Antibody complex based on chitosan and cerium oxide modification, and preparation method and application thereof

Abstract

This invention relates to the field of nanobiotechnology, specifically to an antibody complex based on chitosan and cerium oxide modification, its preparation method, and its application. The complex is formed by the electrostatic self-assembly of chitosan, cerium oxide nanozyme, and Ki67 monoclonal antibody. Specifically, chitosan and cerium oxide nanozyme self-assemble to form a positively charged chitosan / cerium oxide nanozyme complex, which then forms a ternary complex with a negatively charged Ki67. This invention uses cerium oxide nanozyme integrated with a polymeric carrier to replace traditional horseradish peroxidase, achieving one-step detection and overcoming the limitations of the traditional two-step immunohistochemical method. Pathological evaluation of intraoperative frozen sections of tumor tissue can be completed in just 20 minutes, and paraffin section detection is also 40 minutes shorter than traditional methods. The specificity and color intensity are comparable to traditional methods, and the cost per sample is reduced by approximately 89%, making it suitable for rapid pathological diagnosis of tumors.

Description

Technical Field

[0001] This invention relates to the field of nanobiotechnology, specifically to antibody complexes based on chitosan and cerium oxide modification, their preparation methods, and applications. Background Technology

[0002] Immunohistochemistry (IHC) is a technique based on the principle of specific antigen-antibody binding, using chemical labeling technology to locate, characterize, and semi-quantitatively detect specific antigens within tissues or cells. Traditional clinical tumor pathology diagnosis using IHC primarily relies on paraffin sections and multi-stage antibody amplification systems to detect tumor markers. However, this requires multiple pretreatment steps such as fixation and embedding, which is time-consuming when dealing with large numbers of samples, making it difficult to meet the needs of rapid diagnosis. Intraoperative pathology diagnosis currently relies mainly on morphological interpretation through hematoxylin-eosin staining (HE) of frozen sections. However, due to inherent defects such as ice crystal artifacts during tissue processing, misdiagnosis or missed diagnosis is common, placing significant pressure on pathological confirmation. Currently, only a few IHC reagents are available to assist in rapid frozen section pathology diagnosis, but these are expensive and have low clinical application rates. Therefore, developing rapid IHC detection reagents for clinical diagnosis to address these two problems, significantly shortening detection time, is of great practical significance for improving the efficiency of pathological diagnosis.

[0003] Ki67 is a cell cycle-dependent nuclear antigen whose expression level is significantly positively correlated with tumor cell proliferation activity. It has been incorporated into the routine pathological diagnostic system for various solid tumors, such as breast cancer, gastric cancer, and colorectal cancer. Quantitative analysis of the Ki67 labeling index (LI) using IHC technology can provide crucial information for tumor grading, prognostic assessment, and treatment selection. However, existing Ki67 IHC detection reagents have lengthy procedures, typically requiring 30-40 minutes of primary antibody incubation and 20-30 minutes of secondary antibody incubation, which is time-consuming and cannot be applied to intraoperative pathological diagnosis. To address this, researchers have increased antibody incubation temperatures or modified monoclonal antibodies to form polymerase-labeled Fab conjugates, amplifying the detection signal to shorten antibody incubation time. However, these improved methods are complex and require blocking endogenous horseradish peroxidase activity, further prolonging the detection time. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an antibody complex based on chitosan and cerium oxide modification, its preparation method, and its application. This invention uses chitosan, cerium oxide nanozyme, and Ki67 monoclonal antibody as raw materials, and forms a ternary composite structure through electrostatic self-assembly to obtain an antibody complex based on chitosan and cerium oxide modification. Using this antibody complex in an IHC detection system, highly sensitive detection of tumor markers can be achieved within 20 minutes. This solves the problem of existing improved methods having complex operating procedures and requiring the blocking of endogenous horseradish peroxidase activity, thus prolonging the detection time.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a method for preparing an antibody complex based on chitosan and cerium oxide modification, comprising the following steps: S1. Using positively charged chitosan and negatively charged cerium oxide nanozymes as raw materials, a chitosan / cerium oxide nanozyme complex is formed through charge attraction.

[0006] S2. Using Ki67 monoclonal antibody and chitosan / cerium oxide nanozyme complex as raw materials, electrostatic self-assembly was carried out to form a ternary composite structure, resulting in an antibody complex based on chitosan and cerium oxide modification.

[0007] The Ki67 monoclonal antibody was purchased from Guangzhou Mianyi Biotechnology Co., Ltd.

[0008] It should be noted that in this invention, the construction of the chitosan and cerium oxide-modified antibody complex is based on triple electrostatic interactions. First, the chitosan / cerium oxide nanozyme complex is assembled: chitosan carries a positive charge at pH < 6.5, while the cerium oxide nanozyme carries a negative charge due to the ionization of its surface hydroxyl groups. The positive and negative charges attract each other to form the chitosan / cerium oxide nanozyme complex. Zeta potential detection shows that the surface charge of the assembled complex is positive. Second, antibody conjugation occurs: the Ki67 monoclonal antibody has an isoelectric point of approximately 8.5 at pH 7.4, giving it a negative charge. This negatively charged antibody self-assembles with the positively charged chitosan / cerium oxide nanozyme complex through electrostatic interactions, forming a stable ternary structure, which acts as a "signal amplification unit," enhancing detection sensitivity.

[0009] Chitosan, as a natural cationic polymer, differs from most neutral or anionic polysaccharides in that it can bind to negatively charged materials such as cerium oxide nanozymes and proteins such as Ki67 monoclonal antibodies through electrostatic interactions, forming stable multilayer structures or electrostatic complexes, providing a basis for the construction of ternary complexes. Secondly, chitosan can significantly reduce carrier toxicity, improve biocompatibility, and increase delivery efficiency by modifying the surface charge properties of carriers. Therefore, the inherently positively charged nature of chitosan allows for coupling with negatively charged materials and proteins, providing a new strategy for the design of intraoperative rapid diagnostic probes.

[0010] The cerium oxide nanozymes exhibit a variety of enzymatic activities, thanks to the rapid valence state switching mediated by oxygen vacancies in their surface active sites. Compared to natural horseradish peroxidase (HRP), cerium oxide nanozymes maintain better stability and catalytic activity over a wider range of temperature and pH conditions, making them easier to store and use. Furthermore, cerium oxide nanozymes are highly tunable; their structure, size, surface properties, and catalytic activity can be controlled by altering the preparation method, doping with other elements, or combining them with other materials to meet the needs of different applications.

[0011] In a preferred embodiment of the present invention, the mass ratio of cerium oxide nanozyme to chitosan is 1:1 to 8, more preferably 1:4. The mass ratio of the chitosan / cerium oxide nanozyme complex to Ki67 monoclonal antibody is 1:1 to 4, more preferably 1:1, at which point the chitosan / cerium oxide nanozyme complex and Ki67 are optimally conjugated.

[0012] In a preferred embodiment of the present invention, the preparation method of the chitosan / cerium oxide nanozyme complex includes the following steps: S1. Dissolve chitosan in a solvent and protonate the chitosan to obtain a chitosan solution.

[0013] S2. Mix the cerium oxide nanozyme stock solution with water and sonicate for the first time to obtain the cerium oxide nanozyme solution.

[0014] S3. Add the chitosan solution to the phosphate buffer solution, sonicate for the second time, and add the cerium oxide nanozyme solution at the same time. A charge attraction reaction occurs at room temperature. Then, centrifuge and wash to obtain the chitosan / cerium oxide nanozyme complex.

[0015] In a preferred embodiment of the present invention, the mass-to-volume ratio of chitosan to solvent is 1 mg~1.5 mg:1 mL~1.5 mL, more preferably 1 mg:1 mL. The solvent is an acetic acid solution with a volume concentration of 1%~2%, more preferably a 1% volume concentration acetic acid solution. The chitosan solution is a positively charged chitosan solution. Acetic acid is used because the protonation degree of the chitosan amino groups can be maximized under acidic conditions, enhancing the electrostatic adsorption capacity with negatively charged materials. In a specific embodiment, chitosan is dissolved in acetic acid and then vortexed for 30 minutes until completely dissolved.

[0016] In a preferred embodiment of the present invention, the concentration of the cerium oxide nanozyme stock solution is 1 mg / mL to 1.5 mg / mL, more preferably 1 mg / mL. The volume ratio of the cerium oxide nanozyme stock solution to water is 1:6 to 9, more preferably 1:9. The first sonication time is 5 min to 10 min, more preferably 5 min.

[0017] In a preferred embodiment of the present invention, the concentration of the cerium oxide nanozyme solution is 0.1 mg / mL to 0.2 mg / mL, more preferably 0.1 mg / mL. The second sonication time is 10 min to 15 min, more preferably 10 min. The second sonication power is 100 W to 120 W, more preferably 100 W. The purpose of sonication is to eliminate nanoparticle aggregation.

[0018] In a preferred embodiment of the present invention, the electrostatic adsorption reaction time is 1 h to 1.5 h, more preferably 1 h. The volume ratio of chitosan solution to phosphate buffer is 1:5 to 6, more preferably 1:5.

[0019] In a preferred embodiment of the present invention, the centrifugation speed is 12000 rpm, and the time is 10 min to 15 min, more preferably 10 min. Washing is performed twice with acetic acid and water, respectively; the volume concentration of acetic acid is 1% to 2%, more preferably 1%. The purpose of washing is to remove unbound cerium oxide nanozymes.

[0020] In a preferred embodiment of the present invention, the specific method for electrostatic self-assembly to form a ternary composite structure is as follows: S1. Resuspend the chitosan / cerium oxide nanozyme complex in phosphate buffer to obtain a chitosan / cerium oxide nanozyme complex solution.

[0021] S2. Ki67 monoclonal antibody was added to the chitosan / cerium oxide nanozyme complex solution, and an electrostatic self-assembly reaction occurred at room temperature. After centrifugation and washing, a ternary composite structure was formed, and an antibody complex based on chitosan and cerium oxide modification was obtained.

[0022] In a preferred embodiment of the present invention, the CeO2 nanozyme has a particle size of 4 nm to 6 nm, the chitosan has a degree of deacetylation ≥ 85%, and a molecular weight of 20 kDa to 200 kDa. In a specific embodiment, the chitosan has a degree of deacetylation of 92%, and the CeO2 nanozyme has a particle size of 2 nm to 6 nm.

[0023] In a preferred embodiment of the present invention, the electrostatic self-assembly reaction time is 1 h to 1.5 h, more preferably 1 h. The pH of the Ki67 monoclonal antibody is 7.4 to 7.8, more preferably 7.4.

[0024] In a preferred embodiment of the present invention, the centrifugation speed is 15000 rpm and the time is 10 min to 15 min; the washing is performed 3 times with phosphate buffer.

[0025] A third objective of this invention is to provide an application of the above-mentioned chitosan and cerium oxide-modified antibody complex in an immunohistochemical detection reagent.

[0026] In a preferred embodiment of the present invention, an antibody complex based on chitosan and cerium oxide is added to the pretreated slides, incubated, and then stained; the slides are frozen slides or paraffin slides.

[0027] On the one hand, the traditional method for detecting Ki67 protein in paraffin sections using IHC includes the following steps: (1) Gradient dewaxing: Paraffin sections with a thickness of 3 μm were immersed in the following sequence: xylene I for 1 min, xylene II for 1 min, a mixture of xylene and anhydrous ethanol for 30 sec, anhydrous ethanol I for 30 sec, anhydrous ethanol II for 30 sec, 95% ethanol for 30 sec, 85% ethanol for 30 sec, 75% ethanol for 30 sec, 50% ethanol for 30 sec, pure water for 30 sec, and 1×PBS buffer for 1 min, repeated 3 times. Dewaxed sections were obtained. The volume ratio of xylene to anhydrous ethanol in the mixture of xylene and anhydrous ethanol was 1:1.

[0028] (2) Antigen retrieval: First, preheat the 1×Tris-EDTA antigen retrieval solution in a microwave oven on medium heat for 3 minutes. Place the dewaxed sections into the preheated antigen retrieval solution and microwave on high for 3 minutes. Remove the antigen retrieval solution from the microwave oven and cool for 2 minutes, then microwave on low for 15 minutes. Next, once the temperature has dropped below 50°C, wash the sections with 1×PBS buffer for 1 minute, repeating 3 times.

[0029] (3) Add the antibody complex modified with chitosan and cerium oxide and incubate for 10 min.

[0030] (4) Soak the sections in 1×PBS buffer to wash them.

[0031] (5) DAB color development: Dilute the DAB color development solution with pure water at a volume ratio of 1:20, add it to the slice, incubate for 1 min, and observe the color development under a microscope.

[0032] (6) Rinse the tissue with pure water.

[0033] (7) Counterstain with hematoxylin for 1 min to 2 min, then rinse with pure water.

[0034] (8) Use anti-blue solution to perform anti-blueing until the tissue can be clearly seen to change from purple to blue with the naked eye.

[0035] (9) Tissue dehydration, clearing, and fixation: The sections were soaked in the following order: 50% ethanol for 30 seconds, 75% ethanol for 30 seconds, 85% ethanol for 30 seconds, 95% ethanol for 30 seconds, anhydrous ethanol for 30 seconds, a 1:1 mixture of xylene and anhydrous ethanol for 30 seconds, and xylene for 1 minute. Then, neutral resin was added for mounting to achieve rapid detection of Ki67 protein in traditional paraffin sections.

[0036] In this invention, DAB is 3,3'-diaminobenzidine.

[0037] On the other hand, the method for detecting Ki67 protein in intraoperative frozen sections by IHC includes the following steps: (1) Cut fresh tissue into 1cm×1cm×0.5cm tissue blocks, place them in a mold, add embedding agent OCT, and freeze rapidly in an ultra-low temperature freezer.

[0038] (2) Cut the tissue into 5 μm thick sections in a cryostat.

[0039] (3) Fix the tissue sections to be stained with 95% ethanol in a 4°C refrigerator for 2 min.

[0040] (4) Rinse the sections with 1×PBS for 20 seconds.

[0041] (5) Add the antibody complex modified with chitosan and cerium oxide and incubate for 10 min.

[0042] (6) Rinse the sections with 1×PBS for 20 seconds.

[0043] (7) DAB color development: Dilute the DAB color development solution with pure water at a volume ratio of 1:20, incubate for about 1 minute, and observe the color development under a microscope.

[0044] (8) Rinse the slices with pure water for 20 seconds.

[0045] (9) Counterstain with hematoxylin for 1 min to 2 min, then rinse with pure water.

[0046] (10) Tissue dehydration, clearing, and fixation: The sections were soaked in a mixture of 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, xylene:anhydrous ethanol = 1:1, and xylene in that order. The volume ratio of xylene to anhydrous ethanol in the xylene and anhydrous ethanol mixture was 1:1. Neutral resin was then added for mounting to enable rapid detection of Ki67 protein in frozen sections using IHC.

[0047] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides a method for preparing an antibody complex based on chitosan and cerium oxide modification. Using chitosan (CS), cerium oxide nanozyme, and Ki67 monoclonal antibody as raw materials, the complex is formed through electrostatic self-assembly. Specifically, positively charged chitosan and negatively charged cerium oxide nanozyme attract each other to form a positively charged chitosan / cerium oxide nanozyme complex. The positive charge of the chitosan / cerium oxide nanozyme complex originates from the inherent negative charge of cerium oxide; after modification with positively charged chitosan, the complex becomes positively charged overall. Subsequently, the negatively charged Ki67 monoclonal antibody and the positively charged chitosan / cerium oxide nanozyme complex undergo electrostatic self-assembly to form a ternary composite structure, namely the CS-CeO2-Ki67 complex. Chitosan provides the positive charge base, constructs the complex framework, and optimizes biocompatibility. The cerium oxide nanozyme replaces traditional HRP to provide enzyme-like catalytic activity, ensuring stability and practicality, and participating in electrostatic assembly. It is suitable for detection in both frozen and paraffin sections, thus having a wide range of applications. This solves the problem that existing improved methods have complex operating procedures and require blocking endogenous horseradish peroxidase activity, which prolongs the detection time.

[0048] 2. The antibody complex provided by this invention replaces the traditional multi-stage reaction with a one-step incubation, significantly shortening the detection time. The entire process of intraoperative frozen section detection takes ≤20 minutes. For ultra-rapid IHC of frozen sections from common intraoperative breast, lung, and thyroid cancers, only 20 minutes are required, meeting the needs of rapid intraoperative pathological evaluation. The total time for paraffin section detection is ≤60 minutes, far lower than the ≥100 minutes of traditional IHC methods. Furthermore, the antibody complex exhibits no non-specific staining in paraffin sections of various cancers and adjacent tissues; its specificity and staining intensity are comparable to traditional IHC performed with commercially available Ki67 antibodies.

[0049] 3. The cost of single-sample detection of the CS-CeO2-Ki67 complex provided by this invention for IHC is only 0.225 yuan, which is about 89% lower than the 2.5 yuan of traditional IHC. This is beneficial to reducing the cost of clinical diagnosis and increasing the clinical application rate of the detection system. Attached Figure Description

[0050] Figure 1 This diagram illustrates the structure and self-assembly mechanism of the CS-CeO2-Ki67 composite of the present invention.

[0051] Figure 2 The images show the FTIR spectra of the CS-CeO2-Ki67 complexes from Examples 1 to 5 of this invention.

[0052] Figure 3 The UV-Vis spectra of the CS-CeO2-Ki67 complexes of Examples 1 and 6-7 of this invention are shown.

[0053] Figure 4 The image shows the zeta potential diagrams of the CS-CeO2-Ki67 complexes of Examples 1 to 7 of this invention. Figure 4 Figure A shows the Zeta potential diagrams for Examples 1 to 5, and Figure B shows the Zeta potential diagrams for Examples 1 and 6 to 7.

[0054] Figure 5 This image shows a comparison of the color development effects of CS-CeO2-Ki67 complex detection and conventional IHC detection on frozen sections of lung cancer and corresponding adjacent tissues during surgery in accordance with the present invention. Figure 5 Figure a shows lung cancer tissue detected by traditional IHC, Figure b shows lung cancer adjacent tissue detected by traditional IHC, Figure c shows lung cancer tissue detected by CS-CeO2-Ki67 complex, and Figure d shows lung cancer adjacent tissue detected by CS-CeO2-Ki67 complex.

[0055] Figure 6 This image shows a comparison of the color development effects of CS-CeO2-Ki67 complex detection and conventional IHC detection on frozen sections of thyroid cancer and corresponding adjacent tissues during surgery in accordance with the present invention. Figure 6 Figure a shows a thyroid cancer tissue image detected by traditional IHC, Figure b shows a thyroid cancer adjacent tissue image detected by traditional IHC, Figure c shows a thyroid cancer tissue image detected by the CS-CeO2-Ki67 complex, and Figure d shows a thyroid cancer adjacent tissue image detected by the CS-CeO2-Ki67 complex.

[0056] Figure 7 This image shows a comparison of the color development effects of paraffin sections of thyroid cancer and corresponding adjacent tissue detected using the CS-CeO2-Ki67 complex and conventional IHC detection, which are based on the present invention. Figure 7 Figure a shows a thyroid cancer tissue image detected by traditional IHC, Figure b shows a thyroid cancer adjacent tissue image detected by traditional IHC, Figure c shows a thyroid cancer tissue image detected by the CS-CeO2-Ki67 complex, and Figure d shows a thyroid cancer adjacent tissue image detected by the CS-CeO2-Ki67 complex.

[0057] Figure 8 This image shows a comparison of the color development effects of paraffin sections of colorectal cancer and corresponding adjacent tissues detected using the CS-CeO2-Ki67 complex versus conventional IHC detection. Figure 8 Figure a shows a colorectal cancer tissue image detected by traditional IHC, Figure b shows a colorectal cancer adjacent tissue image detected by traditional IHC, Figure c shows a colorectal cancer tissue image detected by the CS-CeO2-Ki67 complex, and Figure d shows a colorectal cancer adjacent tissue image detected by the CS-CeO2-Ki67 complex.

[0058] Figure 9This image shows a comparison of the color development effects of CS-CeO2-Ki67 complex detection and conventional IHC detection on paraffin sections of lung cancer and corresponding adjacent non-cancerous tissues, as presented in this invention. Figure 9 Figure a shows lung cancer tissue detected by traditional IHC, Figure b shows lung cancer adjacent tissue detected by traditional IHC, Figure c shows lung cancer tissue detected by CS-CeO2-Ki67 complex, and Figure d shows lung cancer adjacent tissue detected by CS-CeO2-Ki67 complex. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] It should be noted that the technical terms used in this invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0061] The following specific examples will provide further explanation.

[0062] In this invention, chitosan is abbreviated as CS, cerium oxide is abbreviated as CeO2, and phosphate buffered saline is abbreviated as PBS. In the following text, the concentration of PBS is 0.1M.

[0063] Example 1 A method for preparing an antibody complex based on chitosan and cerium oxide, namely the CS-CeO2-Ki67 complex, includes the following steps: S1. Weigh 10 mg of CS with a molecular weight of 20 kDa and dissolve it in 10 mL of acetic acid solution with a volume concentration of 1%. Vortex for 30 min until completely dissolved to form a positively charged CS solution.

[0064] S2. Take 1 mL of CeO2 aqueous solution with a concentration of 1 mg / mL, add 9 mL of pure water to obtain a CeO2 solution with a concentration of 0.1 mg / mL, and sonicate for 5 min.

[0065] S3. Add 200 μL of CS solution to 1 mL of PBS (pH 7.4). While sonicating at 100 W, add 50 μL of CeO2 solution dropwise. After sonicating for 10 min, stir magnetically at room temperature for 1 h. After the reaction is complete, centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash twice each with 1% acetic acid and pure water. Finally, resuspend the precipitate in 1 mL of PBS (pH 7.4) to obtain the CS-CeO2 complex solution. The mass ratio of CeO2 to CS is 1:4.

[0066] S4. Add 50 μL of Ki67 monoclonal antibody (pH=7.4, 1 mg / mL) to 1 mL of the CS-CeO2 complex solution, vortex to mix, and react at room temperature for 1 h. After the reaction is complete, centrifuge at 15000 rpm for 10 min to collect the precipitate, wash three times with PBS to obtain the CS-CeO2-Ki67 ternary complex. The mass ratio of the CS-CeO2 complex to the Ki67 monoclonal antibody is 1:0.5.

[0067] Example 2 A method for preparing an antibody complex based on chitosan and cerium oxide, namely the CS-CeO2-Ki67 complex, includes the following steps: S1. Weigh 10 mg of CS with a molecular weight of 20 kDa and dissolve it in 10 mL of acetic acid solution with a volume concentration of 1%. Vortex for 30 min until completely dissolved to form a positively charged CS solution.

[0068] S2. Take 1 mL of CeO2 stock solution with a concentration of 1 mg / mL, add 9 mL of pure water to obtain a CeO2 solution with a concentration of 0.1 mg / mL, and sonicate for 5 min.

[0069] S3. Add 100 μL of CS solution to 1 mL of PBS (pH 7.4). While sonicating at 100 W, add 100 μL of CeO2 solution dropwise. After sonicating for 10 min, stir magnetically at room temperature for 1 h. After the reaction is complete, centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash twice each with 1% acetic acid and pure water. Finally, resuspend the precipitate in 1 mL of PBS (pH 7.4) to obtain the CS-CeO2 complex solution. The mass ratio of CeO2 to CS is 1:1.

[0070] S4. Add 50 μL of Ki67 monoclonal antibody (pH=7.4) at a concentration of 1 mg / mL to 1 mL of the CS-CeO2 complex solution, vortex to mix, and react at room temperature for 1 h. After the reaction is complete, centrifuge at 15000 rpm for 10 min to collect the precipitate, wash three times with PBS to obtain the CS-CeO2-Ki67 ternary complex.

[0071] Example 3 A method for preparing an antibody complex based on chitosan and cerium oxide, namely the CS-CeO2-Ki67 complex, includes the following steps: S1. Weigh 10 mg of CS with a molecular weight of 20 kDa and dissolve it in 10 mL of acetic acid solution with a volume concentration of 1%. Vortex for 30 min until completely dissolved to form a positively charged CS solution.

[0072] S2. Take 1 mL of CeO2 stock solution with a concentration of 1 mg / mL, add 9 mL of pure water to obtain a CeO2 solution with a concentration of 0.1 mg / mL, and sonicate for 5 min.

[0073] S3. Add 200 μL of CS solution to 1 mL of PBS (pH 7.4). While sonicating at 100 W, add 100 μL of CeO2 solution dropwise. After sonicating for 10 min, stir magnetically at room temperature for 1 h. After the reaction is complete, centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash twice each with 1% acetic acid and pure water. Finally, resuspend the precipitate in 1 mL of PBS (pH 7.4) to obtain the CS-CeO2 complex solution. The mass ratio of CeO2 to CS is 1:2.

[0074] S4. Add 50 μL of Ki67 monoclonal antibody (pH=7.4) at a concentration of 1 mg / mL to 1 mL of the CS-CeO2 complex solution, vortex to mix, and react at room temperature for 1 h. After the reaction is complete, centrifuge at 15000 rpm for 10 min to collect the precipitate, wash three times with PBS to obtain the CS-CeO2-Ki67 ternary complex.

[0075] Example 4 A method for preparing an antibody complex based on chitosan and cerium oxide, namely the CS-CeO2-Ki67 complex, includes the following steps: S1. Weigh 10 mg of CS with a molecular weight of 20 kDa and dissolve it in 10 mL of acetic acid solution with a volume concentration of 1%. Vortex for 30 min until completely dissolved to form a positively charged CS solution.

[0076] S2. Take 1 mL of CeO2 stock solution with a concentration of 1 mg / mL, add 9 mL of pure water to obtain a CeO2 solution with a concentration of 0.1 mg / mL, and sonicate for 5 min.

[0077] S3. Add 600 μL of CS solution to 1 mL of PBS (pH 7.4). While sonicating at 100 W, add 100 μL of CeO2 solution dropwise. After sonicating for 10 min, stir magnetically at room temperature for 1 h. After the reaction is complete, centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash twice each with 1% acetic acid and pure water. Finally, resuspend the precipitate in 1 mL of PBS (pH 7.4) to obtain the CS-CeO2 complex solution. The mass ratio of CeO2 to CS is 1:6.

[0078] S4. Add 50 μL of Ki67 monoclonal antibody (pH=7.4) at a concentration of 1 mg / mL to 1 mL of the CS-CeO2 complex solution, vortex to mix, and react at room temperature for 1 h. After the reaction is complete, centrifuge at 15000 rpm for 10 min to collect the precipitate, wash three times with PBS to obtain the CS-CeO2-Ki67 ternary complex.

[0079] Example 5 A method for preparing an antibody complex based on chitosan and cerium oxide, namely the CS-CeO2-Ki67 complex, includes the following steps: S1. Weigh 10 mg of CS with a molecular weight of 20 kDa and dissolve it in 10 mL of acetic acid solution with a volume concentration of 1%. Vortex for 30 min until completely dissolved to form a positively charged CS solution.

[0080] S2. Take 1 mL of CeO2 stock solution with a concentration of 1 mg / mL, add 9 mL of pure water to obtain a CeO2 solution with a concentration of 0.1 mg / mL, and sonicate for 5 min.

[0081] S3. Add 800 μL of CS solution to 1 mL of PBS (pH 7.4). While sonicating at 100 W, add 100 μL of CeO2 solution dropwise. After sonicating for 10 min, stir magnetically at room temperature for 1 h. After the reaction is complete, centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash twice each with 1% acetic acid and pure water. Finally, resuspend the precipitate in 1 mL of PBS (pH 7.4) to obtain the CS-CeO2 complex solution. The mass ratio of CeO2 to CS is 1:8.

[0082] S4. Add 50 μL of Ki67 monoclonal antibody (pH=7.4) at a concentration of 1 mg / mL to 1 mL of the CS-CeO2 complex solution, vortex to mix, and react at room temperature for 1 h. After the reaction is complete, centrifuge at 15000 rpm for 10 min to collect the precipitate, wash three times with PBS to obtain the CS-CeO2-Ki67 ternary complex.

[0083] Example 6 A method for preparing an antibody complex based on chitosan and cerium oxide, namely the CS-CeO2-Ki67 complex, includes the following steps: S1. Weigh 10 mg of CS with a molecular weight of 20 kDa and dissolve it in 10 mL of acetic acid solution with a volume concentration of 1%. Vortex for 30 min until completely dissolved to form a positively charged CS solution.

[0084] S2. Take 1 mL of CeO2 stock solution with a concentration of 1 mg / mL, add 9 mL of pure water to obtain a CeO2 solution with a concentration of 0.1 mg / mL, and sonicate for 5 min.

[0085] S3. Add 200 μL of CS solution to 1 mL of PBS (pH 7.4). While sonicating at 100 W, add 50 μL of CeO2 solution dropwise. After sonicating for 10 min, stir magnetically at room temperature for 1 h. After the reaction is complete, centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash twice each with 1% acetic acid and pure water. Finally, resuspend the precipitate in 1 mL of PBS (pH 7.4) to obtain the CS-CeO2 complex solution.

[0086] S4. Add 100 μL of Ki67 monoclonal antibody (pH=7.4, 1 mg / mL) to 1 mL of the CS-CeO2 complex solution, vortex to mix, and react at room temperature for 1 h. After the reaction is complete, centrifuge at 15000 rpm for 10 min to collect the precipitate, wash three times with PBS to obtain the CS-CeO2-Ki67 ternary complex. The mass ratio of the CS-CeO2 complex to the Ki67 monoclonal antibody is 1:1.

[0087] Example 7 A method for preparing an antibody complex based on chitosan and cerium oxide, namely the CS-CeO2-Ki67 complex, includes the following steps: S1. Weigh 10 mg of CS with a molecular weight of 20 kDa and dissolve it in 10 mL of acetic acid solution with a volume concentration of 1%. Vortex for 30 min until completely dissolved to form a positively charged CS solution.

[0088] S2. Take 1 mL of CeO2 stock solution with a concentration of 1 mg / mL, add 9 mL of pure water to obtain a CeO2 solution with a concentration of 0.1 mg / mL, and sonicate for 5 min.

[0089] S3. Add 200 μL of CS solution to 1 mL of PBS (pH 7.4). While sonicating at 100 W, add 50 μL of CeO2 solution dropwise. After sonicating for 10 min, stir magnetically at room temperature for 1 h. After the reaction is complete, centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash twice each with 1% acetic acid and pure water. Finally, resuspend the precipitate in 1 mL of PBS (pH 7.4) to obtain the CS-CeO2 complex solution.

[0090] S4. Add 400 μL of Ki67 monoclonal antibody (pH=7.4, 1 mg / mL) to 1 mL of the CS-CeO2 complex solution. Vortex to mix and react at room temperature for 1 h. After the reaction is complete, centrifuge at 15000 rpm for 10 min to collect the precipitate. Wash three times with PBS to obtain the CS-CeO2-Ki67 ternary complex. The mass ratio of the CS-CeO2 complex to the Ki67 monoclonal antibody is 1:4.

[0091] The structure and properties of the CS-CeO2-Ki67 complexes from Examples 1 to 7 were tested.

[0092] Figure 1 This diagram illustrates the structure and self-assembly mechanism of the CS-CeO2-Ki67 composite of the present invention.

[0093] Figure 2 These are the FTIR spectra of the CS-CeO2-Ki67 complexes from Examples 1 to 5 of this invention. Figure 2 It can be seen that the FTIR spectra of CS, CeO2, and CS-CeO2 are located at 1690 cm⁻¹. -1 The peak at 1729 cm⁻¹ is the vibrational absorption peak of the C=C bond. -1 The peak at 1030 cm⁻¹ corresponds to the vibrational absorption peak of the C=O bond, specifically the -COOH group on CeO₂. -1 and 1545cm -1 Two distinct vibrational absorption peaks are observed, corresponding to the stretching vibration of CO in the primary alcohol and the bending vibration of NH in the amino group, respectively. (511 cm⁻¹) -1 The absorption peak at that point corresponds to the O-Ce group on CeO2. The CeO2-CS spectrum shows characteristic absorption peaks corresponding to CeO2 and CS, respectively, proving that CS and CeO2 have been successfully coupled.

[0094] Figure 3The images show the UV-Vis spectra of the CS-CeO2-Ki67 complexes from Examples 1 and 6-7 of this invention. Figure 3 It can be seen that proteins have light absorption at 280 nm. Ki67, CS-CeO2:Ki67=1:1, CS-CeO2:Ki67=1:2 and CS-CeO2:Ki67=1:4 show different absorption peaks at 280 nm. When the ratio of CS-CeO2 to Ki67 is 1:1, as shown in Example 6, the blue line partially overlaps with the purple line, indicating that the number of Ki67 antibodies coupled to CS-CeO2 reaches its maximum value.

[0095] Figure 4 The image shows the zeta potential diagrams of the CS-CeO2-Ki67 complexes of Examples 1 to 7 of this invention. Figure 4 Figure A shows the Zeta potential diagrams for Examples 1-5, and Figure B shows the Zeta potential diagrams for Examples 1 and 6-7. Figure 4 As shown in Figure A, the Zeta potential diagram reveals that CeO2 has a strong negative charge. After CS modification, the CeO2-CS system becomes positively charged overall. As the size of molecules or dispersed particles decreases, the Zeta potential of the system, regardless of its polarity, increases accordingly, thereby enhancing the system's stability and enabling it to effectively resist aggregation and maintain a good dissolved or dispersed state. When the Zeta potential decreases, regardless of polarity, the attractive force between particles in the system gradually exceeds the repulsive force, leading to the disruption of the dispersed state and subsequent coagulation or aggregation. Therefore, when CeO2:CS = 1:4, the Zeta potential reaches its maximum value, indicating that the CeO2-CS system is the most stable. This ratio of CeO2-CS was subsequently used for conjugation of the Ki67 antibody. Figure 4 As shown in Figure B, when CeO2-CS:Ki67=1:1, the Zeta potential of CS-CeO2-Ki67 reaches its maximum value, and the system is most stable at this time.

[0096] Next, to demonstrate whether the CS-CeO2-Ki67 antibody can be used in intraoperative pathological tissues, this invention uses the CS-CeO2-Ki67 complex to perform IHC on frozen sections of lung cancer, thyroid cancer, and adjacent non-cancerous tissues, with Dako's commercially available Ki67 as a positive control for routine IHC.

[0097] Application Example 1 The application of a CS-CeO2-Ki67 complex in intraoperative frozen section detection includes the following steps: (1) Cut fresh lung cancer and corresponding adjacent tissue into 1cm×1cm×0.5cm tissue blocks, place them in a mold, add 1mL of embedding agent OCT, and freeze rapidly in an ultra-low temperature freezer.

[0098] (2) In a cryostat, lung cancer and the corresponding adjacent tissue were cut into 5 μm thick sections.

[0099] (3) Fix the tissue sections to be stained with fixative, i.e., 95% ethanol, in a refrigerator at 4°C for 2 min.

[0100] (4) Rinse the slides with 1× PBS buffer for 20 seconds.

[0101] (5) The CS-CeO2-Ki67 complex obtained in Example 7 was resuspended in 1 mL of PBS, the concentration was adjusted to 1 μg / mL, and stored at 4 °C in the dark to obtain the CS-CeO2-Ki67 complex solution. The CS-CeO2-Ki67 complex solution was added to the slice and incubated for 10 min.

[0102] (6) Rinse the slides with 1× PBS buffer for 20 seconds.

[0103] (7) DAB color development: Dilute the DAB color development solution with pure water at a volume ratio of 1:20, incubate for about 1 minute, and observe the color development under a microscope.

[0104] (8) Rinse the slices with pure water for 20 seconds.

[0105] (9) Counterstain with hematoxylin for 1 min, then rinse with pure water.

[0106] (10) Tissue dehydration, clearing, and mounting: The sections were soaked in the following order: 50% ethanol for 10 seconds, 75% ethanol for 10 seconds, 85% ethanol for 10 seconds, 95% ethanol for 10 seconds, anhydrous ethanol for 10 seconds, a 1:1 mixture of xylene and anhydrous ethanol for 1 minute, and xylene for 1 minute. Then, neutral resin was added for mounting to achieve rapid detection of Ki67 protein in frozen sections by IHC.

[0107] The proportion of Ki67 positive cells in Application Example 1 was analyzed by microscopy, and the entire process took ≤20 minutes.

[0108] Application Example 2 The difference from Application Example 1 is that the tissue section is a section of thyroid cancer and the corresponding adjacent tissue.

[0109] Application Example 3 The application of a CS-CeO2-Ki67 complex in traditional paraffin section detection includes the following steps: (1) Gradient dewaxing: Paraffin sections of thyroid cancer and corresponding adjacent tissue with a thickness of 3 μm were immersed in the following sequence: xylene I for 1 min, xylene II for 1 min, a 1:1 mixture of xylene and anhydrous ethanol for 30 sec, anhydrous ethanol I for 30 sec, anhydrous ethanol II for 30 sec, 95% ethanol for 30 sec, 85% ethanol for 30 sec, 75% ethanol for 30 sec, 50% ethanol for 30 sec, pure water for 30 sec, and 1×PBS buffer for 1 min, repeated 3 times. Dewaxed sections were obtained.

[0110] (2) Antigen retrieval: First, preheat the 1×Tris-EDTA antigen retrieval solution in a microwave oven on medium heat for 3 minutes. Place the dewaxed sections into the preheated antigen retrieval solution and microwave on high for 3 minutes. Remove the antigen retrieval solution from the microwave oven and cool for 2 minutes, then microwave on low for 15 minutes. Next, once the temperature has dropped below 50°C, wash the sections with 1×PBS buffer for 1 minute, repeating 3 times.

[0111] (3) The CS-CeO2-Ki67 complex prepared in Example 7 was resuspended in 1 mL of PBS, the concentration was adjusted to 1 μg / mL, and stored at 4 °C in the dark to obtain the CS-CeO2-Ki67 complex solution. The CS-CeO2-Ki67 complex solution was added to the slice and incubated for 10 min.

[0112] (4) Soak the sections in 1×PBS buffer to wash them.

[0113] (5) DAB color development: Dilute the DAB color development solution with pure water at a volume ratio of 1:20, add it to the slice, incubate for 1 min, and observe the color development under a microscope.

[0114] (6) Rinse the tissue with pure water.

[0115] (7) Counterstain with hematoxylin for 1 min, then rinse with pure water.

[0116] (8) Use anti-blue solution to perform anti-blueing until the tissue can be clearly seen to change from purple to blue with the naked eye.

[0117] (9) Tissue dehydration, clearing, and mounting: The sections were soaked in the following order: 50% ethanol for 10 seconds, 75% ethanol for 10 seconds, 85% ethanol for 10 seconds, 95% ethanol for 10 seconds, anhydrous ethanol for 10 seconds, a 1:1 mixture of xylene and anhydrous ethanol for 1 minute, and xylene for 1 minute. Then, neutral resin was added for mounting to achieve rapid detection of Ki67 protein in paraffin sections.

[0118] The total time for the above method is ≤60min.

[0119] Application Example 4 The difference from Application Example 3 is that the paraffin sections are sections of colorectal cancer and the corresponding adjacent tissue.

[0120] Application Example 5 The difference from Application Example 3 is that the paraffin sections are sections of lung cancer and the corresponding adjacent tissue.

[0121] Comparative Application Example 1 A method for intraoperative frozen section IHC detection includes the following steps: (1) Cut fresh lung cancer and the corresponding adjacent tissue into 1cm×1cm×0.5cm tissue blocks, place them in a mold, add 1mL of embedding agent OCT, and freeze rapidly in an ultra-low temperature freezer.

[0122] (2) Cut the tissue into 5 μm thick sections in a cryostat.

[0123] (3) Fix the tissue sections to be stained with 95% ethanol in a 4°C refrigerator for 2 min.

[0124] (4) Rinse the sections with 1×PBS for 20 seconds.

[0125] (5) Add Dako Ki67 primary antibody and incubate for 30 min.

[0126] (6) Rinse the sections with 1×PBS for 2 min, repeat three times.

[0127] (7) Add general-purpose poly-HRP secondary antibody and incubate for 20 min.

[0128] (8) Rinse the sections with 1×PBS for 2 min, repeat three times.

[0129] (9) DAB color development: Dilute the DAB color development solution with pure water at a volume ratio of 1:20, incubate for 1 min, and observe the color development under a microscope.

[0130] (10) Rinse the slices with pure water for 20 seconds.

[0131] (11) Counterstain with hematoxylin for 1 min, then rinse with pure water.

[0132] (12) Tissue dehydration, clearing, and mounting: The sections were soaked in the following order: 50% ethanol for 10 seconds, 75% ethanol for 10 seconds, 85% ethanol for 10 seconds, 95% ethanol for 10 seconds, anhydrous ethanol for 10 seconds, a 1:1 mixture of xylene and anhydrous ethanol for 10 seconds, and xylene for 1 minute. Then, neutral resin was added for mounting.

[0133] Comparative Application Example 2 The difference from Comparative Application Example 1 is that lung cancer and its corresponding adjacent tissue are replaced with thyroid cancer and its corresponding adjacent tissue.

[0134] Comparative Application Example 3 A traditional method for IHC detection in paraffin sections includes the following steps: (1) Gradient dewaxing: Thyroid cancer and its adjacent tissue with a thickness of 3 μm were soaked in the following sequence: xylene I for 1 min, xylene II for 1 min, a 1:1 mixture of xylene and anhydrous ethanol for 30 sec, anhydrous ethanol I for 30 sec, anhydrous ethanol II for 30 sec, 95% ethanol for 30 sec, 85% ethanol for 30 sec, 75% ethanol for 30 sec, 50% ethanol for 30 sec, pure water for 30 sec, and 1×PBS buffer for 1 min, repeated 3 times. Dewaxed sections were obtained.

[0135] (2) Antigen retrieval: First, preheat the 1×Tris-EDTA antigen retrieval solution in a microwave oven on medium heat for 3 minutes. Place the dewaxed sections into the preheated antigen retrieval solution and microwave on high for 3 minutes. Remove the antigen retrieval solution from the microwave oven and cool for 2 minutes, then microwave on low for 15 minutes. Next, once the temperature has dropped below 50°C, wash the sections with 1×PBS buffer for 1 minute, repeating 3 times.

[0136] (3) Add Dako Ki67 primary antibody and incubate for 30 min; soak and wash the slides three times with 1×PBS buffer for 2 min each time; add universal poly-HRP secondary antibody and incubate for 20 min.

[0137] (4) Soak the sections in 1×PBS buffer to wash them.

[0138] (5) DAB color development: Dilute the DAB color development solution with pure water at a volume ratio of 1:20, add it to the slice, incubate for 1 min, and observe the color development under a microscope.

[0139] (6) Rinse the tissue with pure water.

[0140] (7) Counterstain with hematoxylin for 1 min, then rinse with pure water.

[0141] (8) Use anti-blue solution to perform anti-blueing until the tissue can be clearly seen to change from purple to blue with the naked eye.

[0142] (9) Tissue dehydration, clearing, and fixation: The sections were soaked in the following order: 50% ethanol for 10 seconds, 75% ethanol for 10 seconds, 85% ethanol for 10 seconds, 95% ethanol for 10 seconds, anhydrous ethanol for 10 seconds, a 1:1 mixture of xylene and anhydrous ethanol for 10 seconds, and xylene for 1 minute. Then, neutral resin was added for mounting.

[0143] The total detection time for the method is 100 minutes.

[0144] Comparative Application Example 4 The difference from Comparative Application Example 3 is that the thyroid cancer and its corresponding adjacent tissue are replaced with intestinal cancer and its corresponding adjacent tissue.

[0145] Comparative Application Example 5 The difference from Comparative Application Example 3 is that the thyroid cancer and its corresponding adjacent tissue are replaced with lung cancer and its corresponding adjacent tissue.

[0146] Figure 5 This image shows a comparison of the color development effects of CS-CeO2-Ki67 complex detection and conventional IHC detection on frozen sections of lung cancer and corresponding adjacent tissues during surgery in accordance with the present invention. Figure 5 Figure a shows lung cancer tissue detected by traditional IHC, Figure b shows lung cancer adjacent tissue detected by traditional IHC, Figure c shows lung cancer tissue detected by CS-CeO2-Ki67 complex, and Figure d shows lung cancer adjacent tissue detected by CS-CeO2-Ki67 complex. Figure 6 This image shows a comparison of the color development effects of CS-CeO2-Ki67 complex detection and conventional IHC detection on frozen sections of thyroid cancer and corresponding adjacent tissues during surgery in accordance with the present invention. Figure 6 Image a shows thyroid cancer tissue detected by traditional IHC; image b shows adjacent tissue of thyroid cancer detected by traditional IHC; image c shows thyroid cancer tissue detected by the CS-CeO2-Ki67 complex; and image d shows adjacent tissue of thyroid cancer detected by the CS-CeO2-Ki67 complex. Figures 5-6 It can be seen that CS-CeO2-Ki67 antibody incubation for 10 minutes can achieve the staining effect of conventional IHC, and the entire staining process takes 20 minutes. The specific reactions produced on lung cancer and thyroid cancer tumor tissues are visually consistent and significantly higher than their expression levels in adjacent normal tissues.

[0147] Figure 7 This image shows a comparison of the color development effects of paraffin sections of thyroid cancer and corresponding adjacent tissue detected using the CS-CeO2-Ki67 complex and conventional IHC detection, which are based on the present invention. Figure 7 Figure a shows a thyroid cancer tissue image detected by traditional IHC, Figure b shows a thyroid cancer adjacent tissue image detected by traditional IHC, Figure c shows a thyroid cancer tissue image detected by the CS-CeO2-Ki67 complex, and Figure d shows a thyroid cancer adjacent tissue image detected by the CS-CeO2-Ki67 complex. Figure 8 This image shows a comparison of the color development effects of paraffin sections of colorectal cancer and corresponding adjacent tissues detected using the CS-CeO2-Ki67 complex versus conventional IHC detection. Figure 8Figure a shows a colorectal cancer tissue image detected by traditional IHC, Figure b shows a colorectal cancer adjacent tissue image detected by traditional IHC, Figure c shows a colorectal cancer tissue image detected by the CS-CeO2-Ki67 complex, and Figure d shows a colorectal cancer adjacent tissue image detected by the CS-CeO2-Ki67 complex. Figure 9 This image shows a comparison of the color development effects of CS-CeO2-Ki67 complex detection and conventional IHC detection on paraffin sections of lung cancer and corresponding adjacent non-cancerous tissues, as presented in this invention. Figure 9 Figure a shows lung cancer tissue detected by traditional IHC; figure b shows adjacent normal tissue of lung cancer detected by traditional IHC; figure c shows lung cancer tissue detected by the CS-CeO2-Ki67 complex; and figure d shows adjacent normal tissue of lung cancer detected by the CS-CeO2-Ki67 complex. Figures 7-9 It can be seen that the specific reactions and staining effects of the CS-CeO2-Ki67 antibody complex detection on various tumor tissues are basically consistent with those of the traditional IHC detection, and are significantly higher than its expression level in adjacent normal tissues.

[0148] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended scope of protection is intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of this invention.

[0149] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of protection of this invention and its equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing an antibody complex based on chitosan and cerium oxide modification, characterized in that, Includes the following steps: Using positively charged chitosan and negatively charged cerium oxide nanozymes as raw materials, a chitosan / cerium oxide nanozyme complex is formed through charge attraction. Using Ki67 monoclonal antibody and chitosan / cerium oxide nanozyme complex as raw materials, electrostatic self-assembly was carried out to form a ternary composite structure, resulting in an antibody complex based on chitosan and cerium oxide modification.

2. The method for preparing the antibody complex based on chitosan and cerium oxide modification according to claim 1, characterized in that, The mass ratio of cerium oxide nanozyme to chitosan is 1:1~8; the mass ratio of chitosan / cerium oxide nanozyme complex to Ki67 monoclonal antibody is 1:1~4.

3. The method for preparing the antibody complex based on chitosan and cerium oxide modification according to claim 1, characterized in that, The preparation method of chitosan / cerium oxide nanozyme complex includes the following steps: Chitosan was dissolved in a solvent and then protonated to obtain a chitosan solution. The cerium oxide nanozyme stock solution was mixed with water to obtain a cerium oxide nanozyme solution. Chitosan solution was added to phosphate buffer, and cerium oxide nanozyme solution was added at the same time. An electrostatic adsorption reaction occurred at room temperature to obtain chitosan / cerium oxide nanozyme complex.

4. The method for preparing the antibody complex based on chitosan and cerium oxide modification according to claim 3, characterized in that, The mass-to-volume ratio of chitosan to solvent is 1 mg~1.5 mg: 1 mL~1.5 mL, and the solvent is an acetic acid solution with a volume concentration of 1%~2%; the concentration of cerium oxide nanozyme stock solution is 1 mg / mL~1.5 mg / mL; the volume ratio of cerium oxide nanozyme stock solution to water is 1:6~9; and the concentration of cerium oxide nanozyme solution is 0.1 mg / mL~0.2 mg / mL.

5. The method for preparing the antibody complex based on chitosan and cerium oxide modification according to claim 3, characterized in that, The volume ratio of chitosan solution to phosphate buffer is 1:5~6; the electrostatic adsorption reaction time is 1h~1.5h.

6. The method for preparing the antibody complex based on chitosan and cerium oxide modification according to claim 1, characterized in that, The specific method for electrostatic self-assembly to form a ternary composite structure is as follows: The chitosan / cerium oxide nanozyme complex was resuspended in phosphate buffer to obtain a chitosan / cerium oxide nanozyme complex solution. Ki67 monoclonal antibody was added to a chitosan / cerium oxide nanozyme complex solution, and an electrostatic self-assembly reaction occurred at room temperature to form a ternary composite structure, resulting in an antibody complex based on chitosan and cerium oxide modification.

7. The method for preparing the antibody complex based on chitosan and cerium oxide modification according to claim 6, characterized in that, The electrostatic self-assembly reaction time was 1 h to 1.5 h, and the pH of the Ki67 monoclonal antibody was 7.4 to 7.

8.

8. An antibody complex based on chitosan and cerium oxide modification, characterized in that, It is prepared by the method described in any one of claims 1 to 7.

9. The application of the antibody complex based on chitosan and cerium oxide modified according to claim 8 in an immunohistochemical detection reagent.

10. The application of the antibody complex based on chitosan and cerium oxide modification according to claim 9 in immunohistochemical detection reagents, characterized in that, An antibody complex based on chitosan and cerium oxide was added to the pretreated sections, incubated, and then stained for color development; the sections were either frozen sections or paraffin sections.

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