High-sensitivity polymer-labeled secondary antibody and preparation method thereof

Abstract

The invention belongs to the technical field of medical examination, and particularly relates to preparation and application of a polymerase-labeled secondary antibody. The preparation method comprises the following steps: activating enzyme by divinyl sulfone (DVS), polymerizing with hyperbranched aminated polyethylene glycol to obtain polymer enzyme, activating the polymer enzyme again, and incubating with a second antibody to obtain a polymer-labeled second antibody. According to the method, the main defects of the secondary antibody in the current market are overcome, the enzyme label density on the antibody is improved, and the number of enzyme molecules labeled on the secondary antibody in unit volume is greatly increased; the volume of the polymer enzyme is reduced; the cell penetrability of the enzyme-labeled secondary antibody is improved, and the sensitivity and the stability are higher.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and particularly relates to a high-sensitivity polymerase-labeled secondary antibody and a preparation method thereof. Background Art

[0002] Secondary antibodies are antibodies that can bind to antibodies, that is, antibodies of antibodies. Their main function is to detect the presence of antibodies and amplify the signal of primary antibodies. It binds to antigen-specific primary antibodies and can detect highly specific amino acid sequences in target proteins. The specificity of secondary antibodies to designated regions or regions of primary antibodies allows multiple secondary antibodies to bind to a single primary antibody, amplifying the signal and increasing the sensitivity of detection. At present, secondary antibodies have been widely used in experiments such as immunoprecipitation (IP), Western Blot (WB), immunohistochemistry (IHC), immunofluorescence (IF) or flow cytometry (FCM), enzyme-linked immunosorbentassay (ELISA), etc., which shows the importance of secondary antibodies in immunological experiments.

[0003] At present, the immunoassay detection systems based on "enzyme-labeled secondary antibody complex" on the market mainly include "antibody-enzyme direct labeling method", "biotin-avidin amplification method" and "chain polymer-enzyme-antibody polymer", specifically: Antibody-enzyme direct labeling method (eg Figure 1 The principle of this method is to directly label the marker enzyme on the secondary antibody through an organic small molecule coupling reagent. [1] Since the labeling enzyme is usually large in size, and the surface area and number of functional groups of the antibody are limited, it is easy to cause the problem of low antibody sensitivity.

[0004] Using biotin-avidin amplification (eg Figure 2 The principle of this method is to use biotin labeling on the secondary antibody, and then link the labeling enzyme with the biotin labeling [2] Although this method can improve the detection sensitivity, since biotin is commonly found in biological tissues, false positives are very likely to occur, which greatly interferes with the experimental results.

[0005] Chain polymer-enzyme-antibody polymer (such as Figure 3 ): This method uses hydrophilic chain polymers (such as dextran [3] , poly-lysine peptide [4]The enzyme is fixed on the skeleton (such as DNA) and then connected to the secondary antibody molecule. This can reduce the effect of the labeled enzyme on the antibody specificity and affinity and improve the detection sensitivity. However, due to the loose chain structure and large size, it is not sensitive enough to detect antigens in the cell nucleus.

[0006] Therefore, it is very important to find an enzyme-labeled secondary antibody with good stability, small size and high sensitivity. Summary of the invention

[0007] 1. Problems to be solved Based on the above reasons, the object of the present invention is to provide a polymerase secondary antibody ( Figure 4 ), which is characterized by overcoming the defects of chain polymer enzymes, reducing the volume of polymer enzymes, and using polyethylene glycol to increase the density of the enzyme label on the antibody, greatly increasing the number of enzyme molecules per unit volume that are linked to the secondary antibody.

[0008] in: Hyperbranched polymers are different from linear polymers in that they have highly branched, three-dimensional ellipsoidal structures and rich surface functional groups. Figure 5 ) contains a multifunctional polyethylene glycol scaffold with a high degree of amino [5] Polyethylene glycol (PEG) is a widely used ether polymer compound with good water solubility, cell permeability and biocompatibility. It can be dissolved in tissue fluid in the body and can be quickly excreted by the body without causing toxic side effects.

[0009] The preparation method of the polymerase secondary antibody is easy to operate and has good controllability.

[0010] This polymerase secondary antibody has good sensitivity.

[0011] The polymerase secondary antibody has good thermal stability.

[0012] Preferably, the polymerase secondary antibody is used in mouse immunohistochemistry.

[0013] Preferably, polymerase secondary antibodies are used in tumor immunohistochemistry.

[0014] (II) Technical solution of the present invention The preparation principle of a polymerase secondary antibody is as follows: a DVS-activated enzyme is polymerized with a hyperbranched amino-polyethylene glycol multi-branched organic molecule to obtain a polymer enzyme, the polymer enzyme is further activated with DVS, and then mixed and incubated with an antibody to obtain a polymer enzyme secondary antibody.

[0015] The method for labeling a polymerase-labeled secondary antibody: 1) Dissolve the enzyme in sodium phosphate buffer at room temperature, add DVS while stirring, and then stir gently at 30°C (room temperature) in the dark for 25-45 minutes; 2) The above reaction solution was purified by Sephadex G-25 column to remove the remaining DVS, and the mobile phase was sodium phosphate buffer (pH 8.0). The brown component, i.e. the activated enzyme, was collected; 3) Concentrating the activated enzyme by centrifugation; 4) Add the hyperbranched amino polyethylene glycol solution to the concentrated activated enzyme while stirring, and the ratio of the number of activated enzyme to the hyperbranched amino polyethylene glycol molecules is 25-30:1. The solution is gently stirred at 30° C. for 15-20 hours; 5) The reaction solution in step 4) was purified by a Sephadex G-25 gel column (Shanghai Xinhu Experimental Equipment Co., Ltd.) and the solution was converted into sodium phosphate buffer (pH 8.0). The first brown component collected was polymer peroxidase; 6) Concentrate the polymer enzyme produced in step 5), add DVS to the solution, the ratio of DVS to enzyme molecules is 15-20:1, and gently stir at 30°C for 3 h; 7) Use Sephadex G-25 separation column to remove the remaining DVS, the mobile phase is PBS buffer (pH 7.1), and collect the brown component, which is the activated polymer enzyme; 8) concentrating the activated polymer enzyme produced in step 7); 9) Add Fab' dropwise to the activated polymer enzyme produced in step 8) at room temperature and continue stirring for 1 h, then incubate at 4°C in the dark for 20-30 hours; 10) Add stabilizer and bactericide to the solution in 9), wherein proclin-300:bovine serum albumin = 15-30:1; 11) The generated polymerase-labeled secondary antibody can be used in immunohistochemistry experiments.

[0016] The enzymes mentioned above include: horseradish peroxidase and / or alkaline phosphatase and / or urease.

[0017] The antibodies mentioned above include: mouse anti-sheep immunoglobulin, rabbit anti-sheep immunoglobulin, goat anti-mouse immunoglobulin, goat anti-rabbit immunoglobulin, horse anti-sheep immunoglobulin, horse anti-mouse immunoglobulin, horse anti-rabbit immunoglobulin, mouse anti-human immunoglobulin, rabbit anti-human immunoglobulin, goat anti-human immunoglobulin, horse anti-human immunoglobulin, etc., as well as active fragments of the above antibodies, such as Fab or (Fab')2 fragments, etc.

[0018] (III) The present invention has beneficial technical effects 1) The present invention uses multi-branched organic small molecules as carriers to tightly connect multiple enzymes to the multi-branched carrier molecules, thereby increasing the compactness between enzyme molecules, making the labeled enzyme more stable and occupying less space; Fab or (Fab')2 fragments with smaller molecular weights are used to connect with the polymer enzyme. While reducing the molecular weight of the overall conjugate, the number and density of the labeled enzymes on the secondary antibody are increased, thereby improving the detection sensitivity.

[0019] 2) The polymer backbone of hyperbranched polyethylene glycol has excellent hydrophilicity and better tissue penetration ability, and the sensitivity of detecting antigens present in the cell nucleus is significantly improved.

[0020] 3) In the following examples, it was found that the secondary antibody could still achieve the same effect as or even better than the commercially available secondary antibody after the dilution factor was increased to 1:500. The present invention greatly reduces the use cost of the secondary antibody. It can be seen that the secondary antibody prepared by the present invention is more conducive to clinical pathological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the synthesis of secondary antibodies by direct antibody-enzyme labeling Figure 2 Schematic diagram of synthesis using biotin-avidin amplification method Figure 3 Schematic diagram of the synthesis of chain polymer-enzyme-antibody polymers Figure 4 Schematic diagram of polymerase synthesis Figure 5 Structural formula of hyperbranched amino polyethylene glycol Figure 6 Polymerase secondary antibody thermal stability experiment (CK20 protein, primary antibody: Jingjie; secondary antibody: Zhongshan Jinqiao) Figure 7 Human liver cancer tissue validation polymer enzyme-labeled secondary antibody reagent (CK7 protein, primary antibody: positive energy; secondary antibody: Zhongshan Jinqiao) Figure 8 Human liver cancer tissue validation polymer enzyme-labeled secondary antibody reagent (CK8&18 protein, primary antibody: positive energy; secondary antibody: Zhongshan Jinqiao) Fig. 9 Human colon cancer tissue validation of polymer enzyme-labeled secondary antibody reagent (CK20 protein, primary antibody: Jingjie; secondary antibody: Zhongshan Jinqiao) Fig.10 Human colon cancer tissue validation of polymer enzyme-labeled secondary antibody reagent (CEA-2C8 protein, primary antibody: Tansheng; secondary antibody: Zhongshan Jinqiao) Fig.11 Human breast cancer tissue validation of multimer enzyme-labeled secondary antibody reagent (CK7 protein, primary antibody: Jingjie; secondary antibody: Zhongshan Jinqiao) Fig.12Human breast cancer tissue validation of multimer enzyme-labeled secondary antibody reagent (CK7 protein, primary antibody: Youen; secondary antibody: Zhongshan Jinqiao) The present invention will be further explained below with reference to the accompanying drawings and embodiments. DETAILED DESCRIPTION Example

[0022] 1) Dissolve 31 mg of horseradish peroxidase (Yingxin Laboratory, CAS No.: 9003-99-0) in 100 μL (concentration 0.31 mg / μL) of sodium phosphate buffer (pH 8.00) at room temperature, add 2 μL of DVS (Sigma Chemical Company, Catalog No.: V3700-5G) while stirring, and then gently stir at room temperature in the dark for 30 minutes; 2) The above reaction solution was purified by Sephadex G-25 column to remove the remaining DVS, and the mobile phase was sodium phosphate buffer (0.2 M sodium phosphate buffer, pH 8.0). The brown component, i.e. the activated enzyme, was collected; 3) Use a centrifugal concentrator to concentrate the activated enzyme to 200 μL; 4) Add 15 μL of 5% aqueous solution of hyperbranched amino polyethylene glycol 5.0 (Polymer Factory, Product No.: PFLDHB-050512) to the concentrated activated enzyme while stirring. Gently stir the solution at 30°C for 16 hours; 5) The reaction solution in step 4) was purified by a Sephadex G-25 gel column and the solution was converted into a sodium phosphate buffer (0.1 M, pH 8.0). The first brown component collected was the polymer peroxidase; 6) Concentrate the polymer peroxidase produced in step 5) to 200 μL by centrifugation, add 2 μL DVS to the solution, and gently stir at 30°C for 3 h; 7) Use Sephadex G-25 separation column to remove the remaining DVS, the mobile phase is PBS buffer (pH 7.1), and collect the brown component, which is the activated polymer peroxidase; 8) Concentrating the activated polymer peroxidase produced in step 7) to 200 μL; 9) 0.5 mg of goat anti-mouse (Fab') 2 chain was added dropwise to the activated stirred multimeric enzyme produced in step 8) at room temperature and continued to stir for 1 h, and then incubated at 4°C in the dark for 24 hours; 10) Add antibacterial agent and stabilizer to the solution in step 9). (0.1% proclin-300, 3 mg bovine serum albumin); (proclin-300 Yingxin Laboratory, Catalog No.: TX01457-50 mL; bovine serum albumin Chongqing Yiheng Biotechnology Co., Ltd., Catalog No.: A8010) 11) The poly-horseradish peroxidase-labeled goat anti-mouse secondary antibody produced can be used for immunohistochemical staining after dilution.

[0023] Preparation of (Fab')2 chain: Dissolve 35 mg of dithiothreitol (McLean, Catalog No.: D806827-5g) in 1 mL of PBS buffer (PH = 7.4), then take 25 μL of dithiothreitol solution and add it dropwise to the stirring 1 mg of goat anti-mouse complete antibody (Abcam, Catalog No.: ab205719). After incubation in a 37°C water bath for 30 minutes, pass the antibody solution through a Sephadex G-25 purification column to remove excess dithiothreitol reducing agent and obtain a pure goat anti-mouse (Fab')2 fragment solution. Example

[0024] 1) Dissolve 31 mg of horseradish peroxidase (Yingxin Laboratory, CAS No.: 9003-99-0) in 100 μL of sodium phosphate buffer (pH 8.00) at room temperature, add 2 μL of DVS (Sigma Chemical Company, Catalog No.: V3700-5G) while stirring, and then gently stir at room temperature in the dark for 30 minutes; 2) The above reaction solution was purified by Sephadex G-25 column to remove the remaining DVS, and the mobile phase was sodium phosphate buffer (0.2 M sodium phosphate buffer, pH 8.0). The brown component, i.e. the activated enzyme, was collected; 3) Use a centrifugal concentrator to concentrate the activated enzyme to 200 μL; 4) Add 15 μL of 5% aqueous solution of hyperbranched amino polyethylene glycol 5.0 (Polymer Factory, Product No.: PFLDHB-050512) to the concentrated activated enzyme while stirring. Gently stir the solution at 30°C for 16 hours; 5) The reaction solution in step 4) was purified by a Sephadex G-25 gel column and the solution was converted into a sodium phosphate buffer (0.1 M, pH 8.0). The first brown component collected was the polymer peroxidase; 6) Concentrate the polymer peroxidase produced in step 5) to 200 μL by centrifugation, add 2 μL DVS to the solution, and gently stir at 30°C for 3 h; 7) Use Sephadex G-25 separation column to remove the remaining DVS, the mobile phase is PBS buffer (pH 7.1), and collect the brown component, which is the activated polymer peroxidase; 8) Concentrating the activated polymer peroxidase produced in step 7) to 200 μL; 9) 0.5 mg of goat anti-rabbit (Fab') 2 chain was added dropwise to the activated stirred multimeric enzyme produced in step 8) at room temperature and continued to stir for 1 h, and then incubated at 4°C in the dark for 24 hours; 10) Add antibacterial agent and stabilizer to the solution in step 9). (0.1% proclin-300, 3 mg bovine serum albumin); (proclin-300 Yingxin Laboratory, Catalog No.: TX01457-50 mL; bovine serum albumin Chongqing Yiheng Biotechnology Co., Ltd., Catalog No.: A8010) 11) The poly-horseradish peroxidase-labeled goat anti-rabbit secondary antibody produced can be used for immunohistochemical staining after dilution.

[0025] Preparation of (Fab')2 chain: Dissolve 35 mg of dithiothreitol (McLean, Catalog No.: D806827-5g) in 1 mL of PBS buffer (PH = 7.4), then take 25 μL of dithiothreitol solution and add it dropwise to the stirring 1 mg of goat anti-rabbit antibody (Abcam, Catalog No.: ab6721) solution. After incubation in a 37°C water bath for 30 minutes, pass the antibody solution through a Sephadex G-25 purification column to remove excess dithiothreitol reducing agent and obtain a pure goat anti-rabbit (Fab')2 fragment solution.

[0026] The polymer enzyme-labeled secondary antibody produced by the present invention is used for immunohistochemical pathological specimen staining. Compared with the existing polymer enzyme secondary antibodies on the market, it has higher sensitivity and better stability. The following examples 1 to 7 all use the same operating steps to compare sensitivity and stability. The enzyme-labeled secondary antibody used is the enzyme-labeled secondary antibody produced in Examples 1 and 2, diluted 1:500 in secondary antibody diluent (Beijing Cool Bolai Biotechnology Co., Ltd., catalog number: SL1390-100mL), and the comparative polymer secondary antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer, item number: PV-8000). The steps of the use example are as follows: 1) Sample slice preparation: The paraffin-embedded tissue slices that had been fixed (Wuhan Saiweier Biotechnology Co., Ltd., Catalog No.: G1101-15ML) were baked in a 60°C constant temperature oven for 2 h and stored for later use.

[0027] 2) Dewaxing: Take the corresponding paraffin-embedded tissue sections (Sangon Biotechnology (Shanghai) Co., Ltd., Catalog No.: A601891-0500) and immerse them in xylene (Chongqing Chuandong Chemical (Group) Co., Ltd.) for 10 minutes each, three times.

[0028] 3) Hydration: Transfer the above sections to 100% alcohol and soak for 5 minutes each, twice; soak in 95% alcohol for 5 minutes once; soak in 75% alcohol for 5 minutes once; soak in purified water for 3 minutes, once.

[0029] 4) Heat repair: Take the above sections, soak them in EDTA (Legen Biotechnology Co., Ltd., Catalog No.: DD0002) antigen repair solution, heat them in a pressure cooker, and maintain the temperature for 3.5 minutes after reaching 120°C. Stop heating and let it cool naturally for 30 minutes. Take out the sections and soak them in PBS (Tianjin Haoyang Huake Biotechnology Co., Ltd., Catalog No.: PB2004Y) buffer for 3 minutes.

[0030] 5) Primary antibody incubation: Remove the sections, spin dry the PBS, add 0.1 mL of the corresponding primary antibody (see the annotations in each figure) on each section to cover the tissue, incubate at room temperature for 60 minutes, spin dry the primary antibody, and soak the sections in PBS buffer three times for 1 minute each time.

[0031] 6) Secondary antibody incubation: Take out the slices, spin dry the PBS, and drop 0.1 mL of the corresponding secondary antibody on each slice to cover the tissue. After incubation at room temperature for 20 minutes, spin dry the secondary antibody and soak the slices in PBS for 3 minutes 3 times.

[0032] 7) Color development: Take out the slices, spin off the PBS, add 0.1 mL of DAB to each slice to cover the tissue on the slice, and incubate at room temperature for 5 minutes. Spin off DAB (Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G1212-200T), and soak the slices in purified water for 1 minute.

[0033] 8) Staining: Take out the above sections, shake off the water, add 0.1 mL of hematoxylin on each section to cover the tissue on the section, incubate at room temperature for 3 minutes, shake off the hematoxylin (Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G1004-100ML), place the sections on a section rack and rinse under tap water for 3 minutes.

[0034] 9) Dehydration: Take out the above sections and soak them in the following liquids for 3 minutes respectively: 95% alcohol, 100% alcohol, 100% alcohol, xylene.

[0035] 10) Sealing: Take out the above sections, blow dry in the fume hood, add one drop (about 0.02 mL) of mounting medium (Shanghai Sangon Biotechnology Co., Ltd., Catalog No.: E675007-0100) on each section, cover the slide, and blow dry in the fume hood (about 5 minutes).

[0036] 11) Film reading: Observe the staining effect under a microscope.

[0037] Application Example 1 Application Example 1 shows that under the same operating steps, the hyperbranched polymer enzyme secondary antibody reagent produced by the present invention (Example 2, 1:500 dilution) was placed at 37°C for six weeks, and the CK20 antigen protein in human colorectal cancer tissue was detected by immunohistochemistry (using Anti-CK20 Rabbit mAb, Jingjie Bio, Catalog No.: ABT-5454). The target antigen can still be clearly and effectively detected. Compared with the secondary antibody reagent stored at the initial 4°C, the effect is not attenuated, indicating that the polymer enzyme-linked secondary antibody reagent produced by the present invention has higher stability ( Figure 6 ).

[0038] Application Example 2 The CK7 protein in human liver cancer tissue was detected by immunohistochemistry using the Cytokeratin 7 Rabbit mAb (Positive Energy, Catalog No.: R381339) primary antibody combined with the 1:500 dilution of the multimeric enzyme secondary antibody in Example 2 of the present invention. The results showed that the staining intensity of the multimeric enzyme secondary antibody of the present invention at a 500-fold dilution was stronger than that of the commercially available secondary antigen solution (HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer Zhongshanjinqiao, Catalog No.: PV-8000). Figure 7 ). This indicates that the polymer enzyme secondary antibody reagent produced by the present invention has higher sensitivity.

[0039] Application Example 3 The CK8 & 18 proteins in human liver cancer tissues were detected by immunohistochemistry using the Cytokeratin 8 / 18 Rabbit mAb (Positive Energy, Catalog No.: R50136) primary antibody combined with the 1:500 dilution of the polymer enzyme secondary antibody in Example 2 of the present invention. The results showed that the staining intensity of the polymer enzyme secondary antibody of the present invention at a 500-fold dilution was stronger than that of the commercially available secondary antigen solution (HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer Zhongshanjinqiao, Catalog No.: PV-8000) ( Figure 8 ). This indicates that the polymer enzyme secondary antibody reagent produced by the present invention has higher sensitivity.

[0040] Application Example 4 Under the same operation steps, the CK20 protein in human colon cancer was detected by immunohistochemistry using Anti-CK20 Rabbit mAb (Jingjie Bio, Catalog No.: ABT-5454) primary antibody and the 1:500 dilution of the polymer enzyme secondary antibody in Example 2 of the present invention. The results showed that the staining intensity of the polymer enzyme secondary antibody of the present invention at a 500-fold dilution was stronger than that of the commercially available secondary antigen solution (HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer Zhongshanjinqiao, Catalog No.: PV-8000) ( Fig. 9 ). This indicates that the polymer enzyme secondary antibody reagent produced by the present invention has higher sensitivity.

[0041] Application Example 5 Under the same operation steps, the CEA Mouse mAb (Tansheng, Catalog No.: FAB-T002-2C8) primary antibody and the 1:500 dilution of the polymer enzyme secondary antibody in Example 1 of the present invention were used to perform immunohistochemistry to detect CEA-2C8 protein in human colon cancer. The results showed that the staining intensity of the polymer enzyme secondary antibody of the present invention at a 500-fold dilution was stronger than that of the commercially available secondary antigen solution (HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer Shanjinqiao, Catalog No.: PV-8000 ( Fig.10 ). This indicates that the polymer enzyme secondary antibody reagent produced by the present invention has higher sensitivity.

[0042] Application Example 6 Under the same operation steps, the CK7 protein in human breast cancer was detected by immunohistochemistry using Anti-CK7 Rabbit mAb (Jingjie Bio, Catalog No.: ABT-5298) primary antibody and the 1:500 dilution of the polymer enzyme secondary antibody in Example 2 of the present invention. The results showed that the staining intensity of the polymer enzyme secondary antibody of the present invention at a 500-fold dilution was stronger than that of the commercially available secondary antibody (HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer Zhongshanjinqiao, Catalog No.: PV-8000) ( Fig.11 ). This indicates that the polymer enzyme secondary antibody reagent produced by the present invention has higher sensitivity.

[0043] Application Example 7 Under the same operation steps, CK7 Mouse mAb (Uen, Catalog No.: PA6628) primary antibody and 1:500 dilution of the polymer enzyme secondary antibody in Example 1 of the present invention were used to perform immunohistochemistry to detect CK7 protein in human breast cancer. The results showed that the staining intensity of the polymer enzyme secondary antibody of the present invention at a 500-fold dilution was stronger than that of the commercially available secondary antibody (HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer Zhongshanjinqiao, Catalog No.: PV-8000) ( Fig.12 ). This indicates that the polymer enzyme secondary antibody reagent produced by the present invention has higher sensitivity.

[0044] References [1] Mason T E, Phifer R F, Spicer S S, et al. An immunoglobulin-enzyme bridge method for localizing tissue antigens[J]. Journal ofHistochemistry & Cytochemistry, 1969, 17(9): 563-569. [2] Hsu S M, Raine L, Fanger H X. Use of avidin-biotin-peroxidasecomplex (ABC) in immunoperoxidase techniques: a comparison between ABC andunlabeled antibody (PAP) procedures[J]. Journal of Histochemistry &Cytochemistry, 1981, 29(4): 577-580. [3] Heras A. Enhanced polymer detection system forimmunohistochemistry[J]. Mod Pathol, 1995, 8: 165A. [4] Shi S R, Guo J, Cote R J, et al. Sensitivity and detectionefficiency of a novel two-step detection system (PowerVision) forimmunohistochemistry[J]. Applied Immunohistochemistry & Molecular Morphology,1999, 7(3): 201. [5] Feliu N, Walter MV, Montañez MI, et al. Stability andbiocompatibility of a library of polyester dendrimers in comparison topolyamidoamine dendrimers[J]. Biomaterials, 2012, 33(7):1970-1981.

Claims

1. A method for preparing a highly sensitive polymerase-labeled secondary antibody, characterized in that: The polymerase secondary antibody comprises a hyperbranched polymer skeleton, a labeling enzyme and a secondary antibody, all of which are connected via a coupling agent.

2. The hyperbranched polymer backbone is a hyperbranched amino polyethylene glycol with a molecular weight of 29080 g / mol, and the marker enzyme is connected to the branched ends and the middle branched structure of the polymer backbone through a coupling agent. The marker enzyme is connected to the secondary antibody through a coupling agent.

3. The polymerase-labeled secondary antibody according to claim 1, characterized in that: The labeling enzyme is horseradish peroxidase and / or alkaline phosphatase and / or urease.

4. The polymerase-labeled secondary antibody according to claim 1, characterized in that: The secondary antibody is a complete antibody or a Fab' or (Fab)2 fragment of the corresponding antibody, including mouse anti-sheep immunoglobulin, rabbit anti-sheep immunoglobulin, goat anti-mouse immunoglobulin, goat anti-rabbit immunoglobulin, horse anti-sheep immunoglobulin, horse anti-mouse immunoglobulin, horse anti-rabbit immunoglobulin, mouse anti-human immunoglobulin, rabbit anti-human immunoglobulin, goat anti-human immunoglobulin, horse anti-human immunoglobulin, etc. The secondary antibody Fab' fragment is a Fab' fragment with a size of 50-55 kDa obtained by cleaving the complete antibody with a dithiothreitol reducing agent.

5. The polymerase-labeled secondary antibody according to claims 1-3, characterized in that: (1) The labeled enzyme is activated by a coupling agent; (2) coupling the activated marker enzyme obtained in step (1) with hyperbranched amino-polyethylene glycol to obtain a hyperbranched complex loaded with the enzyme; (3) The enzyme-loaded hyperbranched complex obtained in step (2) is then activated by a coupling agent and coupled with a secondary antibody to obtain a multimeric enzyme.

6. A polymerase-labeled secondary antibody according to claims 1-4, characterized in that Application of polymerase secondary antibodies in human immunohistochemistry.

7. A polymerase-labeled secondary antibody according to claims 1-4, characterized in that Application of polymerase secondary antibodies in human tumor detection.

Images