HRP-PAMAM-secondary antibody complex as well as preparation method and application thereof

Abstract

The invention provides an HRP-PAMAM-secondary antibody complex as well as a preparation method and application thereof. The preparation method comprises the following steps: activating HRP; the activated HRP and PAMAM are coupled through a Schiff base reaction, and an HRP-PAMAM compound is formed; and dissolving a secondary antibody in a carbonate buffer solution, mixing with the HRP-PAMAM compound, carrying out oscillation reaction, coupling the HRP-PAMAM compound with the secondary antibody through a Schiff base reaction, adding a sodium borohydride solution, terminating the Schiff base reaction, and carrying out ultrafiltration treatment to obtain the HRP-PAMAM-secondary antibody compound. The invention provides a simple, convenient, efficient, stable and sensitive preparation method of an HRP-PAMAM-secondary antibody compound based on Schiff base reaction, the Schiff base reaction is adopted, no additional biological coupling reagent is needed, HRP is activated and then directly coupled with PAMAM, then the HRP-PAMAM compound is coupled with a secondary antibody, and the HRP-PAMAM-secondary antibody compound is prepared. The preparation of the HRP-PAMAM-secondary antibody compound can be completed only by three steps, a new solution is provided for the development of an immunohistochemical secondary antibody detection system, and the HRP-PAMAM-secondary antibody compound has important application value and market prospect.

Description

Technical Field

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

[0002] Immunohistochemistry (IHC) is a technique based on the principle of specific binding between antigens and antibodies. It utilizes labeled specific antibodies to perform in-situ qualitative, quantitative, and qualitative analysis of the distribution and content of certain chemical components in tissue sections. Immunohistochemistry is of great significance in medical research and clinical practice. It can assist doctors in identifying potential tumor markers, pathogens, or abnormal proteins in tissue samples, thereby enabling accurate and personalized diagnosis. It can also help identify new drug targets. Currently, in the domestic immunohistochemistry market of tertiary hospital pathology departments, three foreign companies—Ventana, Leica, and Dako—hold approximately 94% of the market share, essentially monopolizing the domestic immunohistochemistry market. The high price and cost of immunohistochemical secondary antibody reagents highlight the urgent need for high-quality, domestically produced novel immunohistochemical detection systems.

[0003] Currently, the mainstream immunohistochemical secondary antibody detection system on the market is the "enzyme-polymer-antibody" detection system, which achieves signal amplification by coupling multiple antibodies and enzymes onto a polymer. Because it is not affected by endogenous biotin in tissue specimens and has high accuracy, it is widely used in clinical diagnosis. However, most "enzyme-polymer-antibody" preparation methods require highly toxic coupling reagents such as dimethyl sulfoxide (DMSO) and SMCC, resulting in long production cycles, cumbersome operations, poor batch-to-batch production controllability, and a tendency to produce non-specific staining, hindering its further development. Therefore, developing novel "enzyme-polymer-antibody" detection systems using simplified synthesis methods is crucial. Summary of the Invention

[0004] To address the shortcomings of existing immunohistochemical secondary antibody detection systems, such as high production costs, complex processes, and cumbersome operations, this invention aims to provide an HRP-PAMAM-secondary antibody complex, its preparation method, and its applications. This preparation method eliminates the need for additional bioconjugation reagents. Based on the Schiff base reaction, activated HRP is directly conjugated to PAMAM, and then directly linked to the secondary antibody. The reaction can be completed in just three steps, greatly simplifying the production process, saving preparation time and costs. The resulting complex product has a stable molecular structure and higher specificity, enabling precise identification of target molecules and effectively reducing staining background signals. It can improve staining intensity and sensitivity while enhancing the batch-to-batch stability of immunohistochemical secondary antibodies.

[0005] The first aspect of this invention provides a method for preparing an HRP-PAMAM-secondary antibody complex, comprising the following steps: Activate HRP; The activated HRP and PAMAM are coupled via a Schiff base reaction to form an HRP-PAMAM complex. The secondary antibody was dissolved in carbonate buffer and mixed with the HRP-PAMAM complex. The mixture was shaken and reacted to couple the HRP-PAMAM complex with the secondary antibody via a Schiff base reaction. Then, sodium borohydride solution was added to terminate the Schiff base reaction, and ultrafiltration was performed to obtain the HRP-PAMAM-secondary antibody complex.

[0006] In one embodiment of the present invention, activating HRP includes the following steps: dissolving HRP in sodium bicarbonate buffer, adding sodium periodate, allowing the reaction to proceed under light-protected conditions for 1-3 hours to activate the HRP surface with aldehyde groups, then adding ethylene glycol to terminate the reaction, and performing ultrafiltration to obtain an activated HRP solution.

[0007] In one embodiment of the present invention, the ratio of the amount of HRP, sodium periodate and ethylene glycol added is (0.1-0.5 mg): (0.1-5 mg): (2-20) μL: 200 μL.

[0008] In one embodiment of the present invention, activated HRP and PAMAM are coupled via a Schiff base reaction, specifically including the following steps: PAMAM is dissolved in phosphate buffer, mixed with activated HRP solution, and the mixture is shaken to couple activated HRP and PAMAM via a Schiff base reaction. Then, sodium borohydride solution is added to terminate the Schiff base reaction, and ultrafiltration is performed to obtain the HRP-PAMAM complex.

[0009] In one embodiment of the present invention, the mass ratio of activated HRP to PAMAM is (0.1-0.5):(0.001-0.1).

[0010] In one embodiment of the present invention, the PAMAM is a polyamide-amine type dendritic polymer compound.

[0011] In one embodiment of the present invention, PAMAM is one of polyamide-amine dendritic polymer PAMAM 1.0 generation, polyamide-amine dendritic polymer PAMAM 2.0 generation, polyamide-amine dendritic polymer PAMAM 3.0 generation, polyamide-amine dendritic polymer PAMAM 4.0 generation, and polyamide-amine dendritic polymer PAMAM 5.0 generation.

[0012] Preferably, the PAMAM is a polyamide-amine dendritic polymer PAMAM 5.0.

[0013] A second aspect of the present invention provides an HRP-PAMAM-secondary antibody complex, which is prepared by the above-described preparation method.

[0014] A third aspect of the present invention provides an immunohistochemical detection system comprising the above-described HRP-PAMAM-secondary antibody complex.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a simple, efficient, stable, and sensitive method for preparing HRP-PAMAM-secondary antibody complexes based on the Schiff base reaction. The method requires only three steps, reducing the use of coupling agents, simplifying the production process, saving significant time, and enabling precise identification of target molecules. It improves staining intensity while enhancing batch-to-batch stability of immunohistochemical secondary antibodies, providing a new solution for the development of immunohistochemical secondary antibody detection systems. This method has significant application value and market potential. The HRP-PAMAM-secondary antibody complexes obtained using the method provided by this invention have a stable molecular structure and higher specificity, avoiding non-specific staining caused by the secondary antibody.

[0016] 2. The method for preparing the HRP-PAMAM-secondary antibody complex provided by this invention reduces production costs: Traditional methods for preparing immunohistochemical secondary antibody detection systems typically require highly toxic conjugating reagents such as dimethyl sulfoxide (DMSO) and SMCC, involving cumbersome reaction steps and long production cycles. This invention utilizes a Schiff base reaction, eliminating the need for additional biological conjugating reagents. HRP is activated and directly conjugated with PAMAM, and then the HRP-PAMAM complex is conjugated with the secondary antibody. The preparation of the HRP-PAMAM-secondary antibody complex can be completed in only three steps. This method greatly simplifies the production process, shortens the preparation time, and thus significantly reduces production costs, facilitating the large-scale production and application of the HRP-PAMAM-secondary antibody complex.

[0017] 3. The method for preparing the HRP-PAMAM-secondary antibody complex provided by this invention improves product stability and enhances batch-to-batch consistency: The HRP-PAMAM intermediate and HRP-PAMAM-secondary antibody complex prepared by the Schiff base reaction exhibit excellent chemical stability. Specifically, the imine bond formed by the Schiff base reaction can be converted into a stable amine bond through a subsequent reduction reaction, thereby improving product stability and extending shelf life. Furthermore, the method for preparing the HRP-PAMAM-secondary antibody complex provided by this invention is simple to operate and easy to control, thus improving the batch-to-batch consistency of the HRP-PAMAM-secondary antibody complex and ensuring the reliability and reproducibility of immunohistochemical detection results. Figures 16-21 As shown, six different batches of HRP-PAMAM-secondary antibody complexes were prepared using the preparation method provided by this invention, and CK sections of the same lung nodule tissue were simultaneously tested. The results showed that the staining intensity of the different batches of polymeric secondary antibodies was relatively small, proving that the preparation method of this invention has good batch-to-batch stability.

[0018] 4. The HRP-PAMAM-secondary antibody complex provided by this invention has a stable molecular structure and high specificity. PAMAM, as a dendritic polymer, can load multiple HRP molecules and secondary antibody molecules, thereby amplifying the signal and improving staining sensitivity. Simultaneously, the Schiff base reaction used in this invention has high reaction selectivity, effectively reducing non-specific binding and lowering the staining background signal, thus improving the accuracy of immunohistochemical detection results. The results of Application Examples 1, 2, and 3 (see...) Figures 4-15 Immunohistochemical detection using the HRP-PAMAM-secondary antibody complex prepared in this invention can obtain clear staining images with high positive signal intensity and low background signal. Compared with the commercially available DAKO ready-to-use secondary antibody detection system, it has a similar or even better staining effect.

[0019] 5. The HRP-PAMAM-secondary antibody complex provided by this invention has a stable molecular structure and stronger specificity, which can effectively reduce the staining background signal and improve the batch-to-batch stability of immunohistochemical secondary antibodies while improving staining sensitivity.

[0020] 6. The method for preparing HRP-PAMAM complex provided by the present invention produces HRP-PAMAM complex with excellent chemical stability, which improves the stability in use and storage compared with HRP-PAMAM complex prepared by traditional technology. Attached Figure Description

[0021] 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 novel polyantibody preparation method based on the Schiff base reaction; Figure 2 This is a schematic diagram illustrating the principle of enzyme-labeled polymeric antibody detection. Figure 3 Standard curves for the determination of protein content in HRP-PAMAM complex and HRP-PAMAM-IgG complex; Figure 4 The results of CD34 target detection in the skin tissue of the waist bulge are as follows: The polydioxan complex prepared in Example 1. Figure 5 The results of CD34 target detection in the skin tissue of the waist bulge are shown for the polyantibody complex prepared in Example 2. Figure 6 The results of CD34 target detection in the skin tissue of the waist bulge are shown for the polyantibody complex prepared in Example 3. Figure 7 Results of CD34 target detection in skin tissue at the waist bulge using DAKO ready-to-use secondary antibody; Figure 8 The results of CK target detection in lung nodule tissue are shown for the polyantibody complex prepared in Example 1. Figure 9 The results of CK target detection in lung nodule tissue are shown for the polyantibody complex prepared in Example 2. Figure 10 The results of CK target detection in lung nodule tissue are shown for the polyantibody complex prepared in Example 3. Figure 11 Results of CK target detection in lung nodule tissue using DAKO ready-to-use secondary antibody; Figure 12 The results of Ki67 target detection in lung nodule tissue are shown for the polyantibody complex prepared in Example 1. Figure 13 The results of Ki67 target detection in lung nodule tissue are shown for the polyantibody complex prepared in Example 2. Figure 14 The results of Ki67 target detection in lung nodule tissue are shown for the polyantibody complex prepared in Example 3. Figure 15 The results of Ki67 target detection in lung nodule tissue using DAKO ready-to-use secondary antibody; Figure 16 This is a diagram showing the CK section detection results of lung nodule tissue using batch-1 HRP-PAMAM-IgG secondary antibody in Example 4. Figure 17 This is a diagram showing the CK section detection results of lung nodule tissue using the batch-2 HRP-PAMAM-IgG secondary antibody in Example 4. Figure 18 This is a diagram showing the CK section detection results of lung nodule tissue using the third HRP-PAMAM-IgG secondary antibody in Example 4. Figure 19 This is a diagram showing the CK section detection results of lung nodule tissue using the batch of four HRP-PAMAM-IgG secondary antibody in Example 4. Figure 20 This is a diagram showing the CK section detection results of lung nodule tissue using batch 5 HRP-PAMAM-IgG secondary antibody in Example 4. Figure 21 This is a diagram showing the CK section detection results of lung nodule tissue using the batch 6 HRP-PAMAM-IgG secondary antibody in Example 4. Detailed Implementation

[0022] 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 and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0023] In the following embodiments, unless otherwise specified, the raw materials or processing techniques are conventional commercially available materials or conventional processing techniques in the art.

[0024] The Schiff base reaction is a chemical process in which amino and aldehyde groups crosslink to form dynamic covalent imine bonds, and it is a type of click chemistry reaction. Under physiological conditions, the Schiff base reaction can occur in situ between bioactive molecules, cells, and tissues to form hydrogel networks, achieving polymer loading. Biomaterials based on the Schiff base reaction possess advantages such as good conjugation, stable molecular structure, and simple synthesis, making them potential materials for developing novel "enzyme-polymer-antibody" immunohistochemical secondary antibody detection systems. Different types of Schiff base bonds provide biomaterials with tunable mechanical properties and chemical stability, enabling them to meet the needs of different tissues. Although Schiff base materials have been widely used in various biomedical fields such as drug delivery, wound healing, tissue regeneration, and tissue adhesion, research on using the Schiff base reaction to prepare novel immunohistochemical secondary antibody detection systems is currently very limited.

[0025] The method for preparing HRP-PAMAM-secondary antibody complex provided by this invention can be completed in just three steps, reducing the use of coupling agents, simplifying the production process, saving a lot of time, enabling precise identification of target molecules, improving staining intensity while enhancing batch-to-batch stability of immunohistochemical secondary antibodies, providing a new solution for the development of immunohistochemical secondary antibody detection systems, and having significant application value and market prospects.

[0026] Example 1 This embodiment provides an HRP-PAMAM-secondary antibody complex (HRP-PAMAM-IgG complex), and the preparation steps of the HRP-PAMAM-secondary antibody complex are as follows: (1) Activation of HRP: The hydroxyl groups on the surface of HRP were activated to aldehyde groups using NaIO4. 0.384 mg of HRP was weighed and dissolved in 200 μL of 0.1 M NaHCO3 buffer (pH 8.5), shaken to mix, and sonicated for 5 min. 0.384 mg of NaIO4 was weighed and dissolved in 100 μL of NaHCO3 solution, and sonicated to mix. The HRP solution and NaIO4 solution were thoroughly mixed and allowed to stand at room temperature in the dark for 1 h to activate the aldehyde groups on the HRP surface. 4 μL of ethylene glycol was added to the above solution, and the mixture was allowed to stand at room temperature for another 1 h to terminate the reaction. To remove impurities from the solution, the solution was ultrafiltered using a 30 KD ultrafiltration tube, and the product was redispersed in 200 μL of 0.1 M NaHCO3 solution and stored at 4℃ for later use.

[0027] (2) Preparation of the HRP-PAMAM complex: Activated HRP was coupled to PAMAM via a Schiff base reaction. 1 mg of PAMAM was added... 5.0 Dissolved in 1 mL PBS buffer (0.1 M, pH 7.4), and dispersed by sonication to obtain 1 mg / mL PAMAM. 5.0 Solution: Add 10 μL of the above solution to 10 mL of PBS to obtain PAMAM with a concentration of 0.001 mg / mL. 5.0 Solution. Take 200 μL of activated HRP solution and 160 μL of PAMAM. 5.0 The solution (0.001 mg / mL) was shaken to mix thoroughly and reacted at room temperature in the dark for 3 h to ensure complete coupling of PAMAM and HRP. 16 μL of 1 mg / mL NaBH4 solution was added to the above solution, and the reaction was continued for 1 h to terminate the Schiff base reaction. To remove impurities from the product, the solution was ultrafiltered using a 100 KD ultrafiltration tube, and the product was redispersed in 0.1 M NaHCO3 solution and stored at 4°C for later use, yielding the HRP-PAMAM complex.

[0028] (3) Preparation of HRP-PAMAM-IgG complex: The HRP-PAMAM complex was further coupled with the secondary antibody IgG via a Schiff base reaction. 20 µL of IgG carbonate buffer solution (25 mg / mL) was added to the above HRP-PAMAM complex solution, and the mixture was shaken at room temperature for 6 h. At this point, HRP-PAMAM and IgG were directly coupled via the Schiff base reaction. 16 μL of 1 mg / mL NaBH4 solution was added to the above solution, and the reaction was continued for 1 h to terminate the Schiff base reaction. To remove impurities from the product, 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-PAMAM-IgG complex.

[0029] Example 2 This embodiment provides an HRP-PAMAM-secondary antibody complex (HRP-PAMAM-IgG complex), and the preparation steps of the HRP-PAMAM-secondary antibody complex are as follows: (1) Activation of HRP: The hydroxyl groups on the surface of HRP were activated to aldehyde groups using NaIO4. 0.384 mg of HRP was weighed and dissolved in 200 μL of 0.1 M NaHCO3 buffer (pH 8.5), shaken to mix, and sonicated for 5 min. 0.768 mg of NaIO4 was weighed and dissolved in 100 μL of NaHCO3 solution, and sonicated to mix. The HRP solution and NaIO4 solution were thoroughly mixed and allowed to stand at room temperature in the dark for 1 h to activate the aldehyde groups on the HRP surface. 4 μL of ethylene glycol was added to the above solution, and the mixture was allowed to stand at room temperature for another 1 h to terminate the reaction. To remove impurities from the solution, the solution was ultrafiltered using a 30 KD ultrafiltration tube, and the product was redispersed in 200 μL of 0.1 M NaHCO3 solution and stored at 4℃ for later use.

[0030] (2) Preparation of the HRP-PAMAM complex: Activated HRP was coupled to PAMAM via a Schiff base reaction. 1 mg of PAMAM was added... 5.0 Dissolved in 1 mL PBS buffer (0.1 M, pH 7.4), and dispersed by sonication to obtain 1 mg / mL PAMAM. 5.0 Solution: Add 10 μL of the above solution to 10 mL of PBS to obtain PAMAM with a concentration of 0.001 mg / mL. 5.0 Solution. Take 200 μL of activated HRP solution and 160 μL of PAMAM. 5.0The solution (0.001 mg / mL) was shaken to mix thoroughly and reacted at room temperature in the dark for 3 h to ensure complete coupling of PAMAM and HRP. 16 μL of 1 mg / mL NaBH4 solution was added to the above solution, and the reaction was continued for 1 h to terminate the Schiff base reaction. To remove impurities from the product, the solution was ultrafiltered using a 100 KD ultrafiltration tube, and the product was redispersed in 0.1 M NaHCO3 solution and stored at 4°C for later use, yielding the HRP-PAMAM complex.

[0031] (3) Preparation of HRP-PAMAM-IgG complex: The HRP-PAMAM complex was further coupled with the secondary antibody IgG via a Schiff base reaction. 20 µL of IgG carbonate buffer solution (25 mg / mL) was added to the above HRP-PAMAM complex solution, and the mixture was shaken at room temperature for 6 h. At this point, HRP-PAMAM and IgG were directly coupled via the Schiff base reaction. 16 μL of 1 mg / mL NaBH4 solution was added to the above solution, and the reaction was continued for 1 h to terminate the Schiff base reaction. To remove impurities from the product, 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-PAMAM-IgG complex.

[0032] In this embodiment, the HRP-PAMAM-IgG complex prepared above is further used for immunohistochemical detection.

[0033] like Figure 3 As shown in Table 1, Figure 3 Standard curves for protein content determination of HRP-PAMAM and HRP-PAMAM-IgG complexes are shown in Table 1. Table 1 shows the protein content of HRP-PAMAM and HRP-PAMAM-IgG complexes calculated based on the protein content standard curves. The results in Table 1 indicate that the protein content of HRP-PAMAM significantly increased after conjugation with IgG (from 70.94 ug to 81.21 ug), indicating successful conjugation of HRP-PAMAM with IgG.

[0034] Table 1 Example 3 This embodiment provides an HRP-PAMAM-secondary antibody complex (HRP-PAMAM-IgG complex), and the preparation steps of the HRP-PAMAM-secondary antibody complex are as follows: (1) Activation of HRP: The hydroxyl groups on the surface of HRP were activated to aldehyde groups using NaIO4. 0.384 mg of HRP was weighed and dissolved in 200 μL of 0.1 M NaHCO3 buffer (pH 8.5), shaken to mix, and sonicated for 5 min. 1.152 mg of NaIO4 was weighed and dissolved in 100 μL of NaHCO3 solution, and sonicated to mix. The HRP solution and NaIO4 solution were thoroughly mixed and allowed to stand at room temperature in the dark for 1 h to activate the aldehyde groups on the HRP surface. 4 μL of ethylene glycol was added to the above solution, and the mixture was allowed to stand at room temperature for another 1 h to terminate the reaction. To remove impurities from the solution, the solution was ultrafiltered using a 30 KD ultrafiltration tube, and the product was redispersed in 200 μL of 0.1 M NaHCO3 solution and stored at 4℃ for later use.

[0035] (2) Preparation of the HRP-PAMAM complex: Activated HRP was coupled to PAMAM via a Schiff base reaction. 1 mg of PAMAM was added... 5.0 Dissolved in 1 mL PBS buffer (0.1 M, pH 7.4), and dispersed by sonication to obtain 1 mg / mL PAMAM. 5.0 Solution: Add 10 μL of the above solution to 10 mL of PBS to obtain PAMAM with a concentration of 0.001 mg / mL. 5.0 Solution. Take 200 μL of activated HRP solution and 160 μL of PAMAM. 5.0 The solution (0.001 mg / mL) was shaken to mix thoroughly and reacted at room temperature in the dark for 3 h to ensure complete coupling of PAMAM and HRP. 16 μL of 1 mg / mL NaBH4 solution was added to the above solution, and the reaction was continued for 1 h to terminate the Schiff base reaction. To remove impurities from the product, the solution was ultrafiltered using a 100 KD ultrafiltration tube, and the product was redispersed in 0.1 M NaHCO3 solution and stored at 4°C for later use, yielding the HRP-PAMAM complex.

[0036] (3) Preparation of HRP-PAMAM-IgG complex: The HRP-PAMAM complex was further coupled with the secondary antibody IgG via a Schiff base reaction. 20 µL of IgG carbonate buffer solution (25 mg / mL) was added to the above HRP-PAMAM complex solution, and the mixture was shaken at room temperature for 6 h. At this point, HRP-PAMAM and IgG were directly coupled via the Schiff base reaction. 16 μL of 1 mg / mL NaBH4 solution was added to the above solution, and the reaction was continued for 1 h to terminate the Schiff base reaction. To remove impurities from the product, 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-PAMAM-IgG complex.

[0037] Experimental Example The HRP-PAMAM-IgG produced by this invention has higher sensitivity and stability for immunohistochemical staining of pathological specimens than existing multimeric enzyme-labeled secondary antibodies on the market.

[0038] In the following comparative examples, except for the control secondary antibody which used a multimeric enzyme secondary antibody detection system purchased from Dako (catalog number K4001) in the United States, all other reagents were self-produced reagents, and all comparative examples used the same operating procedures. Detailed operating procedures for the examples are as follows: Dewaxing: Take the corresponding tissue sections, first soak them in xylene for 10 min, repeat twice, and then soak them in anhydrous ethanol, ethanol (95%), ethanol (75%) and distilled water for 10 min each.

[0039] Antigen retrieval: The above tissue sections were immersed in EDTA antigen retrieval solution and heated in a water bath. The temperature was maintained at 95 °C for 20 min, and then allowed to cool naturally.

[0040] Blocking: Remove the tissue sections, rinse with PBS buffer for 3 min, repeat 3 times, wipe the liquid around the tissue sections dry, draw circles around the tissue with an oil pen, add 100 µL of peroxidase blocking solution to the surface of the tissue sections to cover the tissue, incubate at room temperature for 20 min, rinse the sections with PBS buffer for 3 min each time, repeat 3 times.

[0041] Primary antibody incubation: After removing the PBS from the surface of the tissue sections, add 150 µL of the corresponding primary antibody to each section to cover the tissue. Incubate at 37 °C for 30 minutes, remove the primary antibody, immerse the sections in PBS buffer for 3 minutes each, and wash 3 times.

[0042] Secondary antibody incubation: After drying the PBS on the surface of the tissue sections, add 150 µL of the corresponding secondary antibody reagent to each section to cover the tissue. After reacting at 37 °C for 20 min, dry the secondary antibody reagent, and wash the sections in PBS for 3 min each, repeating 3 times.

[0043] Developing the color: Shake off the PBS from the surface of the tissue sections, add the developing agent to each section, and incubate at room temperature for 5 min. Then immerse the sections in PBS for 3 min each, repeating 3 times.

[0044] Counterstaining: After drying the PBS on the surface of the tissue sections, add hematoxylin (150 µL) to each section to cover the tissue sections. Incubate at room temperature for 1 min, then dry the hematoxylin and rinse with tap water for 3 min.

[0045] Dehydration: Remove the tissue sections and soak them in anhydrous ethanol and xylene for 3 minutes each, then air dry at room temperature.

[0046] Mounting: Remove the tissue sections, add about 0.02 mL of mounting medium to each section, cover with a coverslip and air dry.

[0047] Review the slides: Observe the staining effect under a microscope.

[0048] Application Example 1 This experiment used skin tissue from the lumbar region, with CD34 as the target. Following the same procedures, experiments were conducted using Example 1, Example 2, Example 3, and DAKO ready-to-use secondary antibody (Catalog No.: K4001). The results showed that Example 2 ( Figure 5 The staining intensity of the polymeric secondary antibody is comparable to that of DAKO ready-to-use secondary antibody. Figure 7 Other Examples 1 Figure 4 ) and Example 3 ( Figure 6 Slightly weaker than DAKO ready-to-use secondary antibody ( Figure 7 ).

[0049] Application Example 2 This experiment used lung nodule tissue, and the detection target was CK. Under the same operating procedures, experiments were conducted using Example 1, Example 2, Example 3, and DAKO ready-to-use secondary antibody (Catalog No.: K4001). The experimental results showed that Example 2 ( Figure 9 The staining intensity of the polymeric secondary antibody is comparable to that of DAKO ready-to-use secondary antibody. Figure 11 Other Examples 1 Figure 8 ) and Example 3 ( Figure 10 Slightly weaker than DAKO ready-to-use secondary antibody ( Figure 11 ).

[0050] Application Example 3 This experiment used lung nodule tissue, and the detection target was Ki67. Under the same operating procedures, experiments were conducted using Example 1, Example 2, Example 3, and DAKO ready-to-use secondary antibody (Catalog No.: K4001). The experimental results showed that Example 2 ( Figure 13 The staining intensity of the polymeric secondary antibody is comparable to that of DAKO ready-to-use secondary antibody. Figure 15 Other Examples 1 Figure 12 ) and Example 3 ( Figure 14 Slightly weaker than DAKO ready-to-use secondary antibody ( Figure 15 ).

[0051] Application Example 4 In this experiment, lung nodule tissues were continuously sliced ​​from the same paraffin block. The target of detection was CK. Under the same operating procedures, the method of Example 2 was used to prepare 6 batches of HRP-PAMAM-IgG secondary antibodies for the experiment (the six batches prepared were batch 1, batch 2, batch 3, batch 4, batch 5, and batch 6). Figures 16-21 The images show the CK section detection results of six different batches of polyantibody complexes simultaneously detecting the same lung nodule tissue. The experimental results show that the staining intensity of different batches of polyantibody is relatively small.

[0052] Results Analysis: The detection results of Application Examples 1, 2, 3, and 4 show that the polyantibody (HRP-PAMAM-IgG) prepared based on the Schiff base reaction yielded a large number of immunohistochemically positive cells with strong positive staining (brownish), comparable to DAKO ready-to-use secondary antibodies. This demonstrates that the accuracy, sensitivity, and repeatability of immunohistochemical detection using the polyantibody (HRP-PAMAM-IgG) prepared based on the Schiff base reaction are excellent.

[0053] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A method for preparing an HRP-PAMAM-secondary antibody complex, characterized in that, Includes the following steps: Activate HRP; The activated HRP and PAMAM are coupled via a Schiff base reaction to form an HRP-PAMAM complex. The secondary antibody was dissolved in carbonate buffer and mixed with the HRP-PAMAM complex. The mixture was shaken and reacted to couple the HRP-PAMAM complex with the secondary antibody via a Schiff base reaction. Then, sodium borohydride solution was added to terminate the Schiff base reaction, and ultrafiltration was performed to obtain the HRP-PAMAM-secondary antibody complex.

2. The method for preparing the HRP-PAMAM-secondary antibody complex according to claim 1, characterized in that, Activating HRP involves the following steps: dissolving HRP in sodium bicarbonate buffer, adding sodium periodate, and allowing the reaction to proceed under light-protected conditions for 1-3 hours to activate the HRP surface and imbue it with aldehyde groups. Then, ethylene glycol is added to terminate the reaction, and ultrafiltration is performed to obtain the activated HRP solution.

3. The method for preparing the HRP-PAMAM-secondary antibody complex according to claim 1, characterized in that, The ratio of HRP, sodium periodate and ethylene glycol added is (0.1-0.5 mg): (0.1-5 mg): (2-20) μL: 200 μL.

4. The method for preparing the HRP-PAMAM-secondary antibody complex according to claim 2, characterized in that, The activated HRP and PAMAM are coupled via a Schiff base reaction, specifically including the following steps: PAMAM is dissolved in phosphate buffer and mixed with the activated HRP solution, the mixture is shaken to react, and the activated HRP and PAMAM are coupled via a Schiff base reaction. Then, sodium borohydride solution is added to terminate the Schiff base reaction, and ultrafiltration is performed to obtain the HRP-PAMAM complex.

5. The method for preparing the HRP-PAMAM-secondary antibody complex according to claim 4, characterized in that, The mass ratio of activated HRP to PAMAM is (0.1-0.5):(0.001-0.1).

6. The method for preparing the HRP-PAMAM-secondary antibody complex according to claim 4, characterized in that, PAMAM is a polyamide-amine type dendritic polymer compound.

7. The method for preparing the HRP-PAMAM-secondary antibody complex according to claim 6, wherein the PAMAM is a polyamide-amine dendritic polymer PAMAM 5.

0.

8. An HRP-PAMAM-secondary antibody complex, characterized in that, The sample was prepared using the preparation method described in any one of claims 1-7.

9. An immunohistochemical detection system, characterized in that, Includes the HRP-PAMAM-secondary antibody complex as described in claim 8.

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