leading to hypothyroidism. Diagnosis of hyperthyroidism and hypothyroidism can be made through the detection of TSAB or TSBAB in immunoglobulins present in serum.

As a result of intensive research to develop a method capable of measuring TSAB or TSBAB activity without error, the present inventors qualitatively or quantitatively measure the activity of TSAB or TSBAB when measuring the degree of phosphorylation of S6 kinase and / or ribosomal S6 protein. The present invention was completed by confirming that it can be measured by.

Accordingly, it is an object of the present invention to provide a method for measuring the activity of a stimulating thyroid hormone receptor (TSAB).

Another object of the present invention is to provide a method for measuring the activity of a blocked thyroid hormone receptor antibody (TSBAB).

Another object of the present invention to provide a kit for measuring the activity of TSAB or TSBAB.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention comprises the steps of (a) obtaining a testing fluid (testing fluid) from the subject of the test object; (b) subjecting the test solution to a culture of thyroid cells; And (c) measuring the phosphorylation of the S6 kinase or ribosomal S6 protein in the treated thyroid cells, thereby providing a method for measuring the activity of a stimulated thyroid stimulating hormone receptor (TSAB).

According to another aspect of the present invention, the present invention provides a method for producing a test fluid, comprising the steps of: (a) obtaining a testing fluid from a subject under test; (b) treating the test solution, and (ii) thyroid stimulating hormone or TSAB to a culture of thyroid cells; And (c) measuring phosphorylation of S6 kinase or ribosomal S6 protein in the treated thyroid cells. Provided is a method for measuring the activity of a blocked thyroid stimulating hormone receptor (TSBAB) comprising the following steps.

The present invention relates to a method for measuring the activity of TSAB causing hyperthyroidism and TSBAB causing hypothyroidism. In particular, the present invention relates to a method for more easily measuring the activity of TSAB and TSBAB. Therefore, the present invention is directed to a biofluid capable of obtaining the activity of TSAB and TSBAB in a subject. In the present invention, it can be applied to various biological fluids (e.g., blood, serum, urine, sweat, etc.), but is preferably blood, more preferably serum, even more preferably an antibody isolated from serum, most preferably Preferably immunoglobulin G (IgG) isolated from serum. Separation of such antibodies can be readily accomplished using certain chromatography methods known in the art (eg, separation using a Protein A column).

On the other hand, the method of the present invention is applied to mammals such as cattle, pigs, sheep, goats, mice and rats, and preferably to humans.

In the method of the present invention, phosphorylation of the S6 kinase or ribosomal S6 protein can be measured by various methods.

For example, γ- ³² p-ATP may be additionally added to the culture in the treatment step of (b), and in step (c), the S6 kinase or ribosomal S6 protein to which ³² p is bound may be measured. .

As a second example, antibodies that specifically bind only to phosphorylated S6 kinase or ribosomal S6 protein (preferably monoclonal antibodies) can be used to specifically measure only phosphorylated S6 kinase or ribosomal S6 protein.

As a third example, the phosphorylated S6 kinase or ribosomal S6 protein is less mobility compared to the non-phosphorylated one in electrophoresis, so the phosphorylation of the S6 kinase or ribosomal S6 protein is measured by observing the mobility retardation phenomenon. You may.

On the other hand, in the second and third examples, step (c) is preferably performed according to the Western blot protocol as a whole (see Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 108-). 121, CRC press). For example, lysing the treated thyroid cell sample to obtain a protein; Denaturing the obtained protein with a solution comprising SDS and 2-mercaptoethanol; SDS-PAGE the denatured protein; Transferring the denatured protein on the SDS-PAGE completed gel to the NC membrane; Reacting the protein on the NC membrane with an anti-S6 kinase or ribosomal S6 protein antibody, or an anti-phosphorylated S6 kinase or ribosomal S6 protein antibody; Reacting the secondary antibody with the primary antibody having the ability to bind to the primary antibody to which the enzyme catalyzing the color reaction is reacted; Inducing a color reaction by adding a substrate of an enzyme bound to the secondary antibody; And it may be carried out by a process comprising the step of measuring the degree of color development developed by the color reaction.

Enzymes that catalyze the color reaction include, but are not limited to, alkaline phosphatase, β-galactosidase, horse radish peroxidase, and the like. In addition, when using alkaline phosphatase, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), ECF substrate, etc. are used as a substrate, and when using a horse radish peroxidase, Chloronaphthol, aminoethylcarbazole, diaminobenzidine, luminol and the like are used as the substrate.

The measurement in step (c) may be carried out by qualitative analysis, such as simply checking the presence or absence of the band and the position of the band, and may also be performed by quantitative analysis by quantifying the intensity of the band. For example, an NC membrane with colored bands can be read directly with a densitometor, or if a luminescent substrate is used (e.g. luminol), developed with a film and the film read with a densitometer and the difference Can be quantified and analyzed. Thus, measurements herein are intended to include both qualitative and quantitative analysis.

When the phosphorylation of S6 kinase or ribosomal S6 protein is measured by the above-described process, it indicates that TSAB activity is present. On the contrary, when phosphorylation is not measured in the method of the second embodiment of the present invention, TSBAB activity is present. It shows. In addition, as described above, when the final result is analyzed quantitatively, the activity of TSAB and TSBAB can be quantitatively represented.

According to another aspect of the present invention, the present invention comprises (i) a kit for measuring the activity of TSAB or TSBAB comprising a thyroid cell line.

In a preferred embodiment, the kit of the present invention further comprises (ii) an antibody against S6 kinase or phosphorylated S6 kinase, and more preferably further comprises (iii) an antibody against ribosomal S6 protein or phosphorylated ribosomal S6 protein. do.

In addition, the kits of the present invention may contain basic reagents for Western blotting (e.g., SDS, electroelution buffer, blocking buffer, secondary antibodies such as horse radish peroxidase-anti-Ig conjugate, PBS, filter paper , NC membranes, etc.)

The method of the present invention can accurately and qualitatively and quantitatively measure the activity of TSAB causing hyperthyroidism and TSBAB causing hypothyroidism in a short time.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Test Method 1: Acquisition of immunoglobulin from patients with hyperthyroidism and hypothyroidism

In 10 patients with hyperthyroidism (Grave's disease) and 1 patient diagnosed with hypothyroidism, 10 ml of blood was obtained prior to treatment, serum was isolated, and a protein A column (Fisher Scientific, Pittsburge, USA) was used. IgG was isolated, lyophilized and dissolved in PBS at a concentration of 10 mg / ml and used in the experiment.

Experimental Method 2: Culture of Thyroid Cells and Treatment of Immunoglobulins Obtained from Patients with Hyperthyroidism and Hypothyroidism

FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) were cultured in 6-well plates, and when the cell density reached about 50-60%, the cells were further cultured for 6 days in a culture medium without thyroid stimulating hormone. Subsequently, the immunoglobulin of Example 1 obtained from normal persons, hyperthyroidism and hypothyroidism was treated with the cultured thyroid cells at a concentration of 5 mg / ml, and then washed twice with cold PBS. The reaction was stopped and cell lysate was obtained on ice.

Experimental method 3: verification of phosphorylation of S6 kinase and ribosomal S6 protein by thyroid autoantibodies

Thyroid cells treated with immunoglobulins from normal, hyperthyroidism and hypothyroidism were treated with 62.5 mM Tris-HCl (pH 6.8 at 25 ° C) with protease inhibitors (Roche Diagnotics, Mannheim, Germany), 2%. It was dissolved in w / v SDS, 10% glycerol, 50 mM DTT, 0.01% w / v bromophenol blue solution and developed on 7.5% SDS-PAGE. After development, the proteins on the SDS-PAGE were transferred to the nitro cellulose membrane (NC membrane-Amersham Biosciences, Uppsala, Sweden) using an electrophoresis device, and the NC membrane to which the proteins were transferred was blocked with blocking buffer (1 × TBS, 0.1% Tween-20). , 5% skim milk powder) for 2 hours and then reacted for 16 hours at 4 ℃ using an antibody against S6 kinase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as the primary antibody. At this time, the primary antibody was diluted in a ratio of 1000: 1, and after the reaction was completed, the NC membrane was washed while shaking three times for 10 minutes with 1 × TBS and 0.1% Tween-20. Anti-rabbit serum (Cell Signaling Technology, Inc., Beverly, MA) bound to horse radish peroxidase as a secondary antibody was diluted to 2000: 1, reacted with NC membrane for 1 hour, and then repeated 3 times for 10 minutes. Washed. Chemiluminescent solutions (ECL Western blotting detection reagents, Amersham Biosciences, Uppsala, Sweden) were mixed 1: 1 and reacted and luminesced on NC membranes and photosensitized with X-ray films to detect total S6 kinase protein. Meanwhile, phosphorylated S6 kinase protein was detected by identifying delayed migration in electrophoresis.

For the measurement of phosphorylated ribosomal S6 protein, total proteins were developed on 15% SDS-PAGE and transferred to nitro cellulose membrane (NC membrane), soaked in blocking buffer for 2 hours, and then in the primary antibody ribosomal S6 protein. Antibodies (Cell Signaling Technology, Inc., Beverly, MA) that specifically detect the phosphorylated proteins in serine 235 and serine 236 were diluted 1000: 1 and reacted at 4 ° C. for 16 hours. After washing three times, the anti-serum serum (Cell Signaling Technology, Inc., Beverly, MA) bound to the secondary antibody hose radish peroxidase was diluted to 2000: 1 and reacted for 1 hour. Ribosome S6 protein was detected by luminescence and photosensitization using luminescent solutions (ECL Western blotting detection reagents, Amersham Biosciences, Uppsala, Sweden).

Experimental Result 1: Activation of S6 Kinase by TSAB

S6 kinases are enzymes that are activated by various growth factors, insulin and hormones in cells. Activation of S6 kinase is determined by phosphorylation of the S6 kinase protein and to date it is known that 8 amino acid sites are phosphorylated. Phosphorylated form of S6 kinase can be confirmed by observing the migration delay phenomenon on SDS-PAGE. As shown in Figure 1, when treated with thyroid stimulating hormone (Thyroid stimulating homone, TSH) to the rat thyroid cell line FRTL-5 thyroid cells can be observed phosphorylation of p70 and p85 type S6 kinase.

In addition, phosphorylation of p70 and p85 type S6 kinase was also observed when TSAB (10 mg / ml) obtained from hyperthyroidism patients was treated with FRTL-5 thyroid cells, a thyroid cell line. Phosphorylation of p70 and p85 S6 kinases by TSAB (10 mg / mL) is inhibited by phosphatidyl inositol-3 kinase inhibitors LY294002 (L) and wortmannin (W), but PDK98059 (PD), a MEK inhibitor Did not inhibit the phosphorylation of S6 kinase by TSAB. This fact indicates that S6 kinase is phosphorylated by TSAB obtained from hyperthyroidism and phosphatidyl inositol-3 kinase is involved in phosphorylation of kinase.

Experimental Results 2: Phosphorylation of Serine 235 and Serine 236 Residues of Ribosome S6 Protein by TSAB

The intracellular matrix of S6 kinase is known as the S6 protein, which is a constitutive protein of 40S ribosomes. The serine 235 and serine 236 amino acid residues of the ribosomal S6 protein are the phosphorylation target sites of S6 kinase, and measuring the phosphorylation status of these sites reflects the activity of the S6 kinase.

Immunoglobulin G (IgG, TSAB) isolated from 10 patients with hyperthyroidism was treated with rat thyroid cells for 30 minutes and the phosphorylation of S6 kinase was observed. As shown in Fig. 2, patients 1, 2, 3, Phosphorylation of S6 kinase can be observed at 4, 6, 9 and 10, and at the same time phosphorylation of ribosomal S6 protein. Culture (CON), immunoglobulin (NP) obtained from normal individuals and immunoglobulin (PM) obtained from hypothyroidism patients could not observe phosphorylation of S6 kinase and phosphorylation of ribosomal S6 protein. Thus, immunoglobulins in patients with hyperthyroidism result in phosphorylation of S6 kinase and also induce phosphorylation of ribosomal S6 protein, a substrate protein of S6 kinase, and this activity is observed in about 70% of patients with hyperthyroidism. Could.

Experimental Result 3: Inhibition of Phosphorylation of Serine 235 and Serine 236 Residues of Ribosome S6 Protein by TSBAB

TSBAB immunoglobulins detected in patients with hypothyroidism inhibit the binding of endogenous thyroid hyperhormones to TSHR, leading to atrophy and hypothyroidism of the thyroid gland.

As shown in FIG. 3, TSH in the rat thyroid cell line FRTL-5 thyroid cells results in phosphorylation of ribosomal S6 protein, wherein TSBAB immunoglobulin isolated from hypothyroidism patients completely inhibits phosphorylation of ribosomal S6 protein by TSH (Lane 2 in FIG. 3). TSBAB also completely inhibited the phosphorylation of ribosomal S6 protein by TSAB. However, it was found that TSBAB did not affect the phosphorylation of ribosomal S6 protein by insulin. Therefore, it can be seen that TSBAB of hypothyroidism specifically inhibits phosphorylation of ribosomal S6 protein by TSH and TSAB.

As described in detail above, the present invention provides a method for measuring the activity of a stimulating thyroid stimulating hormone receptor (TSAB) and a blocked thyroid stimulating hormone receptor (TSBAB). The present invention also provides a kit for measuring the activity of a stimulating thyroid stimulating hormone receptor (TSAB) and a blocked thyroid stimulating hormone receptor (TSBAB). The method of the present invention can accurately and qualitatively or quantitatively measure the activity of TSAB causing hyperthyroidism and TSBAB causing hypothyroidism in a short time.

Claims (10)

Methods for diagnosing hyperthyroidism in mammals other than humans comprising the following steps: (a) obtaining a testing fluid from the subject under test; (b) subjecting the test solution to a culture of thyroid cells; (c) measuring phosphorylation of S6 kinase or ribosomal S6 protein in the treated thyroid cells; And (d) diagnosing hyperthyroidism when S6 kinase or ribosomal S6 protein is phosphorylated. Methods for diagnosing hypothyroidism in non-human mammals comprising the following steps: (a) obtaining a testing fluid from the subject under test; (b) treating said test solution, and (ii) thyroid stimulating hormone or stimulating thyroid stimulating hormone receptor (TSAB) to culture of thyroid cells; (c) measuring phosphorylation of S6 kinase or ribosomal S6 protein in the treated thyroid cells; And (d) diagnosing hypothyroidism if the S6 kinase or ribosomal S6 protein is not phosphorylated. delete delete The method of claim 1 or 2, wherein the test solution is serum. The method according to claim 5, wherein the test solution is an antibody solution isolated from serum. 7. The method of claim 6, wherein the antibody is immunoglobulin G (IgG). A kit for diagnosing hyperthyroidism or hypothyroidism, comprising: (iii) an thyroid cell line and (ii) an antibody against S6 kinase or phosphorylated S6 kinase. delete A kit for diagnosing hyperthyroidism or hypothyroidism, comprising: (iii) an thyroid cell line and (ii) an antibody against ribosomal S6 protein or phosphorylated ribosomal S6 protein.

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