Anti-F-actin antibody detection kit and preparation method thereof

Abstract

The invention relates to a detection kit for an anti-F-actin antibody and a preparation method of the detection kit. The detection kit for the anti-F-actin antibody comprises an antibody detection strip, a labeled antibody and an auxiliary agent, wherein the antibody detection strip is provided with modified hydroxyapatite adsorbed with F-actin antigen protein in a sample application manner; the modified hydroxyapatite is hydroxyapatite which is partially grafted with gamma-polyglutamic acid and partially grafted with hyaluronic acid. According to the antibody detection kit disclosed by the invention, the polylysine is grafted on the hydroxyapatite part, and the binding force of antigen protein and a carrier is remarkably enhanced by the modified hydroxyapatite. The polylysine has relatively strong hydrophilicity and electropositivity, and can interact with negative charges on the surface of the antigen to effectively prevent the antigen from falling off, so that the adhesive force and the detection stability of the antigen protein are improved.

Description

Technical Field

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

[0002] F-actin (fibrous actin), as an important component of the cytoskeleton, is widely involved in various physiological processes such as cell morphology maintenance, movement, and division. Therefore, the study of F-actin is of great significance in the fields of cell biology, medical diagnosis, and biopharmaceuticals. Detecting the presence and activity of F-actin antibodies is a key step in studying the function of F-actin, disease diagnosis, and related drug development. At present, some progress has been made in the detection methods for F-actin antibodies, mainly focusing on enzyme-linked immunosorbent assay (ELISA), Western blot, and immunofluorescence staining. These methods provide certain technical support for the detection of F-actin antibodies, but there are still some shortcomings, especially in terms of the adhesion of antigenic proteins, detection sensitivity, compatibility and stability of the kit.

[0003] In the prior art, some detection methods use solid phase carriers (such as microplates, magnetic beads, etc.) as carriers for antigen fixation, but these solid phase carriers are often limited by surface properties, resulting in weak adhesion of antigen proteins, which in turn affects the accuracy and repeatability of the test results. In addition, the carrier materials in the prior art usually have low biocompatibility, which may have an adverse effect on the binding of antibodies, the sensitivity of the detection reaction, and the stability of the experimental results.

[0004] Although the existing anti-F-actin antibody detection methods have accumulated certain technical experience, the following major technical problems still exist: 1. The antigen protein has weak adhesion. Traditional solid phase carriers often rely on physical adsorption or simple chemical modification to fix antigens. This attachment method is easily affected by the external environment (such as ionic strength, pH, etc.), resulting in the shedding of antigen proteins or non-specific binding, affecting the accuracy and sensitivity of the test results.

[0005] 2. Poor biocompatibility of the carrier. Some existing carrier materials, such as polystyrene and metal materials, usually have low affinity for molecules in the body (such as antibodies, antigens, etc.), which may lead to low efficiency of antibody-antigen binding. In addition, some materials may trigger immune responses or be incompatible with the cell environment, affecting the reliability of the experiment.

[0006] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0007] Based on this, the kit of the present invention effectively improves the adhesion and compatibility of the antigen protein, and at the same time has high sensitivity and stability, providing a more reliable technical means for the accurate detection of F-actin antibodies.

[0008] One object of the present invention is to provide a detection kit for anti-F-actin antibodies, the detection kit for anti-F-actin antibodies comprising: an antibody detection strip, a labeled antibody, and an auxiliary agent;

[0009] Wherein, the antibody detection strip is spotted with modified hydroxyapatite adsorbed with F-actin antigen protein;

[0010] The modified hydroxyapatite is hydroxyapatite partially grafted with gamma-polyglutamic acid and partially grafted with hyaluronic acid.

[0011] Furthermore, the labeled antibody is an acridinium ester labeled antibody.

[0012] Furthermore, the auxiliary agent is selected from one or more of a diluent, a buffer solution, a substrate developer, and a washing solution.

[0013] Furthermore, the substrate solution is selected from BCIP / NBT color developing solution.

[0014] Furthermore, the material of the washing solution is a PBS buffer containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, glycerol, and TritonX-100.

[0015] Furthermore, the material of the diluent is PBS buffer containing sodium chloride and TritonX-100.

[0016] Furthermore, positive controls and negative controls are included.

[0017] Furthermore, the negative control is 8-12 wt % goat serum.

[0018] Furthermore, the positive control is serum containing F-actin antibodies.

[0019] The present invention also provides a method for preparing the detection kit of the anti-F-actin antibody, comprising the following steps:

[0020] S1. adding the hydroxyapatite, γ-polyglutamic acid and hyaluronic acid into a solvent, heating and ultrasonicating, and purifying to obtain the modified hydroxyapatite;

[0021] S2. After mixing the F-actin antigen protein with modified hydroxyapatite, the mixture is coated on a nitrocellulose membrane, and then cut to form an antibody detection strip;

[0022] S3. Packing the antibody detection strip and other components to obtain the anti-F-actin antibody detection kit.

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

[0024] The antibody detection kit of the present invention significantly enhances the binding force between the antigen protein and the carrier by grafting polylysine on the hydroxyapatite part. Polylysine has strong hydrophilicity and positive charge, and can interact with the negative charge on the surface of the antigen, effectively preventing the antigen from falling off, thereby improving the adhesion of the antigen protein and the detection stability. In addition, hyaluronic acid, as a natural polysaccharide, has good biocompatibility and biodegradability. After grafting hyaluronic acid, the modified hydroxyapatite can better adapt to the biological system, reduce nonspecific adsorption, enhance the binding efficiency of the antibody and the antigen, and further improve the sensitivity and accuracy of the detection.

[0025] Since the surface modification of modified hydroxyapatite can effectively improve the adhesion of antigen proteins and the binding efficiency of antibodies, the kit has higher sensitivity when detecting low-concentration antibodies. At the same time, the modified hydroxyapatite has high stability, which can ensure the stability of the performance of the kit during long-term storage or use, further improving the reliability and repeatability of the experiment. 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] F-actin antigen protein was purchased from Nanjing Anyan.

[0031] γ-Polyglutamic acid, 92%, molecular weight 100 kDa, was purchased from Nanjing Xuankai Biotechnology Co., Ltd., China.

[0032] Goat anti-human IgG was purchased from Sigma.

[0033] Example 1

[0034] A detection kit for anti-F-actin antibodies, comprising the following components: 2 antibody detection strips, 2 labeled antibodies (acridinium ester labeled antibodies); 2 washing solutions (the washing solution is made of PBS buffer containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, glycerol, and TritonX-100, with a mass ratio of 9:0.5:0.3:1:0.5:10), 2 diluents (PBS buffer containing sodium chloride and TritonX-100, with a mass ratio of 9:0.5:10), 2 substrate solutions (BCIP / NBT colorimetric solutions), 2 positive controls (F-actin antibodies in human serum), and 2 negative controls (8wt% goat serum);

[0035] The preparation method of the above-mentioned anti-F-actin antibody detection kit comprises the following steps:

[0036] S1. adding the hydroxyapatite, γ-polyglutamic acid and hyaluronic acid (1:4:5, m / m) to a sufficient amount of 65 wt % methanol, heating to 80° C. and ultrasonicating for 4 h, filtering, washing and drying to obtain the modified hydroxyapatite;

[0037] S2. 90ug / ml of F-actin antigen protein was mixed with modified hydroxyapatite (mass ratio 1:1), coated on a nitrocellulose membrane, and cut to form an antibody detection strip;

[0038] S3. According to the above mass fractions, the antibody test strip and other components are packaged and frozen in liquid nitrogen to obtain the anti-F-actin antibody detection kit;

[0039] The method for preparing the acridinium ester labeled antibody comprises the following steps:

[0040] A1. Add goat anti-human IgG to a centrifuge tube, add buffer to 0.3 ml, add acridinium ester, mix immediately, wrap the centrifuge tube with a layer of aluminum foil to avoid light, place it on a rotary mixer, and react at room temperature for 30 minutes;

[0041] A2. Add 1 wt% lysine to a centrifuge tube (the content of lysine is 6% of the total mass of the acridinium ester), keep it away from light, place it on a rotary mixer, mix it at room temperature for 30 minutes, purify it through a Sephadex G-50 column (1×25 cm), add enough glycerol, and store it at -20°C for later use;

[0042] Wherein, the molar ratio of the acridinium ester to goat anti-human IgG is 7.4:1.

[0043] Example 2

[0044] A detection kit for anti-F-actin antibodies, comprising the following components: 2 antibody detection strips, 2 labeled antibodies (acridinium ester labeled antibodies); 2 washing solutions (the washing solution is made of PBS buffer containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, glycerol, and TritonX-100, with a mass ratio of 9:0.5:0.3:1:0.5:10), 2 diluents (PBS buffer containing sodium chloride and TritonX-100, with a mass ratio of 9:0.5:10), 2 substrate solutions (BCIP / NBT colorimetric solutions), 2 positive controls (F-actin antibodies in human serum), and 2 negative controls (8wt% goat serum);

[0045] The preparation method of the above-mentioned anti-F-actin antibody detection kit comprises the following steps:

[0046] S1. adding the hydroxyapatite, γ-polyglutamic acid and hyaluronic acid (1:4:5, m / m) to a sufficient amount of 65 wt % methanol, heating to 80° C. and ultrasonicating for 4 h, filtering, washing and drying to obtain the modified hydroxyapatite;

[0047] S2. 90ug / ml of F-actin antigen protein was mixed with modified hydroxyapatite (mass ratio 1:1), coated on a nitrocellulose membrane, and cut to form an antibody detection strip;

[0048] S3. According to the above mass fractions, the antibody test strip and other components are packaged and frozen in liquid nitrogen to obtain the anti-F-actin antibody detection kit;

[0049] The method for preparing the acridinium ester labeled antibody comprises the following steps:

[0050] A1. Add goat anti-human IgG to a centrifuge tube, add buffer to 0.3 ml, add acridinium ester, mix immediately, wrap the centrifuge tube with a layer of aluminum foil to avoid light, place it on a rotary mixer, and react at room temperature for 30 minutes;

[0051] A2. Add 1 wt% lysine to a centrifuge tube (the content of lysine is 5% of the total mass of the acridinium ester), keep it away from light, place it on a rotary mixer, mix it at room temperature for 30 minutes, purify it through a Sephadex G-50 column (1×25 cm), add enough glycerol, and store it at -20°C for later use;

[0052] Wherein, the molar ratio of the acridinium ester to goat anti-human IgG is 8:1.

[0053] Example 3

[0054] A detection kit for anti-F-actin antibodies, comprising the following components: 2 antibody detection strips, 2 labeled antibodies (acridinium ester labeled antibodies); 2 washing solutions (the washing solution is made of PBS buffer containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, glycerol, and TritonX-100, with a mass ratio of 9:0.5:0.3:1:0.5:10), 2 diluents (PBS buffer containing sodium chloride and TritonX-100, with a mass ratio of 9:0.5:10), 2 substrate solutions (BCIP / NBT colorimetric solutions), 2 positive controls (F-actin antibodies in human serum), and 2 negative controls (8wt% goat serum);

[0055] The preparation method of the above-mentioned anti-F-actin antibody detection kit comprises the following steps:

[0056] S1. adding the hydroxyapatite, γ-polyglutamic acid and hyaluronic acid (1:5:5, m / m) to a sufficient amount of 65 wt % methanol, heating to 80° C. and ultrasonicating for 4 h, filtering, washing and drying to obtain the modified hydroxyapatite;

[0057] S2. 90ug / ml of F-actin antigen protein was mixed with modified hydroxyapatite (mass ratio 1:1), coated on a nitrocellulose membrane, and cut to form an antibody detection strip;

[0058] S3. According to the above mass fractions, the antibody test strip and other components are packaged and frozen in liquid nitrogen to obtain the anti-F-actin antibody detection kit;

[0059] The method for preparing the acridinium ester labeled antibody comprises the following steps:

[0060] A1. Add goat anti-human IgG to a centrifuge tube, add buffer to 0.3 ml, add acridinium ester, mix immediately, wrap the centrifuge tube with a layer of aluminum foil to avoid light, place it on a rotary mixer, and react at room temperature for 30 minutes;

[0061] A2. 1.5 wt% lysine was added to a centrifuge tube (the content of lysine was 6% of the total mass of the acridinium ester), kept in a dark state, placed on a rotary mixer, mixed and reacted at room temperature for 40 min, purified by Sephadex G-50 column (1×25 cm), added with sufficient glycerol, and stored at -20°C for later use;

[0062] Wherein, the molar ratio of the acridinium ester to goat anti-human IgG is 7.4:1.

[0063] Comparative Example 1

[0064] The difference between this comparative example and Example 1 is that: in step S1, hyaluronic acid is replaced with lauric acid of equal mass, and other ingredients and preparation methods are the same as those in Example 1.

[0065] Comparative Example 2

[0066] The difference between this comparative example and Example 1 is that: in step S1, the γ-polyglutamic acid is removed and replaced by hyaluronic acid of equal mass, and the other ingredients and preparation method are the same as those in Example 1.

[0067] Test Case

[0068] The detection kits for anti-F-actin antibodies prepared in Example 1 and Comparative Examples 1-2 were used as test kits for performance testing.

[0069] The anti-F-actin antibody detection kits prepared in Example 1 and Comparative Examples 1-2 were used as kits to detect 30 F-actin antibody-negative sera and 30 F-actin antibody-positive sera;

[0070] Test method:

[0071] L1. Dry the antibody test strip in a 37°C incubator for 30 minutes, place the NC membrane in the test plate, add 200 μl 5% BSA and block in a 37°C incubator for 30 minutes, discard the blocking solution and wash twice with washing solution;

[0072] L2. Antigen incubation: Add 10 μl of serum to be tested diluted with diluent to the test plate, and make negative and positive controls at the same time. Incubate at room temperature for 30 minutes. Set up 3 parallel wells for each sample.

[0073] L3. Secondary antibody incubation: discard the liquid in the test plate, wash with washing solution 5 times × 1 min, add 20 μl labeled antibody, and incubate at room temperature for 30 min;

[0074] L4. Color development: discard the liquid in the test plate, wash with washing solution 5 times × 1 min, add 500 μl alkaline phosphatase-streptavidin, incubate at room temperature for 20 min, discard the liquid in the test plate, wash with washing solution 5 times × 1 min, then add substrate solution, react at room temperature for 20 min, rinse the test plate with running water to terminate the enzyme reaction;

[0075] L5. Take out the test NC membrane strip and blow dry it with a hair dryer, read the result of the antibody test strip, and analyze the result. If obvious brown spots appear, it is positive.

[0076] Table 1 Kit performance test results of Example 1 and Comparative Examples 1-2

[0077] project Example 1 Comparative Example 1 Comparative Example 2 Number of F-actin antibody positive sera detected 29 27 24 Number of F-actin antibody negative sera detected 29 26 25

[0078] According to Table 1, it can be concluded that the anti-F-actin antibody detection kit of Example 1 of the present invention can effectively detect F-actin antibodies, effectively improves the adhesion and compatibility of antigen proteins, and has high sensitivity and stability, providing a more reliable technical means for the accurate detection of F-actin antibodies.

[0079] 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.

[0080] 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 kit for anti-F-actin antibodies, characterized in that: The anti-F-actin antibody detection kit comprises: an antibody detection strip, a labeled antibody, and an auxiliary agent; Wherein, the antibody detection strip is spotted with modified hydroxyapatite adsorbed with F-actin antigen protein; The modified hydroxyapatite is hydroxyapatite partially grafted with gamma-polyglutamic acid and partially grafted with hyaluronic acid.

2. The detection kit for anti-F-actin antibodies according to claim 1, characterized in that: The labeled antibody is an acridinium ester labeled antibody.

3. The detection kit for anti-F-actin antibodies according to claim 1, characterized in that: The auxiliary agent is selected from one or more of a diluent, a buffer, a substrate developer, and a washing solution.

4. The detection kit for anti-F-actin antibodies according to claim 1, characterized in that: The substrate solution is selected from BCIP / NBT color development solution.

5. The detection kit for anti-F-actin antibodies 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, glycerol and TritonX-100.

6. The detection kit for anti-F-actin antibodies according to claim 1, characterized in that: The material of the diluent is PBS buffer containing sodium chloride and TritonX-100.

7. The detection kit for anti-F-actin antibodies according to claim 1, characterized in that: Positive and negative controls were also included.

8. The detection kit for anti-F-actin antibodies according to claim 7, characterized in that: The negative control is 8-12 wt % goat serum.

9. The detection kit for anti-F-actin antibodies according to claim 7, characterized in that: The positive control is serum containing F-actin antibody.

10. A method for preparing a detection kit for anti-F-actin antibodies according to any one of claims 1 to 9, characterized in that: The steps include: S1. adding the hydroxyapatite, γ-polyglutamic acid and hyaluronic acid into a solvent, heating and ultrasonicating, and purifying to obtain the modified hydroxyapatite; S2. After mixing the F-actin antigen protein with modified hydroxyapatite, the mixture is coated on a nitrocellulose membrane, and then cut to form an antibody detection strip; S3. Packing the antibody detection strip and other components to obtain the anti-F-actin antibody detection kit.