Method for measuring autoantibody and measurement reagent

Abstract

To provide a method for measuring a Kv1.4 antibody more simply with high sensitivity, and a measurement reagent.SOLUTION: A method for measuring a Kv1.4 antibody detects and quantifies the Kv1.4 antibody by an immunological method including immunologically binding the Kv1.4 antibody in a sample with an epitope antigen of the Kv1.4 antibody, where the Kv1.4 epitope antigen is composed of an amino acid sequence including 300th amino acid from an N-terminus of Kv1.4.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and a reagent for measuring Kv1.4 antibody. [Background technology]

[0002] Myasthenia gravis (MG) is an autoimmune disease, and autoantibodies such as anti-acetylcholine receptor (AChRAb) antibodies and anti-muscle-specific tyrosine kinase (MuSKAb) antibodies are involved (Non-Patent Document 1). It has been suggested that anti-striated muscle antibodies are involved in MG, which targets cardiac and skeletal muscles (Non-Patent Document 2). Titin, ryanodine receptors, and voltage-dependent K channel (Kv1.4) are known as antigens of anti-striated muscle antibodies, and the significance of each antibody has also been reported (Non-Patent Document 3).

[0003] For Kv1.4 antibodies, immunoprecipitation methods using radioisotopes (RI) (Non-Patent Document 2) and flow cytometry methods using Kv1.4-expressing cells (Patent Document 1) have been developed; however, due to issues such as protection against radiation exposure from RI, the former measurement method is currently used exclusively.

[0004] In the latter method, cells expressing Kv1.4 on the cell membrane of cultured cells are used as an antigen complex. On the other hand, not only Kv1.4 but also many endogenous proteins coexist on the cell membrane of Kv1.4-expressing cells. In samples containing antibodies against these endogenous proteins, it is not possible to accurately measure only Kv1.4 antibodies, and false positives and false negatives remain a problem. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 7074981 [Non-patent literature]

[0006] [Non-Patent Document 1] Sendai Medical Center Medical Journal, 6: 21-26 (2016) [Non-Patent Document 2] Clinical Neurology, 52(11): 1312-1314 (2012) [Non-Patent Document 3] BRAIN and NERVE 63(7) : 70-712 (2011) Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a simpler and more sensitive method and reagent for measuring Kv1.4 antibody. [Means for solving the problem]

[0008] As a result of intensive research, the inventors have identified the specific amino acid sequence (epitope antigen) of the Kv1.4 protein to which the Kv1.4 antibody binds, and have confirmed that more sensitive measurements can be performed by immunological techniques using only the Kv1.4 epitope antigen, thereby completing the present invention.

[0009] The present invention provides the following: (1) A method for measuring a Kv1.4 antibody, comprising detecting and quantifying the Kv1.4 antibody by an immunological method comprising immunologically binding the Kv1.4 antibody in a sample with the Kv1.4 epitope antigen. (2) The aforementioned method, wherein the Kv1.4 epitope antigen consists of 300 amino acids in the N-terminal region of Kv1.4. (3) The above-mentioned measurement method, wherein the immunological technique is an EIA method, an immunoprecipitation method, a flow cytometry method or an immunochromatography method based on an antigen-antibody reaction involving the Kv1.4 epitope antigen. (4) A reagent for measuring Kv1.4 antibody, which comprises the Kv1.4 epitope antigen, for use in the measurement method. Effect of the Invention

[0010] Kv1.4 antibodies can be measured easily and sensitively. [Brief description of the drawings]

[0011] [Figure 1] Amino acid sequence of Kv1.4 epitope antigen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, an embodiment of the present invention will be described. First, the method of the present invention will be described. The method of the present invention is characterized in that the Kv1.4 antibody is quantified by an immunological method comprising immunologically binding the Kv1.4 antibody in a sample to the Kv1.4 epitope antigen.

[0013] The sample to be measured is usually serum or plasma, but is not particularly limited as long as it does not contain any substance that interferes with immunological binding in the immunological method. The conditions for binding in the immunological method can be the same as those for conventional immunological methods. Quantitation based on detection of binding can also be performed under the same conditions as those for conventional immunological methods. Examples of immunological methods include ELISA, flow cytometry, immunostaining, and immunochromatography. For an example of ELISA, see Scientific reports 2020; 10 : 8968. For an example of flow cytometry, see J Vis Exp. 2013; (81): 50935. For an example of immunostaining, see PLoS One. 2014; 9(3): e92698. For an example of immunochromatography, see J. Phys. Conf. Ser. 2019; 1146 : 012019.

[0014] The Kv1.4 epitope antigen includes the N-terminal intracellular domain region of Kv1.4. The molecular structures of these antigens are known as described in Non-Patent Document 2, and their genes have also been identified.

[0015] The Kv1.4 epitope antigen can be obtained by transforming cells, E. coli, or the like with a nucleic acid encoding the antigen. Cultured cells can be used as the cells, for example, 293F cells. For example, the nucleic acid encoding the epitope antigen can be inserted into a transformation vector (e.g., pET-16b), and E. coli can be transformed using the vector, to obtain the epitope antigen expressed in E. coli, or the like.

[0016] For example, based on the relationship between Kv1.4 antibody and the pathology of myasthenia gravis as known from Non-Patent Documents 1 and 2, the measurement results can be used to test and diagnose myasthenia gravis, which is particularly advantageous for distinguishing types of myasthenia gravis.

[0017] A positive result in a test or diagnosis can be determined by comparing it with the value of a healthy person. If the value is significantly higher than the value of a healthy person measured at the same time or measured in advance (e.g., 2.5 SD or higher, or significantly higher by a statistical test), it can be determined to be positive. A positive result also includes a suspicion of myasthenia gravis.

[0018] Next, the measuring reagent of the present invention will be described. The measuring reagent of the present invention is used for carrying out the measuring method of the present invention, and contains a Kv1.4 epitope antigen.

[0019] The Kv1.4 epitope antigen is as explained in relation to the measurement method of the present invention.

[0020] Furthermore, the measuring reagent of the present invention may contain reagents necessary for immunological measurement, such as a positive control, a buffer solution, etc., as necessary, and may be provided as a kit.

[0021] Each component of the measuring reagent of the present invention may be in a solution state or in a dry state such as a freeze-dried product. If it is in a dry state, the measuring reagent of the present invention may further contain a buffer solution or the like for making it into a solution state before use. The amount and form of each component are adjusted according to the conditions of the measuring method.

[0022] The present invention will be described below with reference to examples. In the examples, "%" indicates % by weight unless otherwise specified.

[0023] <Material preparation and methods> (1) Preparation of plasmid for expressing Kv1.4 epitope antigen The Kv1.4 epitope antigen gene sequence shown in SEQ ID NO:1 was prepared by chemical synthesis, inserted into TaKaRa's pCold II plasmid, and prepared so that it could be expressed in E. coli.

[0024] The terminus of the Kv1.4 epitope antigen can be fused with various tag sequences that can be used for purification, detection, etc., and examples thereof include a His tag and a DDDDK tag. In the following examples, a His tag was added to the C-terminal region and used.

[0025] (2) Expression of Kv1.4 epitope antigen E. coli BL21(DE3) strain was transformed with the above plasmid. Expression induction was performed as follows. E. coli transformed with the epitope antigen expression gene was inoculated into 5 mL of LB medium containing 50 ug / mL ampicillin and cultured at 37°C for 16 hours with shaking. Next, 1 mL of culture solution was added to 100 mL of LB medium containing 50 ug / mL ampicillin and cultured at 37°C with shaking. When OD600 reached 0.4-0.6, the culture solution was placed in ice water and cooled for 30 minutes. IPTG was added to a final concentration of 0.1 mM, and the mixture was cultured at 16°C for 16-24 hours to allow expression.

[0026] (3) Purification of Kv1.4 epitope antigen After expression induction, the cells were collected from 40 mL of culture medium by centrifugation (3000 g x 15 min, 4 ° C). The cells were suspended in 2 mL of TBS, and 800 μL of Lysozyme Soln. was added and shaken at 4 ° C for 30 minutes. Then, 1.2 mL of 10% Triton X-100 was added and shaken at 4 ° C for 20 minutes. The mixture was centrifuged (12000 g x 30 min, 4 ° C) to collect the first supernatant. 1 mL of 8 M UREA was added to the precipitate and allowed to stand at room temperature for 30 minutes. The second supernatant was collected by centrifugation (15000 g x 20 min, 4 ° C), and the first and second supernatants were mixed to obtain a crude purified solution. The crude solution was purified by affinity column purification using a His tag fused to the Kv1.4 epitope antigen. Purification was performed using Cytiva's HisTrap FF Crude and Akta Start, following the manufacturer's recommended protocol. The Kv1.4 epitope antigen adsorbed on the column was eluted with a buffer containing 200 mM imidazole and used to prepare an antigen solid-phase plate.

[0027] (4) Preparation of ELISA test reagents The Kv1.4 epitope antigen was immobilized on a commercially available 96-well plate for ELISA. Adsorption to the solid phase can be achieved by commonly used immobilization methods such as physical adsorption and adsorption by covalent bonding with amino groups or carboxyl groups on the surface of the solid phase. In the following examples, ThermoScientific's ImmunoModule (Polysoap) is used as a commercially available 96-well plate for ELISA, but the plate is not limited thereto.

[0028] For immobilization, an epitope antigen solution was prepared in 50 mM carbonate buffer (pH 9.5), dispensed in 100 μL portions into a 96-well plate for ELISA, and allowed to stand at 4° C. for 16 to 24 hours for adsorption. The epitope antigen solution can be 0.01-30 μg / mL, and preferably 0.1-10 μg / mL. In the following examples, 5 μg / mL is used.

[0029] After the adsorption reaction of the epitope antigen, the unadsorbed components were washed off, and then a blocking operation was performed to prevent non-specific reactions. Protein blocking agents (BSA, casein, etc.) and non-protein blocking agents can be used for blocking, and various blocking operation conditions can be used, such as 30 minutes at room temperature, which is commonly used, or 16 hours at 4°C. In the following examples, an aqueous solution of NOF Corp.'s immunoassay blocking reagent N101 diluted 5-fold with pure water was used, and the blocking operation was performed by leaving it at 4°C for 16 to 72 hours. After discarding the blocking solution by decantation, the plate was dried at room temperature for 3 hours to prepare an antigen-immobilized plate.

[0030] (5) ELISA measurement ELISA measurements were performed using the general procedure described below.

[0031] The specimen was diluted 100-fold with buffer, added to the antigen-immobilized plate, and reacted for 1 hour to form a complex of Kv1.4 antibody in the specimen and immobilized Kv1.4 epitope antigen. After washing off unadsorbed proteins, the specimen was reacted with HRP-labeled anti-human Ig antibody to form a complex of Kv1.4 epitope antigen-Kv1.4 antibody-HRP-labeled anti-human Ig antibody. After washing off unreacted HRP-labeled anti-human Ig antibody, the specimen was colored using a HRP substrate such as TMB, and the absorbance at 450 nm (A450) and absorbance at 600 nm (A600) were measured for all specimens. From the measurement results of each specimen, the specific absorbance was calculated using the following formula. Specific absorbance = A450 - A600

[0032] The antibody titer was calculated by dividing the specific absorbance of the sample (sample) by the specific absorbance of the reference sample (control) and multiplying the result by a constant of 3. Antibody titer (sample) = Specific absorbance (sample) / Specific absorbance (control) x 3

[0033] Example 1: Study of the amount of epitope antigen immobilized on a solid phase In preparing the ELISA plate with immobilized epitope antigen, the amount of immobilized epitope antigen was adjusted to 1.0μg, 0.5μg, 0.1μg, 0.05μg, and 0.01μg per well. The specific absorbance was compared using a total of four samples, three positive samples (sample names 15, 358, and 428) and one negative sample (sample name Pool). The results are shown in Table 1. TIFF2025037263000002.tif81170

[0034] It was confirmed that positive and negative could be distinguished regardless of the amount of immobilization. Since it was expected that the effect of non-specific reactions would increase with an increase in the amount of antigen, the amount of immobilization was set to 0.5 μg per well.

[0035] Example 2 Comparison with Cytometric CBA method Using 519 samples that had been measured by the Cytometric CBA method (CBA), the method of the present invention was used to verify the positive-negative concordance rate. For the measurement, the samples were diluted 101-fold with PBS, and 100 μL was added to an ELISA plate. The reaction was allowed to proceed with shaking (600 rpm) at 37°C for 1 hour to form a complex between the immobilized epitope antigen and the autoantibody. After removing unreacted serum components by washing, 100 μL of HRP-labeled anti-human IgG antibody was added as a secondary antibody after diluting 100-fold with secondary antibody diluent. The reaction was allowed to proceed with shaking (600 rpm) at 37°C for 1 hour, and the unreacted secondary antibody was removed by washing. 50 μL of TMB solution, which is the substrate for HRP, was added, and the color reaction was allowed to proceed with shaking (600 rpm) for 15 minutes at 37°C. After adding 100 μL of a reaction stop solution, the absorbance at 450 nm and 600 nm was measured using a plate reader. The measurement results were analyzed using the method described above, with antibody titers of 3.0 or less being considered negative. The results are shown in Table 2. TIFF2025037263000003.tif71165

[0036] The positive agreement rate was 75.4%, and the negative agreement rate was 80.6%. A detailed analysis of the samples in the CBA-negative / ELISA-positive group revealed that these groups contained many samples with very high background levels during CBA measurement, and that the false negative results were due to nonspecific reactions of the human IgG antibodies contained in the samples. Based on this, it was considered that the sensitivity of the ELISA method was increased compared to CBA.

[0037] Example 3 Comparison of measurement results with CBA in 46 cases of myasthenia gravis with myositis and myocarditis For samples from myasthenia gravis complicated with myositis and myocarditis, autoantibodies against Kv1.4 were measured using the CBA method and the method of the present invention (this method). Measurement by this method was performed in the same manner as in Example 2. The results are shown in Tables 3 and 4. TIFF2025037263000004.tif24851TIFF2025037263000005.tif41136 The above results showed that the positive rate was improved and the sensitivity was increased compared to CBA.

Claims

1. A method for measuring a Kv1.4 antibody, comprising detecting and quantifying the Kv1.4 antibody by an immunological method comprising immunologically binding the Kv1.4 antibody in a sample with an epitope antigen of the Kv1.4 antibody.

2. The above-mentioned method, wherein the Kv1.4 epitope antigen comprises an amino acid sequence including up to the 300th amino acid from the N-terminus of Kv1.

4.

3. The above-mentioned measurement method, wherein the above-mentioned immunological technique is an EIA method, an immunoprecipitation method, a flow cytometry method or an immunochromatography method based on an antigen-antibody reaction involving the Kv1.4 epitope antigen.

4. A reagent for measuring Kv1.4 antibody, comprising the Kv1.4 epitope antigen, for use in the measurement method according to any one of claims 1 to 4.

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