Antibody detection kit for neuroparatoma syndrome and preparation method of antibody detection kit

Abstract

The invention relates to an antibody detection kit for neuroparatoma syndrome and a preparation method of the antibody detection kit. The antibody detection kit for neuroparatoma syndrome comprises an antibody detection strip, a cleaning solution, a buffer solution and an auxiliary agent, wherein the antibody detection strip is a nitrocellulose membrane which is coated with antigen proteins containing Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recovery, Ri, SOX1, Titin, Tr (DNER), Yo and Zic4. The invention further discloses a detection method of the antibody detection strip. The antibody detection kit disclosed by the invention can be used for simultaneously detecting 12 autoantibodies related to the neuroparatoma syndrome. The design of simultaneous detection of multiple antibodies greatly improves the comprehensiveness of detection, so that multiple possible pathogenic factors can be covered in single detection, the kit can be used for differential diagnosis of suspected neuroparatoma syndrome patients in professional laboratories, and the problem of missed diagnosis caused by single antibody detection is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of cell detection, in particular to an antibody detection kit for paraneoplastic neurological syndrome and a preparation method thereof. Background Art

[0002] Paraneoplastic syndrome refers to abnormal immune response or other unknown reasons caused by tumor products, which can cause lesions in the nervous, kidney and skin systems, and corresponding clinical manifestations. These manifestations are not directly caused by the location of the primary tumor or metastasis, but indirectly caused by the distant effects of the tumor. The distant organs affected, such as the nervous system, are also called paraneoplastic syndrome of the nervous system. For example, lung cancer and ovarian cancer can show distant effects such as gray matter inflammation and neurodegeneration in the central nervous system. Paraneoplastic syndrome of the nervous system is a group of heterogeneous diseases with nervous system disorders accompanied by tumors or distant metastases of tumors. However, tumors are not the cause of the disease, and abnormal autoimmune reactions are considered to be its potential pathophysiological basis. Specific neuronal antibodies can be detected in most patients with paraneoplastic syndrome. Even if the patient has not yet developed tumor symptoms, the appearance of classic neuronal antibodies, such as anti-Hu antibodies, anti-Yo antibodies, anti-Ri antibodies, anti-CV2 (CRMP5) antibodies, and anti-Amphiphysin antibodies, is highly suggestive of the possibility of occult tumors. In two-thirds of cases, the patient's neurological symptoms may appear five years earlier than the tumor. Therefore, the detection of paraneoplastic syndrome antibodies can detect the presence of tumors in advance.

[0003] The existing detection methods on the market currently require cumbersome preparation steps. The existing detection methods can usually only detect a certain antibody and cannot achieve simultaneous detection of multiple antibodies. It is difficult to fully reflect the antibody spectrum of patients with paraneoplastic syndrome, which limits its wide application in clinical practice.

[0004] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0005] Based on this, the kit of the present invention provides a basis for the diagnosis and differential diagnosis of neurological paraneoplastic syndrome by detecting 12 autoantibodies, including Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4.

[0006] One object of the present invention is to provide an antibody detection kit for paraneoplastic neurological syndrome, wherein the antibody detection kit for paraneoplastic neurological syndrome comprises: an antibody detection strip, a washing solution, a buffer solution, and an auxiliary agent;

[0007] The antibody detection strip is a nitrocellulose membrane coated with antigen proteins containing Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4.

[0008] Furthermore, the auxiliary agent is selected from one or more of enzyme conjugates and substrate solutions.

[0009] Furthermore, the enzyme conjugate is selected from IgG labeled with alkaline phosphatase.

[0010] Furthermore, the substrate solution is selected from BCIP / NBT color developing solution.

[0011] Furthermore, the material of the washing solution is a PBS buffer solution containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and Tween 20.

[0012] Furthermore, the material of the buffer solution is a PBS buffer solution containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, Tween 20 and albumin.

[0013] Furthermore, the fluorescent secondary antibody is stained with fluorescein isothiocyanate.

[0014] Furthermore, positive controls and negative controls are included.

[0015] Furthermore, the negative control is a protein protectant.

[0016] Furthermore, the positive control is a positive goat serum and a protein protective agent containing recombinant antigens of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 proteins.

[0017] The present invention also provides a method for preparing the antibody detection kit for neurological paraneoplastic syndrome, comprising the following steps:

[0018] The antigen proteins of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 were coated onto nitrocellulose membranes respectively and the order was recorded, and the antibody detection strips were formed after cutting;

[0019] The antibody detection strip and other components are packaged to obtain the antibody detection kit for paraneoplastic neurological syndrome.

[0020] The present invention has the following beneficial effects:

[0021] The antibody detection kit of the present invention can simultaneously detect 12 autoantibodies related to neurological paraneoplastic syndrome, including Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 antigen proteins. This design of simultaneous detection of multiple antibodies greatly improves the comprehensiveness of the detection, so that a variety of possible pathogenic factors can be covered in a single detection, and can be used in professional laboratories for differential diagnosis of patients with suspected neurological paraneoplastic syndrome, avoiding the problem of missed diagnosis caused by single antibody detection. In addition, the present invention can simultaneously detect 12 autoantibodies related to neurological paraneoplastic syndrome in the same system. This multiple detection method can comprehensively reflect the antibody spectrum of patients with myasthenia gravis, reduce the sample size and detection time, reduce the detection cost, and improve the detection efficiency and clinical application value. Through the kit, multiple antibody results can be obtained at one time, which is helpful for early detection, accurate typing and targeted treatment of the disease. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0023] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0024] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, the amounts of ingredients used in the specification and claims should be understood to be modified in all instances by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary depending on the desired properties to be obtained by the present invention.

[0025] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques have been fully described in existing literature.

[0026] The antigenic proteins of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 described in this article are known in the art, and their amino acid sequences are known in the art and can be found in professional databases such as NCBI.

[0027] The antigen proteins of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 were purchased from Beijing Jingyi Medical Testing Laboratory Co., Ltd.

[0028] Alkaline phosphatase-labeled IgG was purchased from Shanghai Zeye Biotechnology Co., Ltd.

[0029] Protein protective agent, ABS01-0250, was purchased from Zhengzhou Sairun Biotechnology Co., Ltd.

[0030] Example 1

[0031] A neurological paraneoplastic syndrome antibody detection kit, the neurological paraneoplastic syndrome antibody detection kit comprising the following components: 24 antibody detection strips, 1 washing liquid (the washing liquid is a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and Tween 20, with a mass ratio of 9:1:0.3:0.1:10), 1 buffer (a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, Tween 20, and bovine serum albumin, with a mass ratio of 9:1:0.3:0.1:0.5:10), 1 enzyme-bound 1 sample (IgG labeled with alkaline phosphatase), 1 sample of substrate solution (BCIP / NBT colorimetric solution), 1 sample of positive control (positive goat serum and protein protective agent containing recombinant antigens of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 proteins, with a mass ratio of 1:0.1), 1 sample of negative control (protein protective agent), and 1 sample of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC));

[0032] The antibody detection strip is a nitrocellulose membrane coated with antigen proteins containing Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4.

[0033] The preparation method of the above-mentioned antibody detection kit for neurological paraneoplastic syndrome comprises the following steps:

[0034] The antigen proteins of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 were coated on nitrocellulose membranes at 25°C and 40% humidity, and the order was recorded, and the antibody detection strips were formed after cutting;

[0035] The antibody detection strip and other components are packaged according to the above mass fractions and stored in cold storage to obtain the antibody detection kit for neurological paraneoplastic syndrome.

[0036] Example 2

[0037] A neurological paraneoplastic syndrome antibody detection kit, the neurological paraneoplastic syndrome antibody detection kit comprising the following components: 24 antibody detection strips, 1 washing liquid (the washing liquid is a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and Tween 20, with a mass ratio of 9:1:0.3:0.1:10), 1 buffer (a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, Tween 20, and bovine serum albumin, with a mass ratio of 9:1:0.3:0.1:0.5:10), 1 enzyme-bound 1 sample (IgG labeled with alkaline phosphatase), 1 sample of substrate solution (BCIP / NBT colorimetric solution), 1 sample of positive control (positive goat serum and protein protective agent containing recombinant antigens of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 proteins, with a mass ratio of 1:0.1), 1 sample of negative control (protein protective agent), and 1 sample of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC));

[0038] The antibody detection strip is a nitrocellulose membrane coated with antigen proteins containing Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4.

[0039] The preparation method of the above-mentioned antibody detection kit for neurological paraneoplastic syndrome comprises the following steps:

[0040] The antigen proteins of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 were coated on nitrocellulose membranes at 25°C and 40% humidity, and the order was recorded, and the antibody detection strips were formed after cutting;

[0041] The antibody detection strip and other components are packaged according to the above mass fractions and stored in cold storage to obtain the antibody detection kit for neurological paraneoplastic syndrome.

[0042] Example 3

[0043] A neurological paraneoplastic syndrome antibody detection kit, the neurological paraneoplastic syndrome antibody detection kit comprising the following components: 24 antibody detection strips, 1 washing liquid (the washing liquid is a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and Tween 20, with a mass ratio of 9:1:0.3:0.1:10), 1 buffer (a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, Tween 20, and bovine serum albumin, with a mass ratio of 9:1:0.3:0.1:0.5:10), 1 enzyme-bound 1 sample (IgG labeled with alkaline phosphatase), 1 sample of substrate solution (BCIP / NBT colorimetric solution), 1 sample of positive control (positive goat serum and protein protective agent containing recombinant antigens of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 proteins, with a mass ratio of 1:0.1), 1 sample of negative control (protein protective agent), and 1 sample of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC));

[0044] The antibody detection strip is a nitrocellulose membrane coated with antigen proteins containing Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4.

[0045] The preparation method of the above-mentioned antibody detection kit for neurological paraneoplastic syndrome comprises the following steps:

[0046] The antigen proteins of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 were coated on nitrocellulose membranes at 30°C and 35% humidity, and the order was recorded, and the antibody detection strips were formed after cutting;

[0047] The antibody detection strip and other components are packaged according to the above mass fractions and stored in cold storage to obtain the antibody detection kit for neurological paraneoplastic syndrome.

[0048] Comparative Example 1

[0049] A neurological paraneoplastic syndrome antibody detection kit is prepared according to the preparation method of the embodiment of the Chinese invention patent with publication number CN113252906A to obtain a kit for detecting Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 antigen proteins.

[0050] Test Case

[0051] The antibody detection kits for paraneoplastic syndrome prepared in Example 1 and Comparative Example 1 were used as kits for performance testing.

[0052] The antibody detection kits for neurological paraneoplastic syndrome prepared in Example 1 and Comparative Example 1 were used as kits to detect 30 negative sera of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4, and 30 positive sera of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4;

[0053] Test method:

[0054] A1. Dilute the washing buffer with deionized water (1:20, v / v), incubate at 37°C for 30 min to dissolve the crystals, and store at 2-8°C for later use;

[0055] A2. Add 1 mL of dilution buffer to the incubation tank to ensure that the antibody test strip is completely covered, add 5 μL of negative serum or positive serum to each reaction tank, and then incubate on a shaker at room temperature for 30 minutes;

[0056] A3. Use a pipette to carefully aspirate or gently pour out the liquid in the incubation tank, then add 1 mL of wash buffer and shake for 30 seconds. Repeat 5 times.

[0057] A4. Add 1 mL of enzyme conjugate and substrate solution to each incubation tank and incubate on a shaker for 30 min at room temperature.

[0058] A5. Use a pipette to carefully aspirate or gently pour out the liquid in the incubation tank, then add 1 mL of wash buffer and shake for 30 seconds. Repeat 5 times.

[0059] A6. Add 1 mL of substrate solution to each incubation tank. Incubate for 20 min at room temperature until the bands on the antibody detection strip are clearly visible. Use a pipette to carefully aspirate or gently pour out the liquid in the incubation tank. Then add 1 mL of H2O and shake for 30 s. Repeat 5 times.

[0060] A7. Place the membrane strip on absorbent paper and wait for it to dry completely, then read the result of the antibody test strip and analyze the result.

[0061] Table 1 Kit performance test results of Example 1 and Comparative Example 1

[0062]

[0063] According to Table 1, it can be concluded that the antibody detection kit for neurological paraneoplastic syndrome of Example 1 of the present invention can effectively detect 12 different antibodies, including Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4.

[0064] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0065] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A neurological paraneoplastic syndrome antibody detection kit, characterized in that: The antibody detection kit for neurological paraneoplastic syndrome comprises: an antibody detection strip, a washing solution, a buffer solution, and an auxiliary agent; The antibody detection strip is a nitrocellulose membrane coated with antigen proteins containing Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4.

2. The antibody detection kit for paraneoplastic neurological syndrome according to claim 1, characterized in that: The auxiliary agent is selected from one or more of enzyme conjugates, substrate solution, and fluorescent secondary antibodies.

3. The antibody detection kit for paraneoplastic neurological syndrome according to claim 1, characterized in that: The enzyme conjugate is selected from alkaline phosphatase labeled IgG.

4. The antibody detection kit for paraneoplastic neurological syndrome according to claim 1, characterized in that: The substrate solution is selected from BCIP / NBT color development solution.

5. The antibody detection kit for paraneoplastic neurological syndrome according to claim 1, characterized in that: The material of the washing solution is PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate and Tween 20.

6. The antibody detection kit for paraneoplastic neurological syndrome according to claim 1, characterized in that: The material of the buffer solution is a PBS buffer solution containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, Tween 20 and albumin.

7. The antibody detection kit for paraneoplastic neurological syndrome according to claim 1, characterized in that: Positive and negative controls were also included.

8. The antibody detection kit for paraneoplastic neurological syndrome according to claim 7, characterized in that: The negative control is a protein protectant.

9. The antibody detection kit for paraneoplastic neurological syndrome according to claim 7, characterized in that: The positive control is a positive goat serum and a protein protective agent containing recombinant antigens of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 proteins.

10. The method for preparing the antibody detection kit for paraneoplastic neurological syndrome according to any one of claims 1 to 9, characterized in that: The steps include: The antigen proteins of Amphiphysin, CV2, GAD65, Hu, PNMA2 (Ma2 / Ta), Recoverin, Ri, SOX1, Titin, Tr (DNER), Yo, and Zic4 were coated onto nitrocellulose membranes respectively and the order was recorded, and the antibody detection strips were formed after cutting; The antibody detection strip and other components are packaged to obtain the antibody detection kit for paraneoplastic neurological syndrome.

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