Horseradish peroxidase-labeled secondary antibody compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of chemiluminescence immunoassay, and discloses a horse radish peroxidase-labeled secondary antibody compound and a preparation method and application thereof, the horse radish peroxidase-labeled secondary antibody compound comprises a chitosan compound skeleton, and horse radish peroxidase and a secondary antibody carried on the chitosan compound skeleton; the chitosan compound skeleton is a macromolecular skeleton compounded by a chitosan spherical cross-linking body and dopamine. Through optimization and improvement of a polymer skeleton, the problems of large steric hindrance, low sensitivity and non-specific staining of a traditional polymer can be solved, and further improvement of the sensitivity of a DAB staining method for immunoassay detection is facilitated; the adopted chitosan compound skeleton has abundant amino groups, can bear more labeled enzymes and secondary antibodies, reduces steric hindrance, increases the penetrating power to a cell nuclear membrane, and improves the detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of immunohistochemical analysis, and particularly to a horseradish peroxidase-labeled secondary antibody complex, a preparation method thereof, and an application thereof. Background Art

[0002] Immunoassay is a method for detecting protein biomarkers in a sample based on the principle of "specific binding of antibody and antigen". Immunohistochemical analysis is a detection method based on the above immunoassay. By means of a chemical reaction, the substrate of the labeled antibody is colored to determine whether there is a target antigen in the tissue and its expression.

[0003] The DAB staining method belongs to a kind of immunohistochemical analysis method, also called diaminobenzidine method, which is applicable to immunoblotting or immunohistochemical reactions labeled with horseradish peroxidase HRP. After using a secondary antibody labeled with horseradish peroxidase to bind to the primary antibody to determine the position of specific characteristic substances, a color developer undergoes a chemical reaction under the catalysis of the labeled enzyme, and an insoluble colored compound precipitates in situ, thereby showing the position where the enzyme-labeled secondary antibody exists, that is, the position where the characteristic protein or antigen is located. The color development end product of the DAB staining method can be directly observed under a light microscope, has strong stability, and can be stored for a long time.

[0004] However, the enzyme-labeled secondary antibody uses the detection principle of "chain polymer-enzyme-antibody polymer" to carry the labeled enzyme and the secondary antibody. Due to the limited surface area that can be carried by the chain polymer, the number of antibodies that can be labeled is also limited, and it cannot detect low-abundance antigens well, and the detection sensitivity is also limited. However, if a polymer backbone with a higher degree of polymerization is used, it will cause a large steric hindrance. Therefore, the large-sized enzyme-labeled secondary antibody is not easily permeated into the cell nucleus to bind to nuclear antigens, which will also lead to insufficient detection sensitivity.

[0005] Chinese Patent Invention Publication No. CN111830250A discloses a preparation method of a signal-amplified enzyme-labeled secondary antibody. Using the enzyme-labeled antibody horseradish peroxidase (HRP)-IgG widely used in biochemical analysis as a system and dextran (Dex) as a carrier, a Dex-HRP-IgG conjugate is prepared, and its goal is to increase the HRP / IgG molar ratio, thereby improving the detection sensitivity of the enzyme-labeled antibody. However, the signal amplification effect of the secondary antibody produced by this method is not obvious. Using a polymer backbone with a higher degree of polymerization causes a large steric hindrance, and the detection sensitivity is low. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a horseradish peroxidase-labeled secondary antibody complex, a preparation method thereof, and an application thereof, which can increase the surface area of the polymer backbone for carrying the labeled enzyme and the secondary antibody, improve the number of labelable antibodies, and further optimize the detection sensitivity of the DAB chromogenic reagent.

[0007] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a horseradish peroxidase-labeled secondary antibody complex, which comprises a chitosan complex backbone and horseradish peroxidase and a secondary antibody carried on the chitosan complex backbone; the chitosan complex backbone is a polymer backbone formed by the complexation of a chitosan spherical crosslinking body and dopamine.

[0008] The enzyme-labeled secondary antibody complex is a secondary antibody detection system that amplifies signals by coupling multiple antibodies and enzymes to a polymer. By optimizing and improving the polymer backbone, the present invention can solve the problems of large steric hindrance, low sensitivity, and non-specific staining of traditional polymers. Moreover, the labeled enzyme used is horseradish peroxidase, and a chromogenic end product with obvious color development, high stability, and long-term preservation can be obtained by using the DAB staining method, which is beneficial to further improving the detection sensitivity of immunoassay.

[0009] Specifically, the adopted chitosan complex backbone has rich amino groups, which can carry more labeled enzymes and secondary antibodies. Moreover, it is complexed with dopamine, which also contains amino groups and can undergo self-polymerization reactions, forming an extended branched structure on the chitosan spherical crosslinking body, which can reduce steric hindrance, increase the penetration power of the nuclear membrane, and improve the detection sensitivity.

[0010] Preferably, the preparation method of the chitosan complex backbone comprises the following steps: (1) Add chitosan to an acetic acid aqueous solution, dropwise add a sodium tripolyphosphate aqueous solution while stirring, and then filter to obtain a chitosan spherical crosslinking body; (2) Add the chitosan spherical crosslinking body to ethanol, heat to reflux, add terephthalaldehyde and trehalose, react at a constant temperature, then add sodium perchlorate, stir under light-shielded conditions, and then add ethylene glycol to terminate the reaction, and rotary evaporate to remove the solvent; (3) Then, add dopamine and water at room temperature, add Tris-HCl buffer solution, stir and react to obtain a chitosan complex backbone.

[0011] Chitosan can be cross-linked in the presence of sodium tripolyphosphate to form a spherical cross-linked body. The spherical cross-linked body is used as the substrate of the polymer skeleton, and the surface is rich in amino groups, which can increase the load-bearing surface area of the enzyme and the second antibody. Then, terephthalaldehyde and trehalose are added for graft cross-linking, and chain-like small molecules are formed on the surface of the chitosan cross-linked body. Then, the polyhydroxyl groups on the trehalose are oxidized to polyaldehyde groups, and the aldehyde groups can graft dopamine, coupled with the self-polymerization reaction of dopamine itself, further, the chain-like small molecules will form a cross-linked network structure, and multiple spacer arms are formed on the surface of the chitosan cross-linked body. The spacer arms of the small molecule network structure can also carry enzymes and second antibodies, and multiple branch carriers can increase the compactness between enzyme molecules, making the labeled enzyme more stable, and can achieve flexible immobilization, reduce steric hindrance, and the small-volume enzyme-labeled second antibody can more easily penetrate into the cell nucleus and bind to nuclear antigens, thereby improving the detection sensitivity from many aspects.

[0012] In addition, during the oxidation process of the polyhydroxyl groups on trehalose, the active hydroxyl groups on chitosan can also be oxidized to aldehyde groups, which can also be grafted with dopamine and self-polymerized to form a multi-level branched structure, thereby increasing the number of enzymes and secondary antibodies that can be carried. In addition, when DAB is developed, insoluble colored compounds will be formed and precipitated in situ. The multi-level branched structure can prevent the precipitate from covering other enzyme active sites as much as possible, thereby increasing the number of effective labeling.

[0013] Preferably, in step (1), the concentration of chitosan in the acetic acid aqueous solution is 20-25 g / L; the concentration of the acetic acid aqueous solution is 0.1-0.3 mol / L; the concentration of the sodium tripolyphosphate aqueous solution is 3-4 mg / mL; and the volume ratio of the acetic acid aqueous solution to the sodium tripolyphosphate aqueous solution is 100:2-3.

[0014] Chitosan and sodium tripolyphosphate will undergo ionic cross-linking to form spherical cross-linked bodies. Therefore, the particle size of the spherical cross-linked bodies is affected by the ion concentration during the ionic cross-linking process. If the particle size is too large, it will also affect the steric hindrance of the enzyme-labeled secondary antibody, which is not conducive to improving the detection sensitivity.

[0015] Preferably, in step (2), the mass ratio of the chitosan spherical crosslinked body to ethanol is 1:300-400; the mass ratio of the chitosan spherical crosslinked body to terephthalaldehyde and trehalose is 1:18-20:5-12.

[0016] Trehalose is a non-reducing disaccharide composed of two glucose molecules. Grafting it onto the surface of a chitosan spherical crosslinker can form a spacer arm with a certain spatial structure. Coupled with the grafting and self-polymerization of dopamine, a polymer layer rich in amino groups is formed, which can bind more labeled enzymes and antibody molecules. However, when the grafted spacer arms are too dense, the steric hindrance between them is not conducive to carrying more secondary antibody molecules, and the surface space utilization rate that can be carried cannot reach the highest. Therefore, it is necessary to control the raw material addition amount and reaction conditions to achieve higher detection sensitivity.

[0017] Preferably, in step (2), the mass-volume ratio of the trehalose, sodium perchlorate, and ethylene glycol is 1 g: 0.8 - 1 g: 5 mL.

[0018] Preferably, in step (3), the mass-volume ratio of the trehalose, dopamine, Tris-HCl buffer solution, and water is 1 g: 0.3 - 0.5 g: 3 - 4 mL: 100 mL; the concentration of the Tris-HCl buffer solution is 1 mol / L, and the pH is 7.4; the stirring reaction is carried out at room temperature for 2 - 3 h.

[0019] In a second aspect, the present invention also provides a method for preparing a horseradish peroxidase-labeled secondary antibody complex, comprising the following steps: S1. Add the chitosan complex skeleton to PBS buffer solution, add horseradish peroxidase, and stir and react for 3 - 4 h to obtain a mixture loaded with the enzyme. S2. Add a secondary antibody to the mixture loaded with the enzyme, add EDC, adjust the pH to 5 - 6, carry out dehydration condensation, and separate by a purification column after the reaction to obtain a horseradish peroxidase-labeled secondary antibody complex.

[0020] In the present invention, the chitosan complex skeleton contains rich amino groups and has a large loading surface area, so it can immobilize more horseradish peroxidase and become a good carrier material. Then, it binds to the secondary antibody molecules. EDC is used for activation and coupling reactions, utilizing the surface space of the skeleton to increase the loading rate of the secondary antibody, improve the labeling quantity and density of the antibody, and thus improve the sensitivity of the enzyme-labeled secondary antibody complex.

[0021] Preferably, the mass ratio of the chitosan complex skeleton to the horseradish peroxidase is 1: 5 - 10; the concentration of the horseradish peroxidase in the mixture loaded with the enzyme is 0.5 - 0.8 wt%; the mass ratio of the mixture loaded with the enzyme to the secondary antibody is 20 - 25: 1.

[0022] In a third aspect, the present invention also provides the application of the above-mentioned horseradish peroxidase-labeled secondary antibody complex in a DAB chromogenic reagent.

[0023] Preferably, the DAB chromogenic reagent includes a horseradish peroxidase-labeled secondary antibody complex, a DAB concentrated chromogenic solution, and a DAB substrate buffer solution; the DAB concentrated chromogenic solution includes 3,3-diaminobenzidine tetrachloride; the DAB substrate buffer solution includes hydrogen peroxide.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) By optimizing and improving the polymer backbone, the problems of large steric hindrance, low sensitivity, and non-specific staining of traditional polymers can be solved, which is beneficial to further improving the sensitivity of immunoassay detection by the DAB staining method; (2) The chitosan complex backbone adopted has rich amino groups, can carry more labeled enzymes and secondary antibody molecules, and can form an extended multi-level branched structure on the chitosan spherical cross-linked body. In particular, the spacer arms with a certain spatial structure can reduce steric hindrance while increasing the loading rate, enhance the penetration ability into the nuclear membrane, and improve the detection sensitivity. Specific Embodiments

[0025] The present invention will be further described below in conjunction with embodiments.

[0026] General Embodiment 1. Horseradish Peroxidase-Labeled Secondary Antibody Complex The horseradish peroxidase-labeled secondary antibody complex includes a chitosan complex backbone and horseradish peroxidase and secondary antibody molecules carried on the chitosan complex backbone.

[0027] The preparation method of the above chitosan complex backbone includes the following steps: (1) Chitosan is added to an acetic acid aqueous solution with a concentration of 0.1 - 0.3 mol / L to obtain a chitosan solution with a concentration of 20 - 25 g / L. While stirring, an aqueous sodium tripolyphosphate solution with a concentration of 3 - 4 mg / mL is added dropwise. The volume ratio of the acetic acid aqueous solution to the aqueous sodium tripolyphosphate solution is 100:2 - 3, and then filtration is carried out to obtain a chitosan spherical cross-linked body; (2) The chitosan spherical cross-linked body is added to ethanol according to a mass ratio of 1:300 - 400, heated to reflux, and terephthalaldehyde and trehalose are added. The mass ratio of the chitosan spherical cross-linked body, terephthalaldehyde, and trehalose is 1:18 - 20:5 - 12, and a constant temperature reaction is carried out for 30 - 60 min under reflux; subsequently, sodium perchlorate is added, and stirring is carried out for 20 - 40 min under light-shielded conditions, and then ethylene glycol is added to terminate the reaction. The mass-to-volume ratio of trehalose, sodium perchlorate, and ethylene glycol is 1 g:0.8 - 1 g:5 mL, and then the solvent is removed by rotary evaporation; (3) Next, dopamine and water were added at room temperature, and then Tris-HCl buffer solution (with a concentration of 1 mol / L and a pH of 7.4) was added. The mass-volume ratio of trehalose, dopamine, Tris-HCl buffer solution and water was 1 g: 0.3 - 0.5 g: 3 - 4 mL: 100 mL. After stirring and reacting at room temperature for 2 - 3 h, a chitosan complex skeleton was obtained.

[0028] The preparation method of the above horseradish peroxidase-labeled secondary antibody complex includes the following steps: S1. The chitosan complex skeleton was added to PBS buffer solution (pH = 7.4), and horseradish peroxidase was added. The mass ratio of the chitosan complex skeleton to horseradish peroxidase was 1: 5 - 10. After stirring and reacting for 3 - 4 h, a mixture loaded with the enzyme was obtained, and the concentration of horseradish peroxidase in the mixture loaded with the enzyme was 0.5 - 0.8 wt%. S2. Secondary antibody was added to the mixture loaded with the enzyme. The mass ratio of the mixture loaded with the enzyme to the secondary antibody was 20 - 25: 1. EDC was added, and the pH was adjusted to 5 - 6 for dehydration condensation. After the reaction, separation was carried out through a purification column to obtain the horseradish peroxidase-labeled secondary antibody complex.

[0029] 2. Application (DAB chromogenic reagent) Horseradish peroxidase-labeled secondary antibody complex: The secondary antibody is a mouse-rabbit universal secondary antibody; DAB concentrated chromogenic solution: 3,3-diaminobenzidine tetrachloride solution; DAB substrate buffer solution: A stabilizer solution containing hydrogen peroxide.

[0030] Example 1 The horseradish peroxidase-labeled secondary antibody complex includes a chitosan complex skeleton and horseradish peroxidase and secondary antibody carried on the chitosan complex skeleton.

[0031] The preparation method of the above chitosan complex skeleton includes the following steps: (1) Chitosan was added to an acetic acid aqueous solution with a concentration of 0.2 mol / L to obtain a chitosan solution with a concentration of 25 g / L. While stirring, an aqueous solution of sodium tripolyphosphate with a concentration of 3 mg / mL was added dropwise. The volume ratio of the acetic acid aqueous solution to the aqueous solution of sodium tripolyphosphate was 100: 2.6. Then, filtration was carried out to obtain a chitosan spherical crosslinked body; (2) Add the chitosan spherical crosslinked body to ethanol at a mass ratio of 1:350, heat up to reflux and add terephthalaldehyde and trehalose. The mass ratio of the chitosan spherical crosslinked body, terephthalaldehyde and trehalose is 1:18:9, and react at a constant temperature under reflux for 50 min; then add sodium perchlorate, stir for 30 min under light-shielded conditions, and then add ethylene glycol to terminate the reaction. The mass-volume ratio of trehalose, sodium perchlorate and ethylene glycol is 1 g:0.8 g:5 mL, and then remove the solvent by rotary evaporation; (3) Then, add dopamine and water at room temperature, and then add Tris-HCl buffer solution (concentration is 1 mol / L, pH is 7.4). The mass-volume ratio of trehalose, dopamine, Tris-HCl buffer solution and water is 1 g:0.4 g:3 mL:100 mL. After stirring and reacting at room temperature for 2 h, a chitosan composite framework is obtained.

[0032] The preparation method of the above horseradish peroxidase-labeled secondary antibody complex includes the following steps: S1. Add the chitosan composite framework to PBS buffer solution (pH is 7.4), add horseradish peroxidase. The mass ratio of the chitosan composite framework and horseradish peroxidase is 1:8, stir and react for 3 h to obtain a mixture loaded with the enzyme. The concentration of horseradish peroxidase in the mixture loaded with the enzyme is 0.7 wt%; S2. Add a secondary antibody to the mixture loaded with the enzyme. The mass ratio of the mixture loaded with the enzyme and the secondary antibody is 22:1. Add EDC with a mass 0.1 times that of the secondary antibody, adjust the pH to 6, stir and react for 5 h for dehydration condensation. After the reaction, separate through a purification column to obtain a horseradish peroxidase-labeled secondary antibody complex.

[0033] Example 2 The horseradish peroxidase-labeled secondary antibody complex includes a chitosan composite framework and horseradish peroxidase and a secondary antibody carried on the chitosan composite framework.

[0034] The preparation method of the above chitosan composite framework includes the following steps: (1) Add chitosan to an acetic acid aqueous solution with a concentration of 0.2 mol / L to obtain a chitosan solution with a concentration of 25 g / L. While stirring, dropwise add a sodium tripolyphosphate aqueous solution with a concentration of 3 mg / mL. The volume ratio of the acetic acid aqueous solution and the sodium tripolyphosphate aqueous solution is 100:2.6, and then filter to obtain a chitosan spherical crosslinked body; (2) Add the chitosan spherical crosslinked body to ethanol at a mass ratio of 1:350, heat up to reflux and add terephthalaldehyde and trehalose. The mass ratio of the chitosan spherical crosslinked body, terephthalaldehyde and trehalose is 1:20:11, and react at a constant temperature under reflux for 60 min. Then add sodium perchlorate, stir for 40 min under light-shielded conditions, and then add ethylene glycol to terminate the reaction. The mass-volume ratio of trehalose, sodium perchlorate and ethylene glycol is 1 g:1.0 g:5 mL. After that, rotary evaporate to remove the solvent; (3) Then, add dopamine and water at room temperature, and then add Tris-HCl buffer solution (concentration is 1 mol / L, pH is 7.4). The mass-volume ratio of trehalose, dopamine, Tris-HCl buffer solution and water is 1 g:0.5 g:3 mL:100 mL. Stir and react at room temperature for 2 h to obtain the chitosan composite framework.

[0035] The preparation method of the above horseradish peroxidase-labeled secondary antibody complex includes the following steps: S1. Add the chitosan composite framework to PBS buffer solution (pH = 7.4), add horseradish peroxidase, and the mass ratio of the chitosan composite framework and horseradish peroxidase is 1:10. Stir and react for 4 h to obtain a mixture loaded with the enzyme, and the concentration of horseradish peroxidase in the mixture loaded with the enzyme is 0.6 wt%; S2. Add a secondary antibody to the mixture loaded with the enzyme, and the mass ratio of the mixture loaded with the enzyme and the secondary antibody is 20:1. Add EDC with a mass 0.1 times that of the secondary antibody, adjust the pH to 6, stir and react for 5 h for dehydration condensation. After the reaction, separate through a purification column to obtain the horseradish peroxidase-labeled secondary antibody complex.

[0036] Example 3 The horseradish peroxidase-labeled secondary antibody complex includes a chitosan composite framework and horseradish peroxidase and a secondary antibody carried on the chitosan composite framework.

[0037] The preparation method of the above chitosan composite framework includes the following steps: (1) Add chitosan to an aqueous acetic acid solution with a concentration of 0.3 mol / L to obtain a chitosan solution with a concentration of 20 g / L. While stirring, dropwise add an aqueous sodium tripolyphosphate solution with a concentration of 4 mg / mL. The volume ratio of the aqueous acetic acid solution and the aqueous sodium tripolyphosphate solution is 100:3. Then filter to obtain the chitosan spherical crosslinked body; (2) Add the chitosan spherical cross-linked product to ethanol at a mass ratio of 1:300, heat up to reflux and add terephthalaldehyde and trehalose, where the mass ratio of the chitosan spherical cross-linked product, terephthalaldehyde and trehalose is 1:18:10, and react at a constant temperature under reflux for 50 min; then add sodium perchlorate, stir for 40 min under light-shielded conditions, and then add ethylene glycol to terminate the reaction. The mass-volume ratio of trehalose, sodium perchlorate and ethylene glycol is 1 g:0.8 g:5 mL, and then remove the solvent by rotary evaporation; (3) Then, add dopamine and water at room temperature, and then add Tris-HCl buffer solution (concentration: 1 mol / L, pH: 7.4). The mass-volume ratio of trehalose, dopamine, Tris-HCl buffer solution and water is 1 g:0.3 g:3 mL:100 mL. After stirring and reacting for 2 h, a chitosan composite framework is obtained.

[0038] The preparation method of the above horseradish peroxidase-labeled secondary antibody complex comprises the following steps: S1. Add the chitosan composite framework to PBS buffer solution (pH = 7.4), and add horseradish peroxidase. The mass ratio of the chitosan composite framework to horseradish peroxidase is 1:8, stir and react for 3 h to obtain a mixture loaded with the enzyme, and the concentration of horseradish peroxidase in the mixture loaded with the enzyme is 0.7 wt%; S2. Add a secondary antibody to the mixture loaded with the enzyme. The mass ratio of the mixture loaded with the enzyme to the secondary antibody is 22:1. Add EDC with a mass 0.1 times that of the secondary antibody, adjust the pH to 6, stir and react for 5 h for dehydration condensation, and separate by a purification column after the reaction to obtain the horseradish peroxidase-labeled secondary antibody complex.

[0039] Comparative Example 1 The difference from Example 1 is that dextran T500 is used as the polymer framework of the enzyme-labeled secondary antibody complex.

[0040] The horseradish peroxidase-labeled secondary antibody complex comprises a dextran T500 framework and horseradish peroxidase and a secondary antibody carried on the dextran T500 framework.

[0041] The preparation method of the above horseradish peroxidase-labeled secondary antibody complex comprises the following steps: S1. Add the dextran T500 framework to PBS buffer solution (pH = 7.4), and add horseradish peroxidase. The mass ratio of the dextran T500 framework to horseradish peroxidase is 1:8, stir and react for 3 h to obtain a mixture loaded with the enzyme, and the concentration of horseradish peroxidase in the mixture loaded with the enzyme is 0.7 wt%; S2. Add a secondary antibody to the enzyme-loaded mixture, where the mass ratio of the enzyme-loaded mixture to the secondary antibody is 22:1. Add EDC with a mass 0.1 times that of the secondary antibody, adjust the pH to 6, and stir and react for 5 h for dehydration condensation. After the reaction, separate through a purification column to obtain a horseradish peroxidase-labeled secondary antibody complex.

[0042] Comparative Example 2 The difference from Example 1 is that the chitosan complex skeleton is only a chitosan spherical cross-linked body.

[0043] The preparation method of the chitosan complex skeleton includes the following steps: Add chitosan to an acetic acid aqueous solution with a concentration of 0.2 mol / L to obtain a chitosan solution with a concentration of 25 g / L. While stirring, dropwise add a sodium tripolyphosphate aqueous solution with a concentration of 3 mg / mL, where the volume ratio of the acetic acid aqueous solution to the sodium tripolyphosphate aqueous solution is 100:2.6. Then filter to obtain a chitosan spherical cross-linked body, which is the chitosan complex skeleton.

[0044] Comparative Example 3 The difference from Example 1 is that trehalose is not added to the chitosan complex skeleton.

[0045] The preparation method of the chitosan complex skeleton includes the following steps: (1) Add chitosan to an acetic acid aqueous solution with a concentration of 0.2 mol / L to obtain a chitosan solution with a concentration of 25 g / L. While stirring, dropwise add a sodium tripolyphosphate aqueous solution with a concentration of 3 mg / mL, where the volume ratio of the acetic acid aqueous solution to the sodium tripolyphosphate aqueous solution is 100:2.6. Then filter to obtain a chitosan spherical cross-linked body; (2) Mix the chitosan spherical cross-linked body, dopamine, and water, and then add Tris-HCl buffer solution (with a concentration of 1 mol / L and a pH of 7.4), where the mass-volume ratio of the chitosan spherical cross-linked body, dopamine, Tris-HCl buffer solution, and water is 1 g:0.4 g:3 mL:100 mL. Stir and react at room temperature for 2 h to obtain the chitosan complex skeleton.

[0046] Comparative Example 4 The difference from Example 1 is that dopamine is not added to the chitosan complex skeleton.

[0047] The preparation method of the chitosan complex skeleton includes the following steps: (1) Add chitosan to an acetic acid aqueous solution with a concentration of 0.2 mol / L to obtain a chitosan solution with a concentration of 25 g / L. While stirring, dropwise add a sodium tripolyphosphate aqueous solution with a concentration of 3 mg / mL, where the volume ratio of the acetic acid aqueous solution to the sodium tripolyphosphate aqueous solution is 100:2.6. Then filter to obtain a chitosan spherical cross-linked body; (2) Add the chitosan spherical cross-linked body to ethanol at a mass ratio of 1:350, heat up to reflux and add terephthalaldehyde and trehalose. The mass ratio of the chitosan spherical cross-linked body, terephthalaldehyde and trehalose is 1:18:9. React at a constant temperature under reflux for 50 min. Then add sodium perchlorate, stir for 30 min under light-shielded conditions, and then add ethylene glycol to terminate the reaction. The mass-volume ratio of trehalose, sodium perchlorate and ethylene glycol is 1 g:0.8 g:5 mL. Then remove the solvent by rotary evaporation to obtain a chitosan composite skeleton.

[0048] Comparative Example 5 The difference from Example 1 is that in the chitosan composite skeleton, the addition amounts of trehalose and dopamine are excessive.

[0049] The preparation method of the chitosan composite skeleton includes the following steps: (1) Add chitosan to an acetic acid aqueous solution with a concentration of 0.2 mol / L to obtain a chitosan solution with a concentration of 25 g / L. While stirring, dropwise add an aqueous solution of sodium tripolyphosphate with a concentration of 3 mg / mL. The volume ratio of the acetic acid aqueous solution and the aqueous solution of sodium tripolyphosphate is 100:2.6. Then filter to obtain a chitosan spherical cross-linked body. (2) Add the chitosan spherical cross-linked body to ethanol at a mass ratio of 1:350, heat up to reflux and add terephthalaldehyde and trehalose. The mass ratio of the chitosan spherical cross-linked body, terephthalaldehyde and trehalose is 1:25:15. React at a constant temperature under reflux for 50 min. Then add sodium perchlorate, stir for 30 min under light-shielded conditions, and then add ethylene glycol to terminate the reaction. The mass-volume ratio of trehalose, sodium perchlorate and ethylene glycol is 1 g:1.2 g:5 mL. Then remove the solvent by rotary evaporation; (3) Next, add dopamine and water at room temperature, and then add Tris-HCl buffer solution (concentration: 1 mol / L, pH: 7.4). The mass-volume ratio of trehalose, dopamine, Tris-HCl buffer solution and water is 1 g:0.8 g:4 mL:100 mL. Stir and react at room temperature for 3 h to obtain a chitosan composite skeleton.

[0050] Apply the enzyme-labeled secondary antibody complexes in the examples and comparative examples to the DAB chromogenic reagent, dilute and mix them at 1:500 or 1:1000 respectively to obtain an enzyme-labeled secondary antibody complex dilution, and perform detection under the same conditions to stain the target antigen in the tissue sample. The method is as follows: (1) Immerse the sample slice in tap water for 3 min and then perform antigen retrieval, and then immerse it in tap water again for 3 min. Circle the tissue with an immunohistochemical pen on the sample slice, add 3% hydrogen peroxide, and incubate at room temperature for 10 min. Wash with PBS 3 times, each time for 3 min. (2) Add 2 drops of the primary antibody dilution solution (using mouse anti-human HER2 antibody as the primary antibody) to the sample slice, and completely cover the tissue to be detected. Incubate in a wet box in an incubator at room temperature for 50 min; wash the sample slice 3 times with PBS, 3 min each time; (3) Add 2 drops of the enzyme-labeled secondary antibody complex dilution solution (using rabbit anti-mouse antibody as the secondary antibody) to the sample slice, and completely cover the tissue to be detected. Incubate in a wet box in an incubator at room temperature for 30 min; wash the sample slice 3 times with PBS, 3 min each time; (4) Add the DAB chromogenic dilution solution (DAB concentrated chromogenic solution:DAB substrate buffer = 1:25, prepared freshly) to the sample slice, and completely cover the tissue to be detected. Incubate in an incubator at room temperature for 2 min (according to color change), and rinse with pure water to terminate chromogenesis; (5) Stain the sample slice with hematoxylin for 4 min, and rinse with PBS to blue for 10 s; then soak successively in gradient alcohols (75 wt% ethanol, 95 wt% ethanol, absolute ethanol, absolute ethanol), 30 s each time, and clear with the dewaxing solution / xylene for 5 min; (6) Mount the sample slice with neutral balsam and a coverslip, and read the slice under an optical microscope.

[0051] Table 1 Immunological analysis results of the enzyme-labeled secondary antibody complex dilution solution diluted at 1:500 Note: + indicates the staining intensity, the more + the stronger the staining intensity, (1+) indicates weak staining, positive staining is light yellow, (2+) indicates relatively clear staining, positive staining is yellow, (2.5+) indicates between (2+) and (3+), positive staining is brownish yellow, (3+) indicates clear and obvious staining, positive staining is brownish yellow, (3.5+) indicates very deep and obvious staining, positive staining is brownish yellow; - indicates no staining; + indicates background, the more + the stronger the background; - indicates no background.

[0052] Table 2 Immunological analysis results of the enzyme-labeled secondary antibody complex dilution solution diluted at 1:1000 Note: + indicates the staining intensity, the more + the stronger the staining intensity, (1+) indicates weak staining, positive staining is light yellow, (2+) indicates relatively clear staining, positive staining is yellow, (2.5+) indicates between (2+) and (3+), positive staining is brownish yellow, (3+) indicates clear and obvious staining, positive staining is brownish yellow, (3.5+) indicates very deep and obvious staining, positive staining is brownish yellow; - indicates no staining; + indicates background, the more + the stronger the background; - indicates no background.

[0053] As can be seen from Table 1, through the optimization and improvement of the polymer skeleton, the present invention can solve the problems of large steric hindrance, low sensitivity, and non-specific staining of traditional polymers, which is conducive to further improving the sensitivity of immunological analysis and detection by the DAB staining method. Moreover, as can be seen from Table 2, Comparative Example 1 shows that compared with the conventional polymer skeleton, the chitosan composite skeleton adopted in the present invention has abundant amino groups, can carry more labeled enzymes and secondary antibody molecules, and can form an extended multi-level branched structure. Even at a relatively high dilution factor, high detection sensitivity can still be obtained.

[0054] Comparative Examples 2-4 show that the multi-level branched structure of the chitosan composite skeleton is particularly important for improving detection sensitivity. The spacer arms with a certain spatial structure formed can reduce steric hindrance, carry more labeled enzymes and secondary antibody molecules, and can still obtain relatively high staining intensity at a high dilution factor. Comparative Example 5 shows that the raw material composition ratio for preparing the chitosan composite skeleton will affect the skeleton structure and thus affect the detection sensitivity. When the grafted spacer arms are too dense, they are affected by steric hindrance and are not conducive to carrying more secondary antibody molecules, and the available surface space utilization rate cannot reach the highest, resulting in a lower staining intensity.

[0055] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent structural transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A horseradish peroxidase-labeled secondary antibody complex, characterized in that, The enzyme-labeled secondary antibody complex includes a chitosan complex skeleton and horseradish peroxidase and a secondary antibody carried on the chitosan complex skeleton; the chitosan complex skeleton is a polymer skeleton formed by the compounding of a chitosan spherical crosslinking body and dopamine.

2. The horseradish peroxidase-labeled secondary antibody complex according to claim 1, wherein The preparation method of the chitosan complex skeleton includes the following steps: (1) Add chitosan into an acetic acid aqueous solution, dropwise add a sodium tripolyphosphate aqueous solution while stirring, and then filter to obtain a chitosan spherical crosslinking body; (2) Add the chitosan spherical crosslinking body into ethanol, heat up to reflux and add terephthalaldehyde and trehalose, react at a constant temperature, then add sodium perchlorate, stir under light-shielded conditions, and then add ethylene glycol to terminate the reaction, and rotary evaporate to remove the solvent; (3) Then, add dopamine and water at room temperature, and then add Tris-HCl buffer solution, stir and react to obtain a chitosan complex skeleton.

3. The horseradish peroxidase-labeled secondary antibody complex according to claim 2, wherein In step (1), the concentration of the chitosan in the acetic acid aqueous solution is 20-25 g / L; the concentration of the acetic acid aqueous solution is 0.1-0.3 mol / L; the concentration of the sodium tripolyphosphate aqueous solution is 3-4 mg / mL; the volume ratio of the acetic acid aqueous solution to the sodium tripolyphosphate aqueous solution is 100:2-3.

4. The horseradish peroxidase-labeled secondary antibody complex according to claim 2, wherein In step (2), the mass ratio of the chitosan spherical crosslinking body to ethanol is 1:300-400; the mass ratio of the chitosan spherical crosslinking body, terephthalaldehyde and trehalose is 1:18-20:5-12.

5. The horseradish peroxidase-labeled secondary antibody complex according to claim 2 or 4, characterized in that In step (2), the mass-volume ratio of trehalose, sodium perchlorate and ethylene glycol is 1 g:0.8-1 g:5 mL.

6. The horseradish peroxidase-labeled secondary antibody complex according to claim 2, wherein In step (3), the mass-volume ratio of trehalose, dopamine, Tris-HCl buffer solution and water is 1 g:0.3-0.5 g:3-4 mL:100 mL; the concentration of the Tris-HCl buffer solution is 1 mol / L, and the pH is 7.4; the stirring reaction is stirring at room temperature for 2-3 h.

7. The preparation method of the horseradish peroxidase-labeled secondary antibody complex according to any one of claims 1-6, characterized in that, Including the following steps: S1. Add the chitosan complex skeleton into PBS buffer solution, add horseradish peroxidase, stir and react for 3-4 h to obtain a mixture loaded with the enzyme; S2. Add a secondary antibody into the enzyme-loaded mixture, add EDC, adjust the pH to 5-6, carry out dehydration condensation, and separate by a purification column after the reaction to obtain a horseradish peroxidase-labeled secondary antibody complex.

8. The preparation method according to claim 7, wherein, The mass ratio of the chitosan complex skeleton to horseradish peroxidase is 1:5-10; the concentration of horseradish peroxidase in the enzyme-loaded mixture is 0.5-0.8 wt%; the mass ratio of the enzyme-loaded mixture to the secondary antibody is 20-25:

1.

9. The application of the horseradish peroxidase-labeled secondary antibody complex according to any one of claims 1-6 or the horseradish peroxidase-labeled secondary antibody complex prepared by the preparation method according to any one of claims 7-8 in a DAB chromogenic reagent.

10. The application according to claim 9, wherein The DAB chromogenic reagent includes a horseradish peroxidase-labeled secondary antibody complex, a DAB concentrated chromogenic solution and a DAB substrate buffer solution; the DAB concentrated chromogenic solution includes 3,3-diaminobenzidine tetrachloride; the DAB substrate buffer solution includes hydrogen peroxide.

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