Kit for detecting AChR antibody and preparation method thereof

Abstract

The invention relates to a kit for detecting an AChR antibody and a preparation method thereof. The kit for detecting the AChR antibody comprises an antibody detection strip, a working solution, PBS (Phosphate Buffer Solution), a fluorescent secondary antibody and a mounting medium, wherein the antibody detection plate is a glass slide on which a GABABR antigen and modified chitosan are applied; the modified chitosan is chitosan modified by hyaluronic acid and gamma-polyglutamic acid. By using the modified chitosan, the binding force between the antigen protein and the carrier is obviously enhanced. Chitosan has good biocompatibility, through synergistic modification of hyaluronic acid and gamma-polyglutamic acid, the surface of chitosan shows higher hydrophilicity and stronger charge adsorption capacity, the chitosan can be more stably combined with antigen protein, the falling risk of the antigen protein in the washing and multi-step reaction process is remarkably reduced, and the anti-tumor property of the antigen protein is improved. Therefore, the effective utilization rate and the reaction efficiency of the antigen in detection are improved.

Description

Technical Field

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

[0002] The detection of AChR (acetylcholine receptor) antibodies is of great significance in the field of neuroimmunology, especially in the diagnosis of neuromuscular diseases such as myasthenia gravis. Existing antibody detection technologies, such as enzyme-linked immunosorbent assay (ELISA) and immunofluorescence, usually rely on the fixation of antigens and the binding of antibodies. However, existing technologies still face many challenges in terms of the adhesion and stability of antigen proteins, which affects the sensitivity and accuracy of the test results.

[0003] At present, commonly used antigen fixation methods include physical adsorption, chemical modification and cross-linking agent fixation. These methods usually rely on charge effect, hydrophobic effect or cross-linking reaction to fix the antigen on the carrier surface. However, these methods have some disadvantages. First, the binding force between the antigen and the carrier surface is insufficient, especially in the washing and reaction steps, the antigen is easy to fall off, affecting the accuracy and sensitivity of the detection. Secondly, the stability of the antigen protein is poor and is easily affected by environmental factors such as temperature and pH, resulting in denaturation or inactivation, thereby affecting the reliability of the test results. Finally, the existing antigen fixation methods usually require the use of complex chemical modification or cross-linking steps, which increases the difficulty of operation and production costs.

[0004] These problems indicate that there is still room for improvement in the existing technology in terms of antigen adhesion and stability, especially how to improve the adhesion and resistance to denaturation of antigen proteins on the carrier surface to improve the sensitivity and reliability of detection.

[0005] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0006] Based on this, the kit of the present invention uses modified chitosan as an antigen fixing material, and modifies chitosan in combination with hyaluronic acid and γ-polyglutamic acid, thereby significantly improving the adhesion and stability of the antigen protein. The modified chitosan provides more binding sites, thereby enhancing the binding force between the antigen and the carrier surface. The composite modification of hyaluronic acid and γ-polyglutamic acid not only enhances the adhesion of the antigen, but also improves the stability of the antigen protein, making it less susceptible to denaturation or inactivation during storage and operation. This design solves the problems of insufficient antigen adhesion and poor stability in the prior art, thereby improving the sensitivity and reliability of detection.

[0007] One object of the present invention is to provide a kit for detecting AChR antibodies, wherein the kit comprises: an antibody detection strip, a working solution, PBS, a fluorescent secondary antibody, and a sealing agent;

[0008] Wherein, the antibody detection plate is a glass slide spotted with GABABR antigen and modified chitosan;

[0009] The modified chitosan is chitosan modified by hyaluronic acid and gamma-polyglutamic acid.

[0010] Furthermore, positive controls and negative controls are included.

[0011] Furthermore, the working solution is a PBS buffer solution of bovine albumin.

[0012] Furthermore, the fluorescent secondary antibody is stained with fluorescein isothiocyanate.

[0013] Furthermore, the sealing medium is glycerol.

[0014] Furthermore, the negative control is 8-12 wt % goat serum.

[0015] Furthermore, the positive control is AChR antibody positive serum.

[0016] Specifically, the technical solution effectively improves the adhesion and stability of the antigen protein by adopting modified chitosan material, solves multiple technical bottlenecks in the prior art, thereby improving the sensitivity, stability and reliability of the antibody detection kit, and has broad application prospects.

[0017] The present invention also provides a method for preparing the kit for detecting AChR antibodies, comprising the following steps:

[0018] S1, soaking chitosan in a mixture of hyaluronic acid and γ-polyglutamic acid, heating and ultrasonicating, to obtain modified chitosan;

[0019] S2, mixing the AChR antigen and the modified chitosan, spotting the mixture on a glass slide, and cutting the mixture to form the antibody detection strip;

[0020] S3, packaging the antibody detection strip and other components to obtain the kit for detecting AChR antibodies.

[0021] Furthermore, in step S1, the heating temperature is 75-85°C.

[0022] Furthermore, the preparation process of the technical solution is relatively simple, does not require complex chemical cross-linking steps, reduces potential damage to antigen activity, and reduces production costs. The simplified preparation process makes the technology suitable for large-scale production and has good potential for promotion and application.

[0023] The present invention has the following beneficial effects:

[0024] The present invention significantly enhances the binding force between the antigen protein and the carrier by using modified chitosan. Chitosan itself has good biocompatibility. Through the synergistic modification of hyaluronic acid and γ-polyglutamic acid, its surface exhibits higher hydrophilicity and stronger charge adsorption capacity, and can form a more stable bond with the antigen protein, significantly reducing the risk of antigen protein falling off during washing and multi-step reaction processes, thereby improving the effective utilization rate and reaction efficiency of the antigen in detection.

[0025] Secondly, the introduction of hyaluronic acid and γ-polyglutamic acid not only improves the adhesion of antigen proteins, but also provides a stable microenvironment for antigens. Hyaluronic acid has excellent moisture retention and stability, which can reduce the impact of external temperature and humidity changes on antigen proteins, thereby reducing the probability of their denaturation and inactivation. γ-Polyglutamic acid provides stronger antioxidant capacity and ion buffering function, which can buffer the interference of external pH fluctuations on the activity of antigen proteins. The synergistic effect of the two significantly improves the structural stability and functional integrity of antigen proteins during storage and detection. This improvement effectively extends the shelf life of antigen proteins and ensures the long-term stability of the test kit. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] GABABR antigen, NY-002367P, was purchased from Nanjing Anyan.

[0032] γ-Polyglutamic acid, 92%, molecular weight 100 kDa, was purchased from Nanjing Xuankai Biotechnology Co., Ltd., China.

[0033] Example 1

[0034] A kit for detecting AChR antibodies, comprising the following components: 2 antibody detection strips, 1 working solution (10 g / 100 ml goat serum), 15 PBS (0.01 mol / L), 1 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 2 mounting media (glycerol), 1 positive control (AChR antibody positive serum), and 1 negative control (8 wt % goat serum);

[0035] The preparation method of the above-mentioned kit for detecting AChR antibodies comprises the following steps:

[0036] S1, soaking chitosan in a sufficient amount of a mixed solution of hyaluronic acid and γ-polyglutamic acid (1:1, m / m) (ensuring that the solute is 30wt%), ultrasonically heating at 80° C. for 8 h, filtering, washing, and drying to obtain the modified chitosan;

[0037] S2, mixing a PBS solution of 4 mg / ml MDA5 antigen and modified chitosan (mass ratio is 1:0.6), spotting on a glass slide at a temperature of 25° C. and a humidity of 40%, and cutting to form the antibody detection strip;

[0038] S3. Pack the antibody test strip and other components according to the above mass fractions and store them in cold storage to obtain the kit for detecting AChR antibodies.

[0039] Example 2

[0040] A kit for detecting AChR antibodies, comprising the following components: 2 antibody detection strips, 1 working solution (10 g / 100 ml goat serum), 15 PBS (0.01 mol / L), 1 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 2 mounting media (glycerol), 1 positive control (AChR antibody positive serum), and 1 negative control (8 wt % goat serum);

[0041] The preparation method of the above-mentioned kit for detecting AChR antibodies comprises the following steps:

[0042] S1, soaking chitosan in a sufficient amount of a mixed solution of hyaluronic acid and γ-polyglutamic acid (1:1, m / m) (ensuring that the solute is 30wt%), ultrasonically heating at 80° C. for 10 h, filtering, washing, and drying to obtain the modified chitosan;

[0043] S2, mixing 5 mg / ml of MDA5 antigen in PBS solution and modified chitosan (mass ratio is 1:0.5), spotting on a glass slide at a temperature of 30° C. and a humidity of 40%, and cutting to form the antibody detection strip;

[0044] S3. Pack the antibody test strip and other components according to the above mass fractions and store them in cold storage to obtain the kit for detecting AChR antibodies.

[0045] Example 3

[0046] A kit for detecting AChR antibodies, comprising the following components: 2 antibody detection strips, 1 working solution (10 g / 100 ml goat serum), 15 PBS (0.01 mol / L), 1 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 2 mounting media (glycerol), 1 positive control (AChR antibody positive serum), and 1 negative control (8 wt % goat serum);

[0047] The preparation method of the above-mentioned kit for detecting AChR antibodies comprises the following steps:

[0048] S1, soaking chitosan in a sufficient amount of a mixed solution of hyaluronic acid and γ-polyglutamic acid (1:1, m / m) (ensuring that the solute is 30wt%), ultrasonically heating at 80° C. for 10 h, filtering, washing, and drying to obtain the modified chitosan;

[0049] S2, mixing 5 mg / ml of MDA5 antigen in PBS solution and modified chitosan (mass ratio is 1:0.5), spotting on a glass slide at a temperature of 30° C. and a humidity of 40%, and cutting to form the antibody detection strip;

[0050] S3. Pack the antibody test strip and other components according to the above mass fractions and store them in cold storage to obtain the kit for detecting AChR antibodies.

[0051] Comparative Example 1

[0052] The difference between this comparative example and Example 1 is that in step S1, hyaluronic acid is removed and replaced by an equal mass of γ-polyglutamic acid, and the other components and preparation methods are the same as those in Example 1.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 1 is that: in step S1, γ-polyglutamic acid is removed and replaced by hyaluronic acid of equal mass, and other components and preparation methods are the same as those in Example 1.

[0055] Test Case

[0056] The antibody detection kits prepared in Example 1 and Comparative Examples 1-2 were used as kits for performance testing.

[0057] The antibody detection kits prepared in Example 1 and Comparative Examples 1-2 were used as test kits to detect 30 MDA5-negative sera and 30 MDA5-positive sera respectively;

[0058] Test method:

[0059] A1. Equilibrate room temperature: Take out the slide and place it at room temperature for 30 minutes;

[0060] A2. Sample incubation: Add 100 μl of the diluted serum sample to be tested to each well of the reaction plate in a fume hood, place the slide upside down on the reaction plate, and incubate at room temperature for 1 hour;

[0061] A3. Washing: Immerse the slide in a washing tank filled with PBS for 5 minutes, remove the slide, and wipe off the excess liquid on the edge and back with absorbent paper;

[0062] A4. Secondary antibody incubation: Add 100 μl of fluorescent secondary antibody to each well of a dry and clean reaction plate, quickly invert the slide onto the reaction plate, and incubate at room temperature for 1 hour;

[0063] A5. Washing: Immerse the slide in a washing tank filled with PBS for 5 minutes in a dark environment. Take out the slide and wipe off the excess liquid on the edge and back with absorbent paper.

[0064] A6. Sealing: Add 100 μl of sealing medium to each reaction area and cover with a coverslip;

[0065] A7. Observe the fluorescence signal: Read the green fluorescence result under the microscope, observe with a 20x objective lens, and analyze the result.

[0066] Table 1 Performance test results of antibody detection kits of Example 1 and Comparative Examples 1-2

[0067] project Example 1 Comparative Example 1 Comparative Example 2 Number of MDA5 positive serum samples 29 23 27 Number of MDA5 negative serum samples detected 28 22 25

[0068] It can be concluded from Table 1 that Example 1 of the present invention effectively improves the adhesion and stability of the antigen protein by adopting the modified chitosan material, solves the problems of insufficient antigen adhesion and poor stability in the prior art, and thus improves the sensitivity and reliability of detection.

[0069] 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.

[0070] 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 kit for detecting AChR antibodies, characterized in that: The kit for detecting AChR antibodies comprises: antibody detection strips, working solution, PBS, fluorescent secondary antibody, and sealing agent; Wherein, the antibody detection plate is a glass slide spotted with GABABR antigen and modified chitosan; The modified chitosan is chitosan modified by hyaluronic acid and gamma-polyglutamic acid.

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

3. The detection material for detecting GABABR antibodies according to claim 1, characterized in that: The working solution is PBS buffer containing bovine albumin.

4. The detection material for detecting GABABR antibodies according to claim 1, characterized in that: The fluorescent secondary antibody is stained with fluorescein isothiocyanate.

5. The detection material for detecting GABABR antibodies according to claim 1, characterized in that: The mounting medium is glycerol.

6. The detection material for detecting GABABR antibodies according to claim 2, characterized in that: The negative control is 8-12 wt % goat serum.

7. The kit for detecting AChR antibodies according to claim 2, characterized in that: The positive control is AChR antibody positive serum.

8. The method for preparing a kit for detecting AChR antibodies according to any one of claims 1 to 7, characterized in that: The steps include: S1, soaking chitosan in a mixture of hyaluronic acid and γ-polyglutamic acid, heating and ultrasonicating to obtain modified chitosan; S2, mixing the AChR antigen and the modified chitosan, spotting the mixture on a glass slide, and cutting the mixture to form the antibody detection strip; S3, packaging the antibody detection strip and other components to obtain the kit for detecting AChR antibodies.

9. The method for preparing a kit for detecting AChR antibodies according to claim 8, characterized in that: In step S1, the heating temperature is 75-85°C.