Immunochromatographic test kit for detecting autoantibodies and method for detecting autoantibodies using the same

Abstract

To enable highly accurate, simple and rapid detection of the presence of autoantibodies associated with known neurological diseases in a test subject.SOLUTION: An immunochromatographic test kit according to the present invention is a kit for detecting an autoantibody that binds to a receptor protein present in a subject's central nervous system in relation to a neurological disease, and includes a test strip 1 for developing a specimen, and the test strip 1 is provided with a dropping section 2 for dropping the specimen, a labeling section 3 on which a labeled anti-human IgG antibody is disposed, and a detection section 4 on which an antibody that recognizes and binds to the receptor protein is held, which are arranged in this order, and the specimen is a mixture of the cerebrospinal fluid of the subject and an animal brain extract.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an immunochromatographic test kit for detecting autoantibodies and a method for detecting autoantibodies using the same.

Background Art

[0002] Autoimmune encephalitis typified by anti-N-methyl-D-aspartic acid (NMDA) receptor encephalitis, which is one of the neurological diseases, has recently been clarified to be caused by autoantibodies that specifically target ion channel receptors and the like expressed in the synapses of brain nerve cells, resulting in abnormal behavior, epilepsy, and other severe mental and neurological symptoms. Under such circumstances, many studies have been conducted on methods for detecting autoantibodies against well-known receptors such as NMDA receptors, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, and γ-aminobutyric acid (GABA) receptors. For example, Patent Document 1 presents a method for detecting autoantibodies against NMDA receptors. Specifically, Patent Document 1 presents a method for detecting autoantibodies by contacting a sample suspected of containing autoantibodies against NMDA receptors with an artificially constructed NMDA receptor protein construct and detecting their conjugates.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Currently, methods for detecting autoantibodies against well-known receptors such as NMDA receptors, AMPA receptors, and GABA receptors are being established. For example, by the method as described in Patent Document 1 above, it is possible to detect autoantibodies against NMDA receptors. However, in the method of Patent Document 1, it is necessary to prepare an NMDA receptor protein construct, which is not necessarily simple and requires several days.

[0005] The present invention has been made in view of the above problems, and an object thereof is to enable detection of the presence of autoantibodies related to known neurological diseases in a test subject with high accuracy, simply and in a short time.

Means for Solving the Problems

[0006] In order to achieve the above object, as a result of intensive studies, the present inventors have found that by using an immunochromatographic test kit equipped with an anti-human IgG antibody or the like, with a mixed solution of cerebrospinal fluid in a test subject and an animal brain extract as a sample, the presence of autoantibodies can be detected with high accuracy, simply and in a short time, and thus completed the present invention.

[0007] Specifically, the immunochromatographic test kit according to the present invention is an immunochromatographic test kit for detecting autoantibodies that bind to receptor proteins present in the central nervous system of a subject with respect to a neurological disease, and includes a test strip for developing a specimen. On the test strip, a dropping portion for dropping the specimen, a labeling portion where a labeled anti-human IgG antibody is disposed, and a detection portion where an antibody that recognizes and binds to the receptor protein are arranged in this order, and the specimen is a mixed solution of the cerebrospinal fluid of the subject and an animal brain extract.

[0008] In the immunochromatographic test kit according to the present invention, it is preferable that a reference portion where an antibody that recognizes and binds to the labeled anti-human IgG antibody is held is further arranged on the downstream side of the detection portion on the test strip.

[0009] In the immunochromatographic test kit according to the present invention, it is preferable that the autoantibody is an autoantibody related to autoimmune encephalitis.

[0010] The method for detecting an autoantibody according to the present invention is a method for detecting an autoantibody that binds to a receptor protein present in the central nervous system of a subject with respect to a neurological disease, using the above immunochromatographic test kit, the method comprising: preparing a sample by mixing the cerebrospinal fluid of the subject and a brain extract of an animal; dropping the sample onto the dropping part of the immunochromatographic test kit; and confirming the presence or absence of the autoantibody in the detection part.

Advantages of the Invention

[0011] According to the immunochromatographic test kit for detecting an autoantibody according to the present invention and the method for detecting an autoantibody using the same, the presence of an autoantibody related to a known neurological disease in a test subject can be detected with high precision, simply, and in a short time. Therefore, it is also applicable to the diagnosis of neurological diseases in which the presence of autoantibodies is etiological, and is extremely useful.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0013] Hereinafter, modes for carrying out the present invention will be described. The following description of the preferred embodiments is merely illustrative in nature and is not intended to limit the present invention, its application method, or its uses.

[0014] One embodiment of the present invention relates to a neurological disease and is an immunochromatographic test kit for detecting autoantibodies that bind to receptor proteins present in the central nervous system of a subject to be tested. The immunochromatographic test kit will be described with reference to FIGS. 1 and 2. As shown in FIG. 1, the immunochromatographic test kit includes a test strip 1 for developing a sample to detect autoantibodies. The test strip 1 is provided with a plurality of regions, and from one end (the upper side in FIG. 1) to the other end (the lower side in FIG. 1) in the length direction of the test strip, a dropping part 2, a labeling part 3, a detection part 4, and a reference part 5 are arranged in this order.

[0015] Specifically, as shown in FIG. 2, the test strip 1 includes a sample pad 10, a conjugate pad 20, a membrane 30, and an absorption pad 40 arranged in order from the upstream (the left side in FIG. 2) to the downstream (the right side in FIG. 2) in the length direction of the test strip, that is, in the flow direction of the sample, and they are arranged on a support 50. The sample pad 10 and the conjugate pad 20 are arranged so that at least a part of them overlaps, the conjugate pad 20 and the membrane 30 are arranged so that at least a part of them overlaps, and similarly, the membrane 30 and the absorption pad 40 are arranged so that at least a part of them overlaps. The dropping part 2 is provided on the sample pad 10, the labeling part 3 is provided on the conjugate pad 20, and the detection part 4 and the reference part 5 are provided on the membrane 30.

[0016] In this embodiment, as the sample, a mixed solution of the cerebrospinal fluid of the subject and the brain extract of an animal is used. The subject to be examined is, for example, a mammal, preferably a human. The animal from which the brain extract is collected is, for example, a mammal, and it is preferably the same animal as the above subject, but it is preferably an animal having a receptor protein highly similar to the receptor protein abundantly present in the central nervous system of the subject. Examples of the receptor protein abundantly present in the central nervous system mentioned here include, but are not limited to, proteins such as NMDA receptor, AMPA receptor, and GABA receptor. Also, the high similarity mentioned here refers to the case where an antibody that recognizes the protein of one animal also recognizes the protein of the other animal. In this case, for example, the sequence identity between the amino acid sequence of one protein and the amino acid sequence of the other protein is 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more. When the subject is human, for example, mice, rats, pigs, monkeys, etc. can be used.

[0017] The sample pad 10 is not particularly limited as long as it is a pad made of a material that can accurately and uniformly develop a liquid sample. For example, pads made of materials such as paper, cellulose mixture, nitrocellulose, polyester, acrylonitrile copolymer, glass fiber, rayon, etc. can be used.

[0018] The dropping part 2 provided on the sample pad 10 is provided on one end side (upstream side) in the length direction of the sample pad 10 and is a region where a liquid sample is dropped. No substances such as antibodies are held in the dropping part 2.

[0019] The conjugate pad 20 is not particularly limited as long as it is a pad made of a material that can accurately and uniformly develop a liquid sample. For example, pads made of the same materials as the above-mentioned sample pad 10 can be used.

[0020] The labeling part 3 provided on the conjugate pad 20 is arranged on the other end side (downstream side) of the dropping part 2. When the test subject is a human, a labeled anti-human IgG antibody is arranged in the labeling part 3. As the anti-human IgG antibody, antibodies derived from animals other than humans can be used. For example, a goat anti-human IgG antibody, a rabbit anti-human IgG antibody, a mouse anti-human IgG antibody, etc. can be used. For the labeling of the anti-human IgG antibody, well-known means usually used for antibody labeling can be used. For example, gold colloid labeling, biotin labeling, fluorescent labeling, enzyme labeling, etc. can be used. The arrangement of the labeled anti-human IgG antibody on the labeling part 3 is not particularly limited as long as the antibody is arranged in a form that can move together with the autoantibody in the specimen when the antibody binds to the autoantibody in the specimen. As a method for arranging the antibody on the conjugate pad 20 in the labeling part 3, it can be carried out by a well-known method usually used. For example, after applying the labeled anti-human IgG antibody to the conjugate pad 20 in the labeling part 3 at a predetermined concentration and allowing it to penetrate, it can be carried out by drying at a certain temperature.

[0021] The membrane 30 is not particularly limited as long as it is made of a material that can accurately and uniformly develop a liquid specimen and can fix the antibodies arranged in the detection part 4 and the reference part 5 described later. For example, a nitrocellulose membrane can be used.

[0022] The detection unit 4 provided on the membrane 30 is arranged on the other end side (downstream side) of the labeling unit 3, and the detection unit 4 holds an antibody that recognizes and binds to the receptor protein present in the central nervous system of the test subject. In particular, the receptor protein is a receptor protein related to a neurological disease, preferably a receptor protein related to autoimmune encephalitis. Examples of such receptor proteins include proteins such as NMDA receptor, AMPA receptor, and GABA receptor. Antibodies against such receptor proteins are commercially available and easily obtainable. For example, anti-NMDA receptor antibodies are available from BD Biosciences, Merck, etc. The retention (fixation) of the antibody to the detection unit 4 can be performed, for example, when using a nitrocellulose membrane as the membrane 30, by dropping an antibody at a predetermined concentration onto the detection unit 4 and drying it at a constant temperature. By doing so, the antibody will be fixed to the membrane by hydrophobic bonding or the like.

[0023] The reference unit 5 provided on the membrane 30 is not necessarily provided for the detection of autoantibodies, but it is preferably provided to confirm whether the specimen is developed properly as will be described later. The reference unit 5 is arranged on the other end side (downstream side) of the detection unit 4, and the reference unit 5 holds an antibody that recognizes and binds to the labeled anti-human IgG antibody held by the labeling unit 3. Therefore, when the labeled anti-human IgG antibody held by the labeling unit 3 is a goat anti-human IgG antibody, an anti-goat IgG antibody is used for the antibody held by the reference unit 5. Similarly, when the labeled anti-human IgG antibody held by the labeling unit 3 is a rabbit anti-human IgG antibody, an anti-rabbit IgG antibody is used, and when the labeled anti-human IgG antibody held by the labeling unit 3 is a mouse anti-human IgG antibody, an anti-mouse IgG antibody is used. The retention (fixation) of the antibody to the reference unit 5 can be performed using the same method as the method for fixing the antibody to the detection unit 4 described above.

[0024] The absorption pad 40 is provided for absorbing the specimen that has passed through the membrane 30 downstream. The absorption pad 40 is not particularly limited as long as it is a pad made of a material capable of absorbing the specimen. For example, a cellulose pad can be used.

[0025] The support 50 is provided for supporting the above sample pad 10, conjugate pad 20, membrane 30, and absorption pad 40. The support 50 is not particularly limited as long as it can support them and is made of a material that does not hinder the movement of the specimen. For example, an adhesive sheet made of plastic can be used.

[0026] The immunochromatographic test kit according to this embodiment may include a case member or the like that holds or houses the test strip 1. Further, the immunochromatographic test kit according to this embodiment may include a reagent for causing the label of the labeled anti-human IgG antibody to signal such as luminescence or color development.

[0027] In the immunochromatographic test kit according to this embodiment, the test strip 1 has a configuration including the sample pad 10, conjugate pad 20, membrane 30, and absorption pad 40. However, it is not limited thereto. As long as the dropping part 2, labeling part 3, detection part 4, and reference part 5 can be appropriately provided, a part of them may be omitted or other pads may be added. For example, the conjugate pad 20 may be omitted, and the dropping part 2 and the labeling part 3 may be provided on the sample pad 10. Also, the absorption pad 40 and the support 50 do not necessarily have to be provided.

[0028] Next, a method for detecting autoantibodies using the above immunochromatographic test kit according to an embodiment of the present invention will be described. The method includes a step (a) of preparing a specimen by mixing the cerebrospinal fluid of the subject and the brain extract of an animal, a step (b) of dropping the specimen onto the dropping part of the immunochromatographic test kit, and a step (c) of confirming the presence or absence of the autoantibody at the detection part.

[0029] In step (a), first, cerebrospinal fluid of a subject to be examined and a brain extract of an animal are prepared. Examples of the subject to be examined and the animal from which the brain extract is collected are as described above. The method for collecting the cerebrospinal fluid of the subject is not particularly limited, and it can be collected using a well-known conventional method. The method for collecting the brain extract of the animal is not limited as long as a crude extract containing brain proteins can be obtained. For example, the brain is removed from the head of the animal, a desired site in the brain is cut out, immersed in a predetermined buffer solution, and the resulting solution is homogenized and then centrifuged, etc. After obtaining the brain nerve cell membrane, the cell membrane is solubilized by a conventional method to obtain a crude extract of the brain. In step (a), the obtained cerebrospinal fluid of the subject and the brain extract of the animal are mixed and appropriately diluted or concentrated to prepare a specimen. A solvent suitable for development on the membrane of the immunochromatographic test kit is selected for the specimen.

[0030] Subsequently, in step (b), the above specimen is dropped onto the dropping portion 2 of the sample pad 10 in the immunochromatographic test kit. The dropping amount can be appropriately selected according to the concentration of the specimen, etc. When the specimen is dropped onto the dropping portion 2, due to capillary action, the specimen advances to the downstream side of the sample pad 10 and moves to the conjugate pad 20, the membrane 30, and the absorption pad 40, that is, it is developed so as to pass through the labeling portion 3, the detection portion 4, and the reference portion 5.

[0031] Next, in step (c), the presence or absence of a signal in the detection unit of the immunochromatographic test kit is confirmed. When there is an autoantibody in the cerebrospinal fluid to be tested that binds to the receptor protein present in the central nervous system, the autoantibody binds to the receptor protein in the animal brain extract in the specimen to form a complex. In the labeling unit 3, the autoantibody in the complex binds to the labeled human IgG antibody. Subsequently, together with the specimen, the complex bound to the labeled human IgG antibody moves to the detection unit 4. Since an antibody that binds to the receptor protein is immobilized on the detection unit 4, the receptor protein in the complex binds to the antibody. The excess labeled human IgG antibody that did not bind to the autoantibody binds to the antibody that binds to the labeled anti-human IgG antibody immobilized on the reference unit 5 after passing through the detection unit 4. Thereafter, the presence of the autoantibody can be detected by appropriately developing or emitting light from the label of the labeled human IgG antibody to generate a signal. That is, if the above autoantibody is present in the specimen, the receptor protein that binds to the autoantibody bound to the labeled human IgG antibody is captured in the detection unit 4, resulting in a signal from the label. On the other hand, when the above autoantibody is not present in the specimen, the receptor protein that does not bind to the labeled human IgG antibody is captured in the detection unit 4, and thus no signal from the label can be obtained. Therefore, the presence or absence of the autoantibody in the specimen can be confirmed based on the presence or absence of a signal in the detection unit 4. In addition, in both cases where the above autoantibody is present and absent in the specimen, the excess labeled human IgG antibody reaches the reference unit 5 where the antibody that binds to it is immobilized, and thus a signal from the label can be obtained in the reference unit 5. Therefore, it is also possible to confirm whether the specimen has moved appropriately in the reference unit 5.

Example

[0032] Examples for explaining in detail the immunochromatographic test kit for detecting autoantibodies according to the present invention and the method for detecting autoantibodies using the same are shown below.

[0033] (Preparation of mouse brain extract containing receptor proteins such as NMDA receptor) First, three kinds of mixed anesthesia (Dormitor, Dolmicum, and Betnovate) were injected intraperitoneally at 150 μL into mice (8-week-old male C57Black6j mice), and then sevoflurane was allowed to be smelled to deepen the anesthesia. After confirming that the mice were anesthetized, the mice were decapitated and cut from the neck with surgical scissors. Buffer 1 (5 mM Tris-HCl, pH 8.0, 320 mM sucrose) well cooled with ice was placed in a metal tray on ice, and the heads of the mice were immersed. Then, using pre-cooled ice tweezers and forceps, the brains were removed from the heads of the above mice and immediately placed into Buffer 1 in a beaker on ice to cool the brains well.

[0034] Subsequently, under a dissection microscope, a paper towel was laid on an aluminum block placed on ice, slightly moistened with a small amount of Buffer 1, and then the removed brains were placed thereon. Using a razor blade (Feather Razor FA-10), the brains were cut in half, and one half was placed in Buffer 1 and cooled. Using forceps, regions other than the cerebrum (hippocampus) (cerebellum and brainstem) were removed. 4 mL of Buffer 1 well cooled with ice was placed into a Dounce Tissue Grinder (WHEATON 357542), and the removed forebrain (cerebrum - hippocampus) was placed therein, and homogenized by moving up and down loosely (loose pestle) 10 times and tightly (tight pestle) 10 times on ice.

[0035] The homogenized solution was dispensed into eight 1.5 mL tubes at 500 μL each and centrifuged at 800×g for 15 minutes to precipitate the nuclear fraction. The supernatant was transferred to a 1.5 mL tube, centrifuged at 20000×g for 15 minutes, and then the supernatant was removed, and the precipitate (neuronal membrane fraction: about 20 - 40 mg per tube) was collected. 500 μL of 5 mM Tris-HCl, pH 8.0 was added to the tube containing the precipitate and pipetted, then incubated for 5 minutes and centrifuged at 20000×g for 15 minutes to collect the precipitate (brain neuronal membrane). This operation was repeated once more.

[0036] Weighed out deoxycholic acid and cholesteryl hemisuccinate into two separate 1.5 mL tubes such that for 500 μL, the deoxycholic acid was 1% and the cholesteryl hemisuccinate was 2 mM (0.005 g and 0.0005 g respectively). Added 500 μL of Buffer 2 (50 mM Tris-HCl, pH 8.0, 0.002% n-dodecyl-β-D-maltopyranoside (DDM), 5 μL of protease (cOmplete EDTA free: Roche 11836170001) and phosphatase (Phosphatase Inhibitor Cocktail II (ab201113)) inhibitor (100-fold dilution)) to one tube and pipetted to dissolve the two tubes. Added the solution to the 1.5 mL tube containing the precipitate of the above-mentioned cerebral neuronal membrane, and pipetted with a 1 mL tip to solubilize the cell membrane. This was used as the animal brain extract.

[0037] (Preparation of the cerebrospinal fluid of the subject) The cerebrospinal fluid (CSF) of patients with autoimmune encephalitis (anti-NMDA receptor encephalitis) was received as an anonymized sample from the cerebrospinal fluid of patients with autoimmune encephalitis collected by the clinical research of the Department of Psychiatry, Osaka Acute and Comprehensive Medical Center, Osaka Prefectural Hospital Organization (Clinical Research Support Center). On the other hand, the CSF of healthy subjects was purchased from Innovative Research (Pooled Human Cerebrospinal Fluid (CSF): IRHUCSF1ML).

[0038] (Preparation of specimens) Dissolved 10 μL of the brain extract in 390 μL of buffer (50 mM Tris-HCl pH 8.0, 0.002% n-dodecyl-β-D-maltopyranoside (DDM)), added 20 μL of the above CSF thereto, and mixed well by inverting. Incubated at room temperature for 15 minutes to obtain the specimen for testing.

[0039] (Preparation of immunostrips) Fixation of antibodies to the membrane The nitrocellulose membrane (FF120HP Cytiva: 10547006) was cut into a size of 7 cm, and 0.5 μL each of mouse anti-NMDAR1 antibody (Merck MAB363) diluted 2-fold with 50 mM Tris-HCl pH 8.0 and anti-goat IgG antibody were dropped onto the detection part and the reference part respectively to bind the antibodies, and the membrane was air-dried. After drying, it was immersed in 0.2% iBlock (Invitrogen, T2015) dissolved in TBS-T (50 mM Tris, 150 mM NaCl, 0.1% Tween® 20) and shaken at room temperature for 30 minutes for blocking. Next, it was infiltrated with a rinse buffer (50 mM Tris-HCl pH 8.0, 0.002% DDM) for 5 minutes for replacement, and this was done once again. Then, the strip was placed in a dryer at 50 °C (IWAKI AFO-82) to dry it and used as a membrane for inspection.

[0040] Blocking of the sample pad and conjugate pad The sample pad (2.2 cm × 2.5 cm; CF4 Cytiva: 8114-2250) and the conjugate pad (2.2 cm × 2 cm; Standard 17 Cytiva: 8134-2250) were immersed in 0.2% iBlock dissolved in TBS-T and shaken at room temperature for 30 minutes for blocking. Next, they were infiltrated with a rinse buffer (50 mM Tris-HCl pH 8.0, 0.002% DDM) for 5 minutes for replacement, and this was done once again. Then, the sample pad and the conjugate pad were placed in a dryer at 50 °C to dry them.

[0041] Placement of the antibody on the conjugate pad 10 μL of 40 nm gold colloid-labeled anti-human IgG antibody (jackson: 109-405-098) was dissolved in 290 μL of buffer (50 mM Tris-HCl pH 8.0, 0.002% DDM), and 150 μL of it was allowed to soak into the blocked conjugate pad and then placed in a dryer at 50 °C to dry it.

[0042] The sample pad, conjugate pad, and membrane prepared as described above were arranged so as to partially overlap as shown in Fig. 2 to prepare a test strip.

[0043] (Test) Samples prepared by the above method using the CSF of patients diagnosed with anti-NMDA receptor encephalitis and the CSF of healthy subjects were dropped onto the sample pad of the above test strip, respectively, and the positivity and negativity of the antibody against the NMDA receptor were determined.

[0044] (Results) As shown in Fig. 3, as a result of the above test, in the specimens containing the CSF of encephalitis patients, signals were detected in the detection part (positive). On the other hand, in the specimens containing the CSF of healthy subjects, no signals were detected in the detection part (negative). Also, signals were detected in the reference part in both specimens. That is, it was suggested that by using the immunochromatographic test kit according to the present invention, it is possible to confirm whether the test subject has autoantibodies against receptor proteins present in the central nervous system such as the NMDA receptor. In addition, in the above method, it only takes about several tens of minutes until detection, and the results can be obtained extremely simply and quickly.

[0045] As described above, according to the immunochromatographic test kit according to the present embodiment and the method for detecting autoantibodies using the same, the presence of autoantibodies related to known neurological diseases in the test subject can be detected with high accuracy, simply, and in a short time. Therefore, there is also a possibility that it can be applied to the diagnosis of neurological diseases in which the presence of autoantibodies is the cause, and it is extremely useful.

Explanation of symbols

[0046] 1 Test strip 2 Dropping part 3 Labeling part 4 Detection part 5 Reference part 10 Sample pad 20 Conjugate pad 30 Membrane 40 Absorbent pad 50 Support

Claims

1. An immunochromatographic test kit for detecting autoantibodies that bind to receptor proteins present in the central nervous system of a subject with respect to a neurological disease, including a test strip for developing a sample, on the test strip, a dropping part for dropping the sample, a labeled part where a labeled anti-human IgG antibody is disposed, and a detection part where an antibody that recognizes and binds to the receptor protein is held are arranged in order, The sample is a mixed solution of the cerebrospinal fluid of the subject and an animal brain extract. Immunochromatographic test kit.

2. On the test strip, a reference part where an antibody that recognizes and binds to the labeled anti-human IgG antibody is held is further arranged on the downstream side of the detection part. The immunochromatographic test kit according to claim 1.

3. The autoantibody is an autoantibody related to autoimmune encephalitis. The immunochromatographic test kit according to claim 1.

4. A method for detecting autoantibodies that bind to receptor proteins present in the central nervous system of a subject with respect to a neurological disease, using the immunochromatographic test kit according to any one of claims 1 to 3, a step of preparing a sample by mixing the cerebrospinal fluid of the subject and an animal brain extract, a step of dropping the sample onto the dropping part of the immunochromatographic test kit, and a step of confirming the presence or absence of the autoantibody in the detection part. A method for detecting autoantibodies.

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