Detection material for detecting MDA5 antibody and preparation method thereof

Abstract

The invention relates to a detection material for detecting an MDA5 antibody and a preparation method of the detection material. The detection material for detecting the MDA5 antibody comprises an antibody detection strip, a washing solution and an auxiliary agent, wherein the antibody detection strip is a polystyrene board coated with an MDA5 antigen and modified sodium alginate; the modified sodium alginate is obtained by polymerizing allyl alcohol glycidyl ether modified sodium alginate and an acrylic monomer. Allyl alcohol glycidyl ether modified sodium alginate is polymerized with an acrylic monomer to form a modified sodium alginate material, and the modified sodium alginate material is coated on a polystyrene plate to immobilize MDA5 antigen protein. The modified sodium alginate material can be stably attached to the antigen protein in a chemical binding manner, so that the adhesive force of the antigen is remarkably enhanced. Meanwhile, the sodium alginate has good biocompatibility, the modified sodium alginate is more stable in structure, non-specific adsorption can be effectively reduced, and the sensitivity and accuracy of antibody detection are improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a detection material for detecting MDA5 antibodies and a preparation method thereof. Background Art

[0002] MDA5 (Melanoma Differentiation-Associated protein 5) is a pattern recognition receptor that plays an important role in antiviral immunity. It can recognize viral RNA and activate downstream immune response pathways. In medical diagnosis, the detection of MDA5 antibodies is of great significance because MDA5 antibodies are closely related to the occurrence and development of various autoimmune diseases (such as dermatomyositis, interstitial lung disease, etc.). Therefore, the detection of MDA5 antibodies has a wide range of application value in early diagnosis of the disease, disease monitoring and efficacy evaluation.

[0003] At present, the detection methods of MDA5 antibodies mainly include enzyme-linked immunosorbent assay (ELISA), Western blot and immunofluorescence. These methods provide different degrees of support for the detection of MDA5 antibodies, but there are still some technical problems in the adhesion of antigenic proteins, biocompatibility and stability of detection. Traditional detection methods usually rely on coating antigenic proteins on carriers such as glass slides or microplates, but since the binding between antigens and carriers is mostly physical adsorption, antigens are easily detached or non-specific binding occurs, which in turn affects the sensitivity and accuracy of the detection.

[0004] In the prior art, in order to improve the adhesion of antigen proteins, cross-linking agents or chemical modification methods are usually used to enhance the binding of antigens to carriers. However, these methods may cause antigen structural denaturation and affect the binding efficiency of antibodies and antigens. In addition, some chemical modification methods may reduce the biocompatibility of the detection system, increase the risk of nonspecific binding, and thus affect the reliability of the detection.

[0005] In the existing schemes for detecting MDA5 antibodies, the following technical problems exist:

[0006] Insufficient antigen protein adhesion: Traditional methods rely on physical adsorption or simple chemical fixation. This fixation method may cause the antigen protein to fall off or shift under certain conditions, affecting the sensitivity and stability of the test. Insufficient antigen adhesion will cause large fluctuations in experimental results, making them difficult to repeat, and reducing the accuracy of the test.

[0007] Poor biocompatibility of detection materials: Traditional fixing materials (such as ordinary glass slides, polystyrene, etc.) usually lack sufficient biocompatibility, which may increase nonspecific adsorption or inhibit the binding efficiency of antibodies and antigens, thereby reducing the accuracy of detection. Especially for low-concentration MDA5 antibodies, materials with poor biocompatibility are more difficult to meet the needs of high-sensitivity detection.

[0008] Poor detection stability: Due to the loose attachment of antigen proteins, some existing detection schemes are prone to protein shedding and loss of activity during use or storage, resulting in insufficient stability of the test kit. This instability will make the experimental results unreliable and difficult to meet the needs of high-precision and high-repeatability detection.

[0009] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0010] Based on this, the kit of the present invention forms a modified sodium alginate material by polymerizing sodium alginate modified by allyl alcohol glycidyl ether and acrylic acid monomer, and is coated on a polystyrene plate to fix the MDA5 antigen protein. The modified sodium alginate material can firmly attach the antigen protein by chemical bonding, significantly enhancing the adhesion of the antigen. At the same time, sodium alginate has good biocompatibility, and its structure is more stable after modification, which can effectively reduce nonspecific adsorption and improve the sensitivity and accuracy of antibody detection.

[0011] One object of the present invention is to provide a detection material for detecting MDA5 antibodies, wherein the detection material for detecting MDA5 antibodies comprises: an antibody detection strip, a washing solution, and an auxiliary agent;

[0012] Wherein, the antibody detection strip is a polystyrene plate coated with MDA5 antigen and modified sodium alginate;

[0013] The modified sodium alginate is obtained by polymerizing sodium alginate modified with allyl alcohol glycidyl ether and acrylic acid monomer.

[0014] Furthermore, the auxiliary agent is selected from one or more of a diluent, an enzyme-labeled secondary antibody, a substrate solution, and a buffer solution.

[0015] Furthermore, the enzyme-labeled secondary antibody is goat anti-human IgG labeled with horseradish peroxidase.

[0016] Furthermore, the substrate solution is selected from BCIP / NBT color developing solution.

[0017] Furthermore, the material of the diluent is PBS buffer containing sodium chloride, preservatives and Tween 20.

[0018] Furthermore, positive controls and negative controls are included.

[0019] Furthermore, the negative control is 8 wt % goat serum.

[0020] Furthermore, the positive control is anti-MDA5 antibody positive serum.

[0021] The present invention also provides a method for preparing the detection material for detecting MDA5 antibodies, comprising the following steps:

[0022] S1, adding allyl alcohol glycidyl ether to the sodium alginate solution, heating for reaction, and obtaining an intermediate product;

[0023] S2, mixing acrylic acid, the intermediate product, an emulsifier and an initiator, and heating them for reaction to obtain the modified sodium alginate;

[0024] S3, mixing the MDA5 antigen and modified sodium alginate, coating them on a polystyrene plate, and cutting them to form an antibody detection strip;

[0025] S4, packaging the antibody detection strip and other components to obtain the detection material for detecting MDA5 antibodies.

[0026] Specifically, sodium alginate, as a natural polysaccharide, has good biocompatibility, and its modified structure is more stable, which helps to reduce nonspecific adsorption during the detection process. This can reduce background interference, improve the binding efficiency of antibodies and antigens, thereby increasing the sensitivity and accuracy of the detection, and is suitable for the detection of MDA5 antibodies.

[0027] The present invention also provides a kit, comprising the detection material for detecting MDA5 antibodies.

[0028] The present invention has the following beneficial effects:

[0029] The antibody detection kit of the present invention adopts allyl alcohol glycidyl ether and acrylic acid monomer polymerization by using modified sodium alginate, so that the material surface has stronger chemical binding ability. The modified material can firmly fix the MDA5 antigen protein, thereby significantly improving the adhesion of the antigen on the polystyrene plate, reducing the antigen shedding phenomenon during the detection process, and improving the accuracy and stability of the test results. In addition, the modified sodium alginate material not only enhances the adhesion of the antigen, but also maintains the activity and stability of the antigen, ensuring that the test kit maintains consistent detection performance during long-term storage and multiple uses. This high stability and repeatability provide more reliable technical support for clinical diagnosis, and can effectively improve the repeatability and consistency of the experimental results. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the art. These techniques have been fully described in existing literature.

[0034] The MDA5 antigen described herein is known in the art, and its amino acid sequence is known in the art and can be found in professional databases such as NCBI.

[0035] MDA5 antigen was purchased from Beijing Aibosheng Biotechnology Co., Ltd., catalog number Q9BYX4.

[0036] Horseradish peroxidase-labeled goat anti-human IgG was purchased from Beijing Borsi Technology Co., Ltd.

[0037] Emulsifier, composed of span80 and tween60 in a mass ratio of 1:1.

[0038] Example 1

[0039] A detection material for detecting MDA5 antibodies, comprising the following components: 2 antibody detection strips, 2 washing solutions (the washing solution is a PBS buffer solution containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, and Tween 20, with a pH value of 7.2 and a concentration of 15 mM, and a mass ratio of 9:1:0.3:0.1:10), 2 enzyme-labeled secondary antibodies (horseradish peroxidase-labeled goat anti-human IgG), 1 substrate solution (BCIP / NBT color developing solution), 2 diluents (containing sodium chloride, Proclin 300 preservative and Tween 20, with a pH value of 7.2 and a concentration of 15 mM, and a mass ratio of 9:0.1:3:10), 1 positive control (anti-MDA5 antibody positive serum), and 1 negative control (8wt% goat serum);

[0040] The preparation method of the above-mentioned detection material for detecting MDA5 antibodies comprises the following steps:

[0041] S1. Add 10 parts of sodium alginate to 300 parts of deionized water, adjust the pH to 8.5 with 50wt% sodium hydroxide solution, add 8 parts of allyl alcohol glycidyl ether, heat to 75°C for 8h, cool, adjust the solution pH to 4 with 50wt% glacial acetic acid, add acetone and let stand for 12h, filter after precipitation, let stand for 12h with acetone, filter and dry to obtain an intermediate product;

[0042] S2, heating a mixture containing 0.3 parts of emulsifier and 6 parts of aviation kerosene to 50°C, heating a mixed solution containing 20 parts of acrylic acid, 0.5 parts of intermediate product and 0.05g of ammonium persulfate to 70°C, stirring and reacting for 30 minutes, and then heating to 80°C and stirring and reacting for 30 minutes, adding 0.5g of Tween-80 after cooling and stirring evenly, and removing the solvent to obtain modified sodium alginate;

[0043] S3, after mixing the MDA5 antigen and modified sodium alginate (mass ratio of 1:0.2), respectively coated them on polystyrene plates at a temperature of 25° C. and a humidity of 40%, and cut them into antibody detection strips;

[0044] S4. Pack the antibody test strip and other components according to the above mass fractions and store them in cold storage to obtain the detection material for detecting MDA5 antibodies.

[0045] Example 2

[0046] A detection material for detecting MDA5 antibodies, comprising the following components: 2 antibody detection strips, 2 washing solutions (the washing solution is a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, and Tween 20, with a pH value of 7.2 and a concentration of 15 mM, and a mass ratio of 9:1:0.3:0.1:12), 2 enzyme-labeled secondary antibodies (horseradish peroxidase-labeled goat anti-human IgG), 1 substrate solution (BCIP / NBT color developing solution), 2 diluents (containing sodium chloride, Proclin 300 preservative and Tween 20, with a pH value of 7.4 and a concentration of 15 mM, and a mass ratio of 9:0.1:3:15), 1 positive control (anti-MDA5 antibody positive serum), and 1 negative control (8wt% goat serum);

[0047] The preparation method of the above-mentioned detection material for detecting MDA5 antibodies comprises the following steps:

[0048] S1. Add 10 parts of sodium alginate to 300 parts of deionized water, adjust the pH to 8.5 with 50wt% sodium hydroxide solution, add 8 parts of allyl alcohol glycidyl ether, heat to 75°C for 8h, cool, adjust the solution pH to 4 with 50wt% glacial acetic acid, add acetone and let stand for 12h, filter after precipitation, let stand for 12h with acetone, filter and dry to obtain an intermediate product;

[0049] S2, heating a mixture containing 0.3 parts of emulsifier and 6 parts of aviation kerosene to 50°C, heating a mixed solution containing 20 parts of acrylic acid, 0.5 parts of intermediate product and 0.05g of ammonium persulfate to 70°C, stirring and reacting for 30 minutes, and then heating to 80°C and stirring and reacting for 30 minutes, adding 0.5g of Tween-80 after cooling and stirring evenly, and removing the solvent to obtain modified sodium alginate;

[0050] S3, after mixing the MDA5 antigen and modified sodium alginate (mass ratio of 1:0.5), respectively coated them on polystyrene plates at a temperature of 25° C. and a humidity of 40%, and cut them into antibody detection strips;

[0051] S4. Pack the antibody test strip and other components according to the above mass fractions and store them in cold storage to obtain the detection material for detecting MDA5 antibodies.

[0052] Example 3

[0053] A detection material for detecting MDA5 antibodies, comprising the following components: 2 antibody detection strips, 2 washing solutions (the washing solution is a PBS buffer solution containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, and Tween 20, with a pH value of 7.2 and a concentration of 15 mM, and a mass ratio of 9:1:0.3:0.1:10), 2 enzyme-labeled secondary antibodies (horseradish peroxidase-labeled goat anti-human IgG), 1 substrate solution (BCIP / NBT color developing solution), 2 diluents (containing sodium chloride, Proclin 300 preservative and Tween 20, with a pH value of 7.2 and a concentration of 15 mM, and a mass ratio of 9:0.1:3:10), 1 positive control (anti-MDA5 antibody positive serum), and 1 negative control (8wt% goat serum);

[0054] The preparation method of the above-mentioned detection material for detecting MDA5 antibodies comprises the following steps:

[0055] S1. Add 10 parts of sodium alginate to 300 parts of deionized water, adjust the pH to 8.5 with 50wt% sodium hydroxide solution, add 8 parts of allyl alcohol glycidyl ether, heat to 75°C for 8h, cool, adjust the solution pH to 4 with 50wt% glacial acetic acid, add acetone and let stand for 12h, filter after precipitation, let stand for 12h with acetone, filter and dry to obtain an intermediate product;

[0056] S2, heating a mixture containing 0.3 parts of emulsifier and 6 parts of aviation kerosene to 50°C, heating a mixed solution containing 20 parts of acrylic acid, 0.5 parts of intermediate product and 0.05g of ammonium persulfate to 70°C, stirring and reacting for 30 minutes, and then heating to 80°C and stirring and reacting for 30 minutes, adding 0.5g of Tween-80 after cooling and stirring evenly, and removing the solvent to obtain modified sodium alginate;

[0057] S3, after mixing the MDA5 antigen and modified sodium alginate (mass ratio of 1:0.3), respectively coated them on polystyrene plates at a temperature of 25° C. and a humidity of 35%, and cut them into antibody detection strips;

[0058] S4. Pack the antibody test strip and other components according to the above mass fractions and store them in cold storage to obtain the detection material for detecting MDA5 antibodies.

[0059] Comparative Example 1

[0060] Comparative Example 1

[0061] The difference between this comparative example and Example 1 is that acrylic acid is removed in step S2, and only the intermediate product is used for reaction; other components and preparation methods are the same as those in Example 1.

[0062] Test Case

[0063] The detection materials for detecting MDA5 antibodies prepared in Example 1 and Comparative Example 1 were used as a kit for performance testing.

[0064] The detection materials for detecting MDA5 antibodies prepared in Example 1 and Comparative Example 1 were used as kits to detect 30 MDA5 antibody sera and 30 MDA5 antibody positive sera respectively;

[0065] Test method:

[0066] A1. Place the antibody test strip at 4°C overnight, wash with PBST, and repeat 5 times;

[0067] A2. Blocking: Add 400ul of 5wt% BSA to each well of a polystyrene plate (96-well plate), incubate at room temperature for 2h, then wash with PBST, repeat 5 times;

[0068] A3. Add 100ul of serum to each well and incubate at room temperature for 1 hour;

[0069] A4. Add enzyme-labeled secondary antibody, incubate at room temperature for 30 min, wash with PBST, and repeat 5 times;

[0070] A5. Add substrate solution and color for 15 minutes;

[0071] A6. Stop the reaction, read the results of the antibody test strip, and use a 450 / 630nm microplate reader to read and analyze the results.

[0072] Table 1 Kit performance test results of Example 1 and Comparative Example 1

[0073] project Example 1 Comparative Example 1 Number of MDA5 antibody positive sera detected 29 24 Number of MDA5 antibody negative sera detected 28 26

[0074] It can be concluded from Table 1 that the detection material for detecting MDA5 antibodies in Example 1 of the present invention not only improves the adhesion and biocompatibility of the MDA5 antigen, but also optimizes the stability and sensitivity of the detection, providing an efficient and reliable technical guarantee for the accurate detection of the MDA5 antibody.

[0075] 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.

[0076] 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 detection material for detecting MDA5 antibodies, characterized in that: The detection materials for detecting MDA5 antibodies include: antibody detection strips, washing liquid, and auxiliary agents; Wherein, the antibody detection strip is a polystyrene plate coated with MDA5 antigen and modified sodium alginate; The modified sodium alginate is obtained by polymerizing sodium alginate modified with allyl alcohol glycidyl ether and acrylic acid monomer.

2. The detection material for detecting MDA5 antibodies according to claim 1, characterized in that: The auxiliary agent is selected from one or more of a diluent, an enzyme-labeled secondary antibody, a substrate solution, and a buffer solution.

3. The detection material for detecting MDA5 antibodies according to claim 2, characterized in that: The enzyme-labeled secondary antibody is goat anti-human IgG labeled with horseradish peroxidase.

4. The detection material for detecting MDA5 antibodies according to claim 2, characterized in that: The substrate solution is selected from BCIP / NBT color development solution.

5. The detection material for detecting MDA5 antibodies according to claim 2, characterized in that: The material of the diluent is PBS buffer containing sodium chloride, preservatives and Tween 20.

6. The detection material for detecting MDA5 antibodies according to claim 1, characterized in that: Positive and negative controls were also included.

7. The detection material for detecting MDA5 antibodies according to claim 6, characterized in that: The negative control was 8 wt % goat serum.

8. The detection material for detecting MDA5 antibodies according to claim 6, characterized in that: The positive control is anti-MDA5 antibody positive serum.

9. The method for preparing the detection material for detecting MDA5 antibodies according to any one of claims 1 to 8, characterized in that: The steps include: S1, adding allyl alcohol glycidyl ether to the sodium alginate solution, heating for reaction, and obtaining an intermediate product; S2, mixing acrylic acid, the intermediate product, an emulsifier and an initiator, and heating them for reaction to obtain the modified sodium alginate; S3, mixing the MDA5 antigen and modified sodium alginate, coating them on a polystyrene plate, and cutting them to form an antibody detection strip; S4, packaging the antibody detection strip and other components to obtain the detection material for detecting MDA5 antibodies.

10. A kit, characterized in that: The invention comprises the detection material for detecting MDA5 antibody according to any one of claims 1 to 8.