An HRP-colloidal gold-antibody complex, its preparation method and application

Abstract

This invention discloses an HRP-colloidal gold-antibody complex and its preparation method. The HRP-colloidal gold-antibody complex comprises colloidal gold nanoparticles, an antibody, and horseradish peroxidase. The antibody is linked with multiple thiol groups introduced through chemical modification. The colloidal gold nanoparticles and the antibody are connected through gold-sulfur bonds formed by some of the thiol groups on the antibody. The horseradish peroxidase is linked with a maleimide group introduced through chemical modification. The horseradish peroxidase and the antibody are connected through thioether bonds formed by the addition reaction of the maleimide group with the remaining thiol groups on the antibody that have not formed gold-sulfur bonds. The HRP-colloidal gold-antibody complex provided by this invention achieves a balance between speed and sensitivity in rapid intraoperative detection scenarios. The composite antibody structure is stable, non-specific adsorption is reduced, and the reaction can be completed in only one step, reducing the incubation time of the secondary antibody.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to an HRP-colloidal gold-antibody complex, its preparation method, and its application. Background Technology

[0002] Intraoperative rapid pathological diagnosis typically relies on frozen section technology to complete tissue sampling, slide preparation, and result interpretation within a short time. To further assist intraoperative surgical decision-making, immunohistochemistry (IHC) is often performed on frozen sections in clinical practice to determine tumor origin, assess surgical margins, or locate key molecular markers. However, intraoperative frozen immunohistochemistry has significant limitations: firstly, the intraoperative decision-making time window is extremely limited, requiring the immune reaction and staining process to be as short as possible; secondly, frozen sections have a relatively loose tissue structure and more significant endogenous background interference, making them prone to non-specific adsorption; thirdly, conventional testing procedures usually involve multiple antigen-antibody incubation and washing steps, making it difficult to balance probe binding stability and final signal interpretability within an extremely shortened process. Therefore, intraoperative frozen IHC requires both "extreme speed" and "high signal-to-noise ratio and high repeatability," both of which are often difficult to achieve simultaneously with current technologies.

[0003] Current frozen IHC primarily relies on conventional "primary antibody + secondary antibody" systems or "polymer enzyme-labeled macromolecule" systems (e.g., using dextran or dendritic polymers as a backbone to link large amounts of horseradish peroxidase (HRP)). The former requires multiple incubations and washes, which is time-consuming and cannot meet the requirements for rapid intraoperative diagnosis; the latter, although it can amplify the signal, has a loose organic polymer macromolecule backbone structure and significant steric hindrance, resulting in low penetration into cells or tissue sections. In addition, traditional polymer enzyme-labeled probes often use random coupling methods, and the coupling sites are uncontrollable. This not only easily leads to performance fluctuations due to batch-to-batch differences, but also masks the antigen-binding sites of antibodies or the catalytic active sites of enzymes, increasing background staining and causing difficulties in clinical interpretation or even the risk of false negatives / false positives.

[0004] To overcome the limitations of macromolecular polymers, metal nanomaterials (such as colloidal gold) have been introduced into the field of immunolabeling. Traditional colloidal gold labeling mainly relies on the electrostatic adsorption between the isoelectric point of proteins and gold particles. However, this electrostatic binding force is weak, and antibodies easily detach from the gold particle surface during the rigorous washing process of IHC, leading to severe signal attenuation. In recent years, thiolation has been introduced into this field as a protein modification method, improving the fixation strength of antibodies on the gold particle surface through the coordination bond (Au-S bond) formed between the thiol group (-SH) on the antibody and the gold atom on the metal surface. However, existing thiolated gold labeling complexes are usually only used as a "colloidal gold-antibody" binary structure. Due to the lack of subsequent enzymatic amplification effect, the sensitivity of their physical color development is far from meeting the requirements for detecting low-abundance antigens in frozen sections. If free HRP is directly and blindly mixed in the existing system for random physical coupling, it will repeat the problems of cross-linking aggregation, loss of activity, and increased nonspecific background.

[0005] In summary, how to overcome the bottleneck of random coupling in the preparation stage and construct a novel composite antibody probe that uses metal nanoparticles as a rigid carrier, integrates the highly specific recognition of antibodies and the efficient enzymatic amplification function of HRP, and has a defined and stable chemical covalent bond between each component, thereby achieving rapid and highly sensitive immunohistochemical detection of intraoperative frozen sections in a "one-step" method, is a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing immunohistochemical detection systems in frozen section pathological diagnosis, such as cumbersome and time-consuming detection steps, weak signals from low-abundance antigens, and the high randomness of traditional enzyme-linked immunosorbent assay (ELISA) conjugates affecting antigen binding activity, this invention provides an HRP-colloidal gold-antibody complex, its preparation method, and its application. This HRP-colloidal gold-antibody complex achieves a balance between speed and sensitivity in rapid intraoperative detection scenarios. The composite antibody structure is stable, non-specific adsorption is reduced, the reaction can be completed in a single step, reducing the incubation time for secondary antibodies, making it suitable for frozen section tissue environments, and improving batch consistency. It provides a new and efficient immunoassay tool for point-of-care diagnosis of frozen sections.

[0007] To achieve the above objectives, the first aspect of the present invention provides an HRP-colloidal gold-antibody complex, wherein the HRP-colloidal gold-antibody complex comprises colloidal gold nanoparticles, an antibody, and horseradish peroxidase; The antibody is attached with multiple thiol groups introduced through chemical modification; The colloidal gold nanoparticles are connected to the antibody through gold-sulfur bonds formed by some of the thiol groups on the antibody; The horseradish peroxidase is attached with a chemically modified maleimide group, and the horseradish peroxidase and the antibody are connected by a thioether bond formed by the addition reaction of the maleimide group with the remaining thiol group on the antibody that has not formed a gold-sulfur bond.

[0008] The N-hydroxysuccinimide ester terminus of the SATA molecule specifically binds to the free primary amino group on the antibody surface, thereby introducing a protective thiol group; SMCC introduces a maleimide group by binding to the primary amino group on the HRP surface. Both ultimately form an extremely stable thioether covalent bond through click chemistry.

[0009] The complex provided by this invention forms a ternary molecular micro-topological network with colloidal gold as a rigid carrier, antibodies as directional bridging centers, and multiple horseradish peroxidases covalently loaded outwards.

[0010] In one embodiment of the present invention, the multiple thiol groups on the antibody are introduced by modifying and deprotecting the antibody with N-succinimide-S-acetylthioacetate; The maleimide group on the horseradish peroxidase is introduced after activating the horseradish peroxidase with succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester.

[0011] In one embodiment of the present invention, the particle size of the colloidal gold nanoparticles is 10~50 nm.

[0012] In one embodiment of the present invention, the colloidal gold nanoparticles have a particle size of 20 nm.

[0013] A second aspect of the present invention provides a method for preparing the above-mentioned HRP-colloidal gold-antibody complex, comprising the following steps: S1. The antibody was modified by thiolation with N-succinimide-S-acetylthioacetate and then deprotected to expose free thiol groups to obtain thiolated antibody. S2. The thiolized antibody is mixed with the colloidal gold nanoparticle solution and reacted to allow the thiolized antibody to bind to the colloidal gold nanoparticles through gold-sulfur bonds, thereby obtaining a colloidal gold-antibody complex. S3. Horseradish peroxidase was activated by succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester to give it maleimide groups on its surface, thus obtaining HRP with maleimide groups. S4. The HRP activated in step S3 is mixed with the colloidal gold-antibody complex obtained in step S2 and coupled to form a thioether bond by reacting the maleimide group with the remaining free thiol group on the colloidal gold-antibody complex. After purification, the HRP-colloidal gold-antibody complex is obtained.

[0014] The preparation method provided by this invention adopts a sequential coupling strategy of first constructing a "gold-sulfur" rigid core and then grafting enzyme molecules through thiol-maleimide click chemistry. This sequence cleverly utilizes the steric hindrance distribution on the surface of antibody molecules, which greatly avoids antibody detachment caused by free HRP macromolecules directly competing for binding sites on the colloidal gold surface, thereby achieving extremely high probe stability and enzyme loading.

[0015] In one embodiment of the present invention, obtaining the thiolized antibody in step S1 specifically includes the following steps: S11. Prepare an antibody solution with a concentration of 0.5~10 mg / mL using phosphate buffer; S12. Dissolve N-succinimide-S-acetylthioacetate in dimethyl sulfoxide to prepare a SATA solution with a concentration of 10~100 mM. S13. Mix the antibody solution with a SATA solution whose volume is 1 / 100 to 1 / 10 of the antibody solution volume, and react in the dark at 4°C to 30°C for 30 minutes to 2 hours. S14. Add hydroxylamine buffer to the reaction system and react at room temperature or 4°C for 1-3 hours to carry out the deprotection reaction and specifically remove the acetyl protecting group. S15. After the reaction is complete, the reaction product is loaded onto a desalting column for desalting. Phosphate buffer is used as the mobile phase to collect the thiolized antibody and adjust the concentration to 1~50 mg / mL.

[0016] In one embodiment of the present invention, in steps S13 and S14, the ratio of the added mass of SATA, the added mass of antibody, and the added volume of hydroxylamine buffer is (0.1-0.5µg):(0.1-5mg):(2~20µL).

[0017] In one embodiment of the present invention, obtaining the colloidal gold-antibody complex in step S2 specifically includes the following steps: S21. The pH value of the colloidal gold nanoparticle solution was adjusted using a 0.1 mM potassium carbonate solution. S22. Mix the thiolized antibody obtained in step S1 with the colloidal gold nanoparticle solution after adjusting the pH value in step S21, and react at room temperature for 10-30 minutes. S23. After the reaction is complete, the reaction product is ultrafiltered using an ultrafiltration tube and dispersed in phosphate buffer to obtain the colloidal gold-antibody complex for later use.

[0018] In one embodiment of the present invention, the ratio of the volume of the colloidal gold nanoparticle solution to the mass of the added thiolized antibody is (1~50 µL): (0.1~5 mg).

[0019] In one embodiment of the present invention, the acquisition of HRP with maleimide groups in step S3 specifically includes the following steps: S31. Dissolve HRP in PBS buffer with a pH of 7.0-8.0 to prepare an HRP solution with a concentration of 1-20 mg / mL; S32. Add SMCC powder to the HRP solution and react at room temperature or 4°C for 1-3 hours. S33. After the reaction is complete, the reactants are loaded onto a desalting column for desalting. PBS buffer is used as the mobile phase, and the filtrate is collected to obtain the HRP with maleimide groups.

[0020] 10. The preparation method according to claim 4, characterized in that the coupling of the HRP-colloidal gold-antibody complex in step S4 specifically includes the following steps: S41. Mix the HRP with maleimide groups obtained in step S3 with the colloidal gold-antibody complex obtained in step S2, and react at 4°C in the dark for 16-24 hours. S42. Load the reaction product into a desalting column for preliminary filtration and desalting, using PBS buffer as the mobile phase, and collect the desalting product. S43. The desalted product collected in step S42 is concentrated by ultrafiltration using an ultrafiltration centrifuge tube to remove free unreacted HRP and impurities. The product is then resuspended in PBS buffer and the concentration is adjusted to 0.5~10 mg / mL to obtain the HRP-colloidal gold-antibody complex in probe form.

[0021] A third aspect of the present invention provides the application of the above-described HRP-colloidal gold-antibody complex or the complex prepared by the above method in immunoassay.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. In the HRP-colloidal gold-antibody complex provided by this invention, the antibody serves as the connecting hub, eliminating the need for traditional sodium periodate methods or random physical adsorption. One end of the complex forms a high-strength coordination bond (Au-S junction) with gold atoms via the thiol groups on the antibody; the other end utilizes unreacted thiol groups to undergo a highly efficient click chemical addition reaction with maleimide groups on activated HRP, forming a covalent thioether bond. This directional coupling completely solves the cross-linking and aggregation problem caused by random sites, preserving antigen-binding activity and enzyme catalytic activity while ensuring the complex is not only resistant to rigorous immunoassays but also exhibits excellent batch-to-batch uniformity.

[0023] 2. In the HRP-colloidal gold-antibody complex provided by this invention, traditional polymerase macromolecular probes often suffer from poor penetration (large steric hindrance) in frozen tissue sections due to their loose backbone. This invention uses rigid inorganic colloidal gold of approximately 20 nm as the core, avoiding macromolecular chain entanglement. The large specific surface area effect of colloidal gold enables high-density HRP loading, and combined with HRP's own highly efficient enzymatic catalytic ability, it ensures excellent tissue penetration and extremely high signal abundance when targeting weak or low-abundance antigens in frozen tissues.

[0024] 3. The HRP-colloidal gold-antibody composite provided by this invention integrates the dual functions of "target recognition" and "catalytic color development." Under extremely tight intraoperative time windows, target staining can be completed in a single, simple incubation step. This completely eliminates the cumbersome secondary antibody incubation and multiple washing cycles of the conventional "primary antibody + secondary antibody" process, reducing the core antibody reaction time from over 20 minutes to approximately 10 minutes. Simultaneously, it eliminates non-specific background staining caused by secondary antibody cross-adsorption at the source, providing a cleaner, higher signal-to-noise ratio pathological view for margin determination and tumor origin assessment. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of a method for preparing HRP-colloidal gold-antibody complexes based on thiolation reaction; Figure 2 This is a schematic diagram illustrating the detection principle of HRP-colloidal gold-antibody complex. Figure 3 Conceptual diagram for the preparation of HRP-colloidal gold-antibody complex; Figure 4 The results of CD34 target detection in intraoperative frozen hemangioma tissue of the HRP-colloidal gold-antibody complex prepared in Example 1 are shown. Figure 5The results of CD34 target detection in intraoperative frozen hemangioma tissue of the HRP-colloidal gold-antibody complex prepared in Example 2 are shown. Figure 6 For comparison, the CD34 target detection results of DAKO ready-to-use secondary antibody in intraoperative frozen hemangioma tissue; Figure 7 The results of CK-PAN target detection are shown in the intraoperative frozen skin tissue of the HRP-colloidal gold-antibody complex prepared in Example 1. Figure 8 The results of CK-PAN target detection are shown in the intraoperative frozen skin tissue of the HRP-colloidal gold-antibody complex prepared in Example 2. Figure 9 The results of CK-PAN target detection were obtained for the comparative DAKO ready-to-use secondary antibody in intraoperative frozen skin tissue. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes without departing from the concept of the present invention.

[0027] To make the technical solution of this invention clearer and more understandable, avoid redundancy, and ensure consistency in terminology, the full Chinese definitions of the English abbreviations of certain compounds, reagents, and technical terms used in this application specification and claims are as follows: HRP: Horseradish peroxidase. SATA: N-succinimidyl-S-acetylthioacetate. SMCC: Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. DMSO: Dimethyl sulfoxide; PBS: Phosphate-Buffered Saline; DAB: 3,3'-Diaminobenzidine; OCT: Optimal Cutting Temperature compound, a special embedding gel for frozen sections; K2CO3: Potassium carbonate; CD34: Leukocyte differentiation antigen 34 (a target of vascular endothelial markers). CK-PAN: Broad-spectrum cytokeratin (epithelial cell marker target).

[0028] In the following embodiments, unless otherwise specified, the reagents, raw materials, or processing techniques used are all commercially available materials or conventional processing techniques in the art. Those skilled in the art should understand that these embodiments are only used to illustrate the applicability and efficiency of the present invention and are not intended to limit the scope of protection of the claims of the present invention.

[0029] The present invention will be further described in detail below with reference to specific embodiments. Example 1

[0030] This embodiment provides an HRP-colloidal gold-antibody complex, the preparation method of which includes the following steps: S1. Thiolization modification of the antibody: Dissolve 2 mg SATA in 150 μL of dimethyl sulfoxide (DMSO) and vortex to mix. Mix the SATA solution (0.6 μL) with the antibody solution (200 μL) thoroughly and incubate at 4°C for 2 h to allow the antibody to carry thiol groups protected by acetyl groups. Add 20 μL of hydroxylamine buffer to the above solution and continue to incubate at 4°C for 2 h to specifically remove the acetyl protecting group and expose the active free thiol group (-SH). To purify the product, desalt the above solution using a desalting column, and store the resulting flow-through at 4°C for later use.

[0031] S2. Preparation of colloidal gold-antibody complex: The thiol-modified antibody was coupled to colloidal gold via a gold-thiol reaction. 400 μL of 20 nm colloidal gold solution was mixed with the thiolized antibody solution prepared in step 1 and incubated at room temperature for 30 minutes to form the colloidal gold-antibody complex. To remove impurities from the product, the solution was ultrafiltered using a 100 KD ultrafiltration tube, and the product was redispersed in PBS buffer solution and stored at 4°C for later use, yielding the colloidal gold-antibody complex.

[0032] S3. Maleimide Activation of HRP: 2.332 mg of HRP was dissolved in 291 μL of PBS buffer to obtain an HRP solution. Separately, 2.562 mg of heterobifunctional crosslinking agent SMCC powder was dissolved in the above HRP solution. The reaction was carried out at 4°C in the dark for 2 hours, allowing the N-hydroxysuccinimide ester end of the SMCC molecule to react with the primary amino group on the HRP surface, thereby introducing maleimide groups onto the HRP, resulting in an HRP-maleimide solution. After the reaction was complete, unreacted SMCC was rapidly removed using a desalting column to obtain pure activated HRP.

[0033] S4. Preparation of HRP-colloidal gold-antibody complex: HRP and the colloidal gold-antibody complex were covalently linked using thioether bonds. The colloidal gold-antibody complex obtained in step 2 was mixed with the HRP-maleimide solution obtained in step 3, and reacted at 4°C in the dark for 16-24 hours. To remove impurities from the product, it was filtered through a desalting column to rapidly remove small molecule impurities from the reaction system. The above solution was ultrafiltered using a 100 KD ultrafiltration tube, and the product was redispersed in 0.1 M PBS solution and stored at 4°C for later use, yielding the HRP-colloidal gold-IgG complex. Example 2

[0034] This embodiment provides an HRP-colloidal gold-antibody complex, the preparation method of which includes the following steps: S1. Thiolization modification of the antibody: Dissolve 2 mg SATA in 150 μL of dimethyl sulfoxide (DMSO) and vortex to mix. Mix the SATA solution (1 μL) with the antibody solution (200 μL) thoroughly and incubate at 4°C for 2 h to allow the antibody to carry thiol groups protected by acetyl groups. Add 20 μL of hydroxylamine buffer to the above solution and continue to incubate at 4°C for 2 h to specifically remove the acetyl protecting group and expose the active free thiol group (-SH). To purify the product, desalt the above solution using a desalting column, and store the resulting flow-through at 4°C for later use.

[0035] S2. Preparation of colloidal gold-antibody complex: The thiol-modified antibody was coupled to colloidal gold via a gold-thiol reaction. 600 μL of 20 nm colloidal gold solution was mixed with the thiolized antibody solution prepared in step 1 and incubated at room temperature for 30 minutes to form a colloidal gold-antibody complex. To remove impurities from the product, the solution was ultrafiltered using a 100 KD ultrafiltration tube, and the product was redispersed in PBS buffer solution and stored at 4°C for later use, yielding the colloidal gold-antibody complex.

[0036] S3. Maleimide Activation of HRP: 2.469 mg of HRP was dissolved in 291 μL of PBS buffer to obtain an HRP solution. Separately, 2.715 mg of heterobifunctional crosslinking agent SMCC powder was dissolved in the above HRP solution. The reaction was carried out at 4°C in the dark for 2 hours, allowing the N-hydroxysuccinimide ester end of the SMCC molecule to react with the primary amino group on the HRP surface, thereby introducing maleimide groups onto the HRP, resulting in an HRP-maleimide solution. After the reaction was complete, unreacted SMCC was rapidly removed using a desalting column to obtain pure activated HRP.

[0037] S4. Preparation of HRP-colloidal gold-antibody complex: HRP and the colloidal gold-antibody complex were covalently linked using thioether bonds. The colloidal gold-antibody complex obtained in step 2 was mixed with the HRP-maleimide solution obtained in step 3, and reacted at 4°C in the dark for 16-24 hours. To remove impurities from the product, it was filtered through a desalting column to rapidly remove small molecule impurities from the reaction system. The above solution was ultrafiltered using a 100 KD ultrafiltration tube, and the product was redispersed in 0.1 M PBS solution and stored at 4°C for later use, yielding the HRP-colloidal gold-IgG complex.

[0038] Comparative Example Polymer enzyme-linked immunosorbent assay (ELISA) system (catalog number K4001) manufactured by DAKO Corporation, USA.

[0039] Test case The complexes prepared in Examples 1 and 2 of this invention were subjected to rigorous comparison testing with the comparative example (DAKO system) under the same clinical intraoperative frozen immunohistochemical setting. The specific procedures are as follows: (1) General frozen tissue sectioning and pretreatment: Standard intraoperative frozen specimens (hemangioma tissue, skin tissue) were obtained, embedded in OCT glue, and placed in a -20℃ cryostat to cut thin film sections with a thickness of 5-7 μm. After being attached to a glass slide, the sections were immediately fixed in formalin for 1-2 minutes. The sections were then washed 3 times with PBS buffer. 100 µL of peroxidase blocking solution was added and the sections were incubated at room temperature for 1 min to block endogenous enzyme interference. The sections were then washed 3 times with PBS.

[0040] (2) Colorimetric Group 1: The "One-Step" Detection Process of the Invention (Experimental Groups 1 and 2 of Examples) After drying the surface buffer of the slide, add approximately 100 µL of reagent containing the probe solution from Example 1 or Example 2 (for easy complex formation) to cover the tissue. Incubate at 37°C for approximately 10 minutes (during this stage, due to the probe's structural characteristics, antigen-antibody recognition and specific binding, as well as HRP loading, are completed instantaneously and simultaneously). After washing with PBS for 1 min, add DAB chromogenic solution and incubate at room temperature for 2 min. Then perform routine washing, hematoxylin counterstaining for 1 min, washing with water, dehydration with ethanol and xylene, and mounting. The chromogenic process and slide preparation are extremely quick.

[0041] (3) Colorimetric Group 2: Comparative "Two-Step" Detection Procedure (DAKO Control Group) Since this is a control system, it requires two standard incubation steps. After the above pretreatment, add the standard primary antibody and incubate for 7 minutes, then wash for 1 minute; subsequently, add the secondary antibody system from the DAKO kit and incubate for 15 minutes; after three washes, proceed to the DAB staining and counterstaining steps as before. This process alone, including the core antigen antibody incubation and intermediate washing steps, takes more than 25 minutes.

[0042] Test results: Application Example 1 (Target CD34, hemangioma tissue sample): Figure 4 The probe colorimetric image prepared in Example 1 is shown below. Figure 5 This is the probe colorimetric image from Example 2. Figure 6 The image shows the chromogenic pattern of the DAKO kit. Comparison revealed that, due to the elimination of secondary antibody incubation (one-step method), the signal amplification source provided by the probe in this embodiment of the invention through precise chemical covalent coupling is extremely stable. Vascular endothelium in the tissue showed significant positive staining (dark brown). Among them, Example 2, with optimized components (… Figure 5 The color richness and edge sharpness perfectly match the extremely time-consuming DAKO two-step system. Figure 6 Example 1 is sufficient to meet the requirements of clinical image reading and diagnosis, and no non-specific background staining caused by cross-contamination of secondary antibodies was found in the entire image.

[0043] Application Example 2 (Target CK-PAN, skin tissue sample): Figure 7 (Example 1) Figure 8 (Example 2) and Figure 9 (DAKO comparison) demonstrates validation of a broader antigen expression system. Targeting keratinocytes in basal cells and squamous epithelium, Example 2 ( Figure 8 The positive signal presented is a deep brown color, with precise positioning and no diffusion. Its performance level has fully reached the gold standard product level that requires high investment and time. Figure 9 ).

[0044] In summary, the HRP-colloidal gold-antibody complex structure provided by this invention enables clear, highly sensitive, and clean colorimetric results to be obtained in a single step by relying on the dual redirection anchoring of gold atoms-thiol groups and thiol-maleimide, eliminating the need for traditional secondary antibody amplification procedures. This significantly reduces the diagnostic time of frozen sections (reducing molecular binding time by more than half) and has revolutionary application value for intraoperative pathological diagnosis.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An HRP-colloidal gold-antibody complex, characterized in that, The HRP-colloidal gold-antibody complex comprises colloidal gold nanoparticles, an antibody, and horseradish peroxidase; The antibody is attached with multiple thiol groups introduced through chemical modification; The colloidal gold nanoparticles are connected to the antibody through gold-sulfur bonds formed by some of the thiol groups on the antibody; The horseradish peroxidase is attached with a chemically modified maleimide group, and the horseradish peroxidase and the antibody are connected by a thioether bond formed by the addition reaction of the maleimide group with the remaining thiol group on the antibody that has not formed a gold-sulfur bond.

2. The HRP-colloidal gold-antibody complex according to claim 1, characterized in that, The multiple thiol groups on the antibody were introduced by modifying and deprotecting the antibody with N-succinimide-S-acetylthioacetate; The maleimide group on the horseradish peroxidase is introduced after activating the horseradish peroxidase with succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester.

3. The HRP-colloidal gold-antibody complex according to claim 1 or 2, characterized in that, The colloidal gold nanoparticles have a particle size of 10~50 nm.

4. The HRP-colloidal gold-antibody complex according to claim 3, characterized in that, The colloidal gold nanoparticles have a particle size of 20 nm.

5. A method for preparing the HRP-colloidal gold-antibody complex as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. The antibody was modified by thiolation with N-succinimide-S-acetylthioacetate and then deprotected to expose free thiol groups to obtain thiolated antibody. S2. The thiolized antibody is mixed with the colloidal gold nanoparticle solution and reacted to allow the thiolized antibody to bind to the colloidal gold nanoparticles through gold-sulfur bonds, thereby obtaining a colloidal gold-antibody complex. S3. Horseradish peroxidase was activated by succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester to give it maleimide groups on its surface, thus obtaining HRP with maleimide groups. S4. The HRP activated in step S3 is mixed with the colloidal gold-antibody complex obtained in step S2 and coupled to form a thioether bond by reacting the maleimide group with the remaining free thiol group on the colloidal gold-antibody complex. After purification, the HRP-colloidal gold-antibody complex is obtained.

6. The preparation method according to claim 5, characterized in that, The acquisition of the thiolized antibody in step S1 specifically includes the following steps: S11. Prepare an antibody solution with a concentration of 0.5~10 mg / mL using phosphate buffer; S12. Dissolve N-succinimide-S-acetylthioacetate in dimethyl sulfoxide to prepare a SATA solution with a concentration of 10~100 mM. S13. Mix the antibody solution with a SATA solution whose volume is 1 / 100 to 1 / 10 of the antibody solution volume, and react in the dark at 4°C to 30°C for 30 minutes to 2 hours. S14. Add hydroxylamine buffer to the reaction system and react at room temperature or 4°C for 1-3 hours to carry out the deprotection reaction and specifically remove the acetyl protecting group. S15. After the reaction is complete, the reaction product is loaded onto a desalting column for desalting. Phosphate buffer is used as the mobile phase to collect the thiolized antibody and adjust the concentration to 1~50 mg / mL.

7. The preparation method according to claim 6, characterized in that, In steps S13 and S14, the ratio of the added mass of SATA, the added mass of antibody, and the added volume of hydroxylamine buffer is (0.1-0.5 µg): (0.1-5 mg): (2-20 µL).

8. The preparation method according to claim 5, characterized in that, The process of obtaining the colloidal gold-antibody complex in step S2 specifically includes the following steps: S21. The pH value of the colloidal gold nanoparticle solution was adjusted using a 0.1 mM potassium carbonate solution. S22. Mix the thiolized antibody obtained in step S1 with the colloidal gold nanoparticle solution after adjusting the pH value in step S21, and react at room temperature for 10-30 minutes. S23. After the reaction is complete, the reaction product is ultrafiltered using an ultrafiltration tube and dispersed in phosphate buffer to obtain the colloidal gold-antibody complex for later use.

9. The preparation method according to claim 8, characterized in that, The ratio of the volume of the colloidal gold nanoparticle solution to the mass of the added thiolized antibody is (1~50 µL): (0.1~5 mg).

10. The preparation method according to claim 5, characterized in that, The acquisition of HRP with maleimide groups in step S3 specifically includes the following steps: S31. Dissolve HRP in PBS buffer with a pH of 7.0-8.0 to prepare an HRP solution with a concentration of 1-20 mg / mL; S32. Add SMCC powder to the HRP solution and react at room temperature or 4°C for 1-3 hours; S33. After the reaction is complete, the reactants are loaded onto a desalting column for desalting. PBS buffer is used as the mobile phase, and the filtrate is collected to obtain the HRP with maleimide groups.

11. The preparation method according to claim 5, characterized in that, The conjugation of the HRP-colloidal gold-antibody complex in step S4 specifically includes the following steps: S41. Mix the HRP with maleimide groups obtained in step S3 with the colloidal gold-antibody complex obtained in step S2, and react at 4°C in the dark for 16-24 hours. S42. Load the reaction product into a desalting column for preliminary filtration and desalting, using PBS buffer as the mobile phase, and collect the desalting product. S43. The desalted product collected in step S42 is concentrated by ultrafiltration using an ultrafiltration centrifuge tube to remove free unreacted HRP and impurities. The product is then resuspended in PBS buffer and the concentration is adjusted to 0.5~10 mg / mL to obtain the HRP-colloidal gold-antibody complex in probe form.

12. The application of an HRP-colloidal gold-antibody complex as described in any one of claims 1-5, or a complex prepared by the method described in any one of claims 5-11, in immunoassay.

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