An enzyme-labeled secondary antibody polymer for immunohistochemical staining

Abstract

The invention discloses an enzyme-labeled secondary antibody polymer for immunohistochemical staining. The enzyme-labeled secondary antibody polymer is formed by combining a secondary antibody with a polymer backbone, the polymer is labeled with a marker enzyme, the polymer backbone is polymethacrylate-2-hydroxyethyl ester, and the marker enzyme is connected to polymethacrylate-2-hydroxyethyl ester through a coupling agent. The present invention utilizes polymethacrylate-2-hydroxyethyl ester to modify proteins, reduce immunogenicity and antigenicity, and can also weaken the hydrolysis of proteases and increase the solubility of protein molecules. The enzyme-labeled secondary antibody polymer is applied to immunohistochemical staining and has high specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to an enzyme-labeled secondary antibody polymer for immunohistochemical staining. Background Art

[0002] Immunohistochemical Staining (IHC) is a technology that uses chemical reactions to locate and observe antigen-antibody reactions on tissue samples. It is based on the principle of "antigen-antibody specific binding" to detect proteins in biological samples and then locate, qualitatively and quantitatively analyze them. It has the advantages of simple operation, high sensitivity and strong specificity. It is widely used in the field of immune detection, especially immunohistochemistry (IHC) staining in hospital pathology departments, third-party pathology testing institutions, pathology teaching and research departments, and pathology research laboratories, providing pathologists with evidence-based medical evidence for diagnosing and identifying tumors.

[0003] There are four main types of enzyme-labeled secondary antibodies on the market: 1) Antibody-glutaraldehyde-enzyme direct labeling method, that is, a single secondary antibody and HRP enzyme are directly coupled and labeled by glutaraldehyde. Since the molecular weight of the labeled enzyme is usually large, and the reagent reaction conditions are not easy to control during the labeling process, each antibody can only label a certain number of enzymes, which can easily cause problems such as insufficient detection sensitivity or reduced antibody specificity. 2) Avidin-biotin-complex amplification method, namely the ABC (Avidin Biotin Complex) method. Biotin is a small organic molecule with a small volume, which can be labeled on more secondary antibody molecules and has little effect on the specificity and affinity of the antibody. By utilizing the high affinity between biotin and avidin, the secondary antibody labeled with biotin can be combined with the enzyme molecule labeled with avidin to form a complex. Compared with the direct method, the ABC method can greatly improve the sensitivity of detection, but since biotin is widely present in biological tissues, non-specific staining is very likely to occur, resulting in obvious background staining and interfering with the interpretation of experimental results. 3) Chain polymer-enzyme-antibody polymer, namely the dextran method. Due to the chain structure of dextran, the enzyme molecule can be labeled on its skeleton first, and then connected to the secondary antibody molecule. This labeling can reduce the effect of the enzyme on the specificity and affinity of the antibody and improve the detection sensitivity. However, due to the loose overall structure of the chain polymer-enzyme-antibody polymer and the large volume, the concentration of the enzyme is not high enough, and the sensitivity for detecting some intracellular antigens is still insufficient. Moreover, the chain enzyme is a hydrophilic group, and the lipophilicity of the double lipid structure of the cell membrane and cytoplasm is not strong enough, and the binding strength to the cell membrane and cytoplasm is weak, which affects the sensitivity and specificity of the detection. 4) The method of dendritic PAMA polymerization is characterized by large or small molecular weight. Because the number of amino groups increases, the number of enzyme targets that can be linked also increases, which can improve the specificity and sensitivity of the reaction. However, the preparation process is quite complicated, and the degree of polymerization of the polymer is not easy to control, resulting in impure intermediates that bring non-specific background to the staining, and cannot fully meet clinical needs. Therefore, it is of great significance to obtain an enzyme-labeled secondary antibody with good stability, strong specificity, high sensitivity and clean background. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide an enzyme-labeled secondary antibody polymer, which uses poly(2-hydroxyethyl methacrylate) to modify the protein, reduce the immunogenicity and antigenicity, weaken the hydrolysis of proteases, increase the solubility of protein molecules, and thus improve the specificity and sensitivity of the enzyme-labeled secondary antibody.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] An enzyme-labeled secondary antibody polymer is formed by combining a secondary antibody with a polymer backbone. The enzyme-labeled secondary antibody polymer is labeled with a marker enzyme, the polymer backbone is polymethacrylate-2-hydroxyethyl ester, and the marker enzyme is connected to the polymethacrylate-2-hydroxyethyl ester through a coupling agent.

[0007] Preferably, the molecular weight of the poly(2-hydroxyethyl methacrylate) is 2500-7500 kDa, more preferably 5000 kDa.

[0008] Preferably, the labeling enzyme is linked to the end of the poly(2-hydroxyethyl methacrylate) branch chain.

[0009] Preferably, the secondary antibody is selected from secondary antibody Fab' fragments.

[0010] Preferably, the secondary antibody Fab' fragment is a secondary antibody Fab' fragment having a molecular weight of 50-75 kDa formed by hydrolyzing the secondary antibody with pepsin to obtain a F(ab')2 fragment and then cleaving it with 2-mercaptoethylamine.

[0011] Preferably, the secondary antibody Fab' fragment is selected from one or more of horse anti-mouse IgG-Fab' fragment, horse anti-rabbit IgG-Fab' fragment, mouse anti-goat IgG-Fab' fragment, rabbit anti-goat IgG-Fab' fragment, horse anti-goat IgG-Fab' fragment, rabbit anti-mouse IgG-Fab' fragment, goat anti-mouse IgG-Fab' fragment, goat anti-rabbit IgG-Fab' fragment, donkey anti-mouse IgG-Fab' fragment, donkey anti-rabbit IgG-Fab' fragment, mouse anti-donkey IgG-Fab' fragment, rabbit anti-donkey IgG-Fab' fragment, horse anti-donkey IgG-Fab' fragment, donkey anti-horse IgG-Fab' fragment, camel anti-goat IgG-Fab' fragment or camel anti-donkey IgG-Fab' fragment.

[0012] Preferably, the labeling enzyme is selected from one or more of horseradish peroxidase (HRP enzyme), alkaline phosphatase (AP enzyme), glucosidase or β-galactosidase.

[0013] Preferably, the coupling agent is selected from one or more of N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (sulfo-SMCC), succinimidyl 3-(2-pyridyldisulfide)-propionate (SPDP) or N-succinimidyl-S-acetylthioacetate (SATA).

[0014] The present invention also provides a method for preparing the above-mentioned enzyme-labeled secondary antibody polymer, comprising the following steps:

[0015] Preparing an enzyme complex: linking the labeled enzyme via a first coupling agent to form an enzyme complex;

[0016] Activating the polymer skeleton: activating poly(2-hydroxyethyl methacrylate) with a second coupling agent to obtain an activated polymer skeleton;

[0017] Preparation of product by coupling reaction: the enzyme complex, activated polymer backbone and secondary antibody are subjected to coupling reaction to obtain enzyme-labeled secondary antibody polymer.

[0018] Preferably, in the above preparation method, the first coupling agent and the second coupling agent are the same or different.

[0019] Preferably, the first coupling agent is selected from sulfo-SMCC.

[0020] Preferably, the second coupling agent is selected from SPDP.

[0021] Preferably, in the above preparation method, the molar ratio of the enzyme complex, the activated polymer backbone and the secondary antibody is (1~15): (1~10):1; more preferably (4~15): (2~8):1; most preferably 4:2:1.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The enzyme-labeled secondary antibody polymer described in the present invention is a small molecule enzyme-labeled secondary antibody, which is used for immunohistochemical staining and can effectively improve the specificity and sensitivity of immunohistochemical staining.

[0024] 2. The enzyme-labeled secondary antibody polymer described in the present invention uses poly(2-hydroxyethyl methacrylate) as the polymer backbone. After being connected to the protein secondary antibody, the modification of the N-terminus, C-terminus and certain amino acid side chains of the protein can change the biochemical properties of the protein, reduce the immunogenicity and antigenicity, weaken the hydrolysis of proteases, and increase the solubility of protein molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of KI-67 staining using the enzyme-labeled secondary antibody polymer of the present invention and a commercially available enzyme-labeled secondary antibody are shown.

[0026] Figure 2 The results are compared in sensitivity and specificity of KI-67 staining using the enzyme-labeled secondary antibody polymer of the present invention and commercially available enzyme-labeled secondary antibodies.

[0027] Figure 3 The figure shows the results of CK (pan) staining using the enzyme-labeled secondary antibody polymer of the present invention and a commercially available enzyme-labeled secondary antibody.

[0028] Figure 4The results are compared in sensitivity and specificity of CK (pan) staining using the enzyme-labeled secondary antibody polymer of the present invention and commercially available enzyme-labeled secondary antibodies.

[0029] Figure 5 The results are shown in Figure 2, which are the results of CD3 staining using the enzyme-labeled secondary antibody polymer of the present invention and a commercially available enzyme-labeled secondary antibody.

[0030] Figure 6 The results are compared in sensitivity and specificity of CD3 staining using the enzyme-labeled secondary antibody polymer of the present invention and commercially available enzyme-labeled secondary antibodies.

[0031] Figure 7 It is the difference in absorbance between the enzyme-labeled secondary antibody polymer of the present invention and the commercially available enzyme-labeled secondary antibody.

[0032] Figure 8 The effect of different polymer backbones on the sensitivity of immunohistochemical staining.

[0033] Fig. 9 This is the effect of the molecular weight of poly(2-hydroxyethyl methacrylate) on the sensitivity of immunohistochemical staining. DETAILED DESCRIPTION

[0034] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0035] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0036] The embodiment of the present invention provides an enzyme-labeled secondary antibody polymer, which is a polymer formed by combining a secondary antibody with a polymer backbone, wherein the polymer is labeled with a labeling enzyme, wherein the polymer backbone is poly(2-hydroxyethyl methacrylate), and the labeling enzyme is connected to the poly(2-hydroxyethyl methacrylate) via a coupling agent.

[0037] In the present invention, poly(2-hydroxyethyl methacrylate) is used as a polymer skeleton. Poly(2-hydroxyethyl methacrylate) is a high polymer with oil-water amphiphilicity that can dissolve in water and organic matter at the same time. Poly(2-hydroxyethyl methacrylate) also has a hydroxyl end that can introduce carboxyl, amino and other active groups. After coupling with proteins (such as goat anti-mouse IgG, goat anti-rabbit IgG, horse anti-goat IgG, camel anti-goat IgG, etc.) by solid phase or liquid phase method, the N-terminus, C-terminus and certain amino acid side chains of the polypeptide can be modified, thereby changing the properties of the protein, such as changing the size of protein / polypeptide molecules, hydrophobicity and charge, etc., increasing protein water solubility and stability, etc., and reducing immunogenicity and antigenicity, weakening the hydrolysis of proteases, and increasing the solubility of protein molecules. In the present invention, poly(2-hydroxyethyl methacrylate) contains a large number of hydroxyl groups that are oxidized to form aldehyde groups, some of which are connected to the amino groups in the marker enzyme, and some of which are connected to the secondary antibody to form a stable conjugate. In a specific embodiment, the marker enzyme is connected to the end of the poly(2-hydroxyethyl methacrylate) branch chain.

[0038] Preferably, the secondary antibody described in the embodiment of the present invention is a secondary antibody Fab' fragment, and the molecular weight of the secondary antibody Fab' fragment is selected from 50-75 kDa.

[0039] In some embodiments, the secondary antibody Fab' fragment is a secondary antibody Fab' fragment having a molecule of 50-75 kDa formed by hydrolyzing the secondary antibody with pepsin to obtain a F(ab')2 fragment and then cleaving it with 2-mercaptoethylamine.

[0040] Preferably, the secondary antibody Fab' fragment used in some embodiments is selected from but not limited to one or more of horse anti-mouse IgG-Fab' fragment, horse anti-rabbit IgG-Fab' fragment, mouse anti-goat IgG-Fab' fragment, rabbit anti-goat IgG-Fab' fragment, horse anti-goat IgG-Fab' fragment, rabbit anti-mouse IgG-Fab' fragment, goat anti-mouse IgG-Fab' fragment, goat anti-rabbit IgG-Fab' fragment, donkey anti-mouse IgG-Fab' fragment, donkey anti-rabbit IgG-Fab' fragment, mouse anti-donkey IgG-Fab' fragment, rabbit anti-donkey IgG-Fab' fragment, horse anti-donkey IgG-Fab' fragment, donkey anti-horse IgG-Fab' fragment, camel anti-goat IgG-Fab' fragment or camel anti-donkey IgG-Fab' fragment.

[0041] Preferably, the labeling enzyme described in the embodiment of the present invention is selected from one of horseradish peroxidase (HRP enzyme), alkaline phosphatase (AP enzyme), glucosidase or beta-galactosidase.

[0042] Preferably, the coupling agent described in the embodiment of the present invention is selected from one or more of N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt (sulfo-SMCC), succinimidyl 3-(2-pyridyldisulfide)-propionate (SPDP) or N-succinimidyl-S-acetylthioacetate (SATA).

[0043] The present invention also provides a method for preparing an enzyme-labeled secondary antibody polymer, comprising the following steps:

[0044] Preparing an enzyme complex: linking the labeled enzyme via a first coupling agent to form an enzyme complex;

[0045] Activating the polymer skeleton: activating poly(2-hydroxyethyl methacrylate) with a second coupling agent to obtain an activated polymer skeleton;

[0046] Preparation of product by coupling reaction: the enzyme complex, activated polymer backbone and secondary antibody are subjected to coupling reaction to obtain enzyme-labeled secondary antibody polymer.

[0047] Preferably, in the preparation method described in the embodiment of the present invention, the first coupling agent and the second coupling agent are the same or different.

[0048] Preferably, the first coupling agent is selected from sulfo-SMCC.

[0049] Preferably, the second coupling agent is selected from SPDP.

[0050] Preferably, in the preparation method described in the embodiment of the present invention, the molar ratio of the enzyme complex, the activated polymer backbone and the secondary antibody is (1~15): (1~10):1; more preferably (4~15): (2~8):1; most preferably 4:2:1.

[0051] In the embodiment of the present invention, the preparation method further comprises placing the obtained enzyme-labeled secondary antibody polymer into a dialysis bag, dialyzing it with phosphate buffer (pH 7.4) for 12 h, changing the dialysis solution every 4 h, then adding the antibody diluent to mix, and packaging it at 4°C for storage for later use.

[0052] The following are specific embodiments of the present invention. In the following embodiments, the reagents, raw materials, parameters, etc. used are explained as follows:

[0053] In the embodiments of the present invention, the poly(2-hydroxyethyl methacrylate) (nCH2=C(CH3)COOCH2CH2OH) used is from Merck, SKU: 477028.

[0054] In the present invention, the horseradish peroxidase used is from Merck, SKU: SRE0082, packaging specification: 30k units.

[0055] In the examples of the present invention, the goat anti-mouse secondary antibody IgG used is from Merck, SKU: AP124.

[0056] In the examples of the present invention, the goat anti-rabbit secondary antibody IgG used is from Merck, SKU: 40839.

[0057] In the embodiment of the present invention, the refrigerated temperature range is 2-8° C., and the room temperature range is 15-35° C. Overnight means between 12-18 hours.

[0058] Example 1

[0059] This embodiment provides an enzyme-labeled secondary antibody polymer, which is prepared by the following method:

[0060] 1. Preparation of enzyme complex:

[0061] (1) Dissolve HRP in 0.1 M sodium phosphate buffer (containing 0.3 M sodium chloride, pH 7.2) to a concentration of 10 mg / mL;

[0062] (2) Dissolve sodium periodate in deionized water to a concentration of 0.1 M.

[0063] (3) Rapidly mix the solutions of steps (1) and (2) above and react at 37°C for 15 min.

[0064] (4) Filter the solution in (3) through a gel column G50, and then centrifuge (R = 3000 rpm);

[0065] (5) Collect the centrifuged liquid and measure the absorbance at 403 nm. If the absorbance is zero, it is optimal. Otherwise, filter and centrifuge again using a G50 column until the absorbance is zero. Adjust the concentration of iodinated HRP to 20 mg / mL, dry it in a freezer, and store it in a -20°C refrigerator for future use.

[0066] (6) Dissolve the above HRP lyophilized powder in phosphate buffer and adjust the concentration to 20-50 mg / mL.

[0067] (7) Add 15 mg of sulfo-SMCC (Thermofisher) to the solution in (6), shake to mix, and incubate at 37°C for 30 min.

[0068] (8) The liquid in (7) was purified by filtration using a desalting resin column. The filtrate was brown in color. The filtrate was then dialyzed overnight using phosphate buffer and the concentration was adjusted to 20 mg / mL to obtain an HRP enzyme complex.

[0069] (ii) Activation of polymer skeleton:

[0070] (1) Dissolve 10 mg of poly(2-hydroxyethyl methacrylate) in 1 mL of 10 mM sodium phosphate buffer (containing 0.15 M sodium chloride, pH 7.5);

[0071] (2) Add 100 μL of 0.1 M SPDP solution and react at room temperature for 60-90 min, keeping away from light;

[0072] (3) Add 100 μL of glycerol and react for 15-30 min at room temperature in the dark;

[0073] (4) Use G50 column for resin desalting, repeat twice;

[0074] (5) Add 0.05-0.1% bovine serum solution and store in a refrigerator at 4°C for a long time to obtain (ii) poly(2-hydroxyethyl methacrylate) coupled SPDP intermediate.

[0075] (III) Preparation of F(ab')2 of goat anti-mouse IgG (Merck):

[0076] (1) Dissolve 20 mg of goat anti-mouse IgG in 2 mL of sodium phosphate buffer (pH 7.2-7.6) and add appropriate amount of EDTA to maintain an EDTA concentration of 10 mM;

[0077] (2) Add 6 mg of TCEP (tri(2-carboxyethyl)phosphine, Thermofisher) at a molar equivalent of 3:1, mix well, and incubate at 37°C with shaking for 180 min.

[0078] (3) Purify with a desalting column, collect 0.5 mL of solution, and measure the absorbance at 285 nm. If the absorption peak meets the requirements, it is acceptable. If not, proceed as follows:

[0079] A: Add 10 μL of the collection solution onto the microtiter plate and add bovine serum albumin working solution;

[0080] B: Incubate at 37°C for 60-90 min until the color changes to brown;

[0081] C: Measure the absorption peak at 562 nm. It is OK if the absorption peak meets the requirements.

[0082] (IV) Preparation of products by coupling reaction:

[0083] (1) Mix the above HRP enzyme complex, poly(2-hydroxyethyl methacrylate)-coupled SPDP intermediate, and goat anti-mouse IgG (Merck) F(ab')2 liquid at a molar ratio of 4:2:1 and shake well. Incubate in a 37°C incubator for 60-90 min or at 4°C overnight.

[0084] (2) Filter twice using a G50 column, and dialyze the filtrate against sodium phosphate buffer (pH 7.2-7.6) once every 6 hours for a total of three times to obtain dialysate;

[0085] (3) Add bovine serum albumin working solution to the dialysate and store it in a 4°C refrigerator to obtain enzyme-labeled secondary antibody polymer.

[0086] Example 2

[0087] This embodiment provides an enzyme-labeled secondary antibody polymer, which is prepared by the following method:

[0088] 1. Preparation of enzyme complex:

[0089] (1) Dissolve HRP in 0.1 M sodium phosphate buffer (containing 0.3 M sodium chloride, pH 7.6) to a concentration of 20 mg / mL;

[0090] (2) Dissolve sodium periodate in deionized water to a concentration of 0.1 M.

[0091] (3) The solutions of steps (1) and (2) were mixed quickly and reacted at 37°C for 15 min;

[0092] (4) Filter the solution in (3) through a gel column G50, and then centrifuge (R = 3000 rpm);

[0093] (5) Collect the centrifuged liquid and measure the absorbance at 403 nm. If the absorbance is zero, it is optimal. Otherwise, filter and centrifuge again using a G50 column until the absorbance is zero. Adjust the concentration of iodinated HRP to 20 mg / mL, dry it in a freezer, and store it in a -20°C refrigerator for future use.

[0094] (6) Dissolve the above HRP lyophilized powder in phosphate buffer and adjust the concentration to 20-50 mg / mL. The higher the concentration of the enzyme, the higher the reaction sensitivity;

[0095] (7) Add 15 mg of sulfo-SMCC (Thermofisher) to the solution in (6), shake to mix, and incubate at 37°C for 30 min.

[0096] (8) The liquid in (7) was purified by filtration using a desalting resin column. The filtrate was brown in color. The filtrate was then dialyzed overnight using phosphate buffer and the concentration was adjusted to 20 mg / mL to obtain an HRP enzyme complex.

[0097] (ii) Activation of polymer skeleton:

[0098] (1) Dissolve 10 mg of poly(2-hydroxyethyl methacrylate) in 1 mL of 10 mM sodium phosphate buffer (containing 0.15 M sodium chloride, pH 7.5);

[0099] (2) Add 100 μL of 0.1 M SPDP solution and react at room temperature for 60-90 min, keeping away from light;

[0100] (3) Add 100 μL of glycerol and react for 15-30 min at room temperature in the dark;

[0101] (4) Use G50 column for resin desalting, repeat twice;

[0102] (5) Add 0.05-0.1% bovine serum solution and store in a refrigerator at 4°C for a long time to obtain (ii) poly(2-hydroxyethyl methacrylate) coupled SPDP intermediate.

[0103] (III) Preparation of F(ab')2 of goat anti-rabbit IgG (Merck):

[0104] (1) Dissolve 20 mg of goat anti-rabbit IgG in 2 mL of sodium phosphate buffer (pH 7.2-7.6) and add appropriate amount of EDTA to maintain an EDTA concentration of 10 mM;

[0105] (2) Add 6 mg of TCEP (tri(2-carboxyethyl)phosphine, Thermofisher) at a molar equivalent of 3:1, mix well, and incubate at 37°C with shaking for 180 min.

[0106] (3) Purify with a desalting column, collect 0.5 mL of solution, and measure the absorbance at 285 nm. If the absorption peak meets the requirements, it is acceptable. If not, proceed as follows:

[0107] A: Add 10 μL of the collection solution onto the microtiter plate and add bovine serum albumin working solution;

[0108] B: Incubate at 37°C for 60-90 min until the color changes to brown;

[0109] C: Measure the absorption peak at 562 nm. It is OK if the absorption peak meets the requirements.

[0110] (IV) Preparation of products by coupling reaction:

[0111] (1) Mix the above HRP enzyme complex, poly(2-hydroxyethyl methacrylate)-coupled SPDP intermediate, and goat anti-rabbit IgG (Merck) F(ab')2 liquid at a molar ratio of 4:2:1 and shake well. Incubate in a 37°C incubator for 60-90 min or at 4°C overnight.

[0112] (2) Filter twice using a G50 column, and dialyze the filtrate against sodium phosphate buffer (pH 7.2-7.6) once every 6 hours for a total of three times to obtain dialysate;

[0113] (3) Add bovine serum albumin working solution to the dialysate and store it in a 4°C refrigerator to obtain enzyme-labeled secondary antibody polymer.

[0114] Application Example 1

[0115] This application example uses the enzyme-labeled secondary antibody polymer (Baisheng) prepared in Example 1 as an example for application in immunohistochemical staining, wherein the enzyme-labeled secondary antibody of Leica is DS9800, and the enzyme-labeled secondary antibody of Dako is k5007. The difference in absorbance between the above three enzyme-labeled secondary antibodies is shown in Figure 7 .

[0116] The specific steps are as follows:

[0117] 1. Dewaxing and hydration

[0118] (1) Select paraffin-embedded tissue for sectioning.

[0119] (2) Soak the tissue sections in xylene for 8 min × 3 times, changing the xylene each time to ensure that the dewaxing is complete.

[0120] (3) Place the tissue sections in a wet box and add 200 μL of anhydrous ethanol for 3 min 3 times.

[0121] (4) Place the tissue sections in a wet box and add 200 μL of 95% ethanol for 3 min 3 times.

[0122] (5) Place the tissue sections in a wet box and add 200 μL of 75% ethanol for 3 min 3 times.

[0123] (6) Soak the tissue sections in deionized water for 3 min.

[0124] 2. Heat antigen retrieval

[0125] Add 500 mL of deionized water to the pressure cooker, place the tissue sections in a heat-resistant plastic box on a slide rack, add 120 mL of EDTA antigen repair solution (pH 9) to the plastic box, cover the pressure cooker, heat until steam comes out, keep it for 3 minutes, then stop heating and cool it naturally for 30 minutes. Open the lid and transfer the tissue sections to deionized water and soak them for 5 minutes × 2 times.

[0126] 3. Endogenous peroxidase blocking

[0127] (1) Rinse tissue sections with 200 μL of PBS buffer, spin dry the PBS buffer, and repeat three times.

[0128] (2) Add 200 μL of endogenous peroxidase blocking agent (3% hydrogen peroxide solution) and incubate at room temperature for 5 min. Repeat twice.

[0129] (3) Rinse tissue sections with 200 μL of PBS buffer.

[0130] (4) Soak the tissue sections in PBS buffer for 5 min × 2 times.

[0131] 4. Primary Antibody Incubation

[0132] (1) Shake off the PBS buffer, wipe the edge of the tissue with a paper towel, and draw circles along the edge of the tissue with a water-blocking pen.

[0133] (2) Add 50-150 μL of the corresponding primary antibody to the tissue, ensuring that the primary antibody solution covers the entire tissue.

[0134] (3) Incubate in a wet box at room temperature for 30 min.

[0135] (4) Rinse tissue sections with PBS buffer.

[0136] (5) Soak the tissue sections in PBS buffer for 5 min × 3 times.

[0137] 5. Secondary Antibody Incubation

[0138] (1) Spin off the PBS buffer.

[0139] (2) Add 50-150 μL (depending on the size of the tissue) of secondary antibody polymer reagent to the tissue, making sure the secondary antibody solution covers the entire tissue.

[0140] (3) Incubate in a wet box at room temperature for 10 min.

[0141] (4) Rinse tissue sections with PBS buffer.

[0142] (5) Soak the tissue sections in PBS buffer for 5 min × 3 times.

[0143] 6. DAB color development

[0144] (1) Prepare DAB colorimetric solution: add 50 μL of DAB (B) substrate solution dropwise into 950 μL of DAB (A) buffer and mix well for later use.

[0145] (2) Spin off the PBS buffer.

[0146] (3) Add 150 μL of DAB colorimetric solution onto the tissue.

[0147] (4) Incubate in a wet box at room temperature for 3 min.

[0148] (5) Rinse tissue sections with deionized water 3 times.

[0149] (6) Soak the tissue sections in deionized water for 5 min × 2 times.

[0150] 7. Hematoxylin turns blue

[0151] (1) Dry the deionized water.

[0152] (2) Add 50-150 μL of hematoxylin stain solution to the tissue.

[0153] (3) Incubate in a wet box at room temperature for 5 min.

[0154] (4) Rinse tissue sections with deionized water 3 times.

[0155] (5) Soak the tissue sections in deionized water for 5 min × 2 times.

[0156] 8. Dehydration, transparency, and sealing

[0157] (1) Place the tissue sections in a wet box and add 200 μL of 75% ethanol for 3 min 3 times.

[0158] (2) Place the tissue sections in a wet box and add 200 μL of 95% ethanol for 3 min 3 times.

[0159] (3) Place the tissue sections in a wet box and add 200 μL of anhydrous ethanol for 3 min 3 times.

[0160] (4) Soak the tissue sections in xylene for 10 min × 2 times.

[0161] (5) Remove the tissue slices from the solvent, lay them flat, add neutral resin on the tissue, and cover them with a coverslip.

[0162] 9. Observe the tissue morphology under a microscope, determine and record the staining results. Staining intensity: -, 1+, 2+, 3+; nonspecific staining: -, 1+, 2+, 3+.

[0163] 10. Experimental results:

[0164] (1) Comparison of KI-67 staining results is shown in Table 1. Figure 1 and Figure 2

[0165] Table 1 KI-67 staining results using different enzyme-labeled secondary antibodies

[0166]

[0167] From Table 1, Figure 1 and Figure 2 It can be seen that compared with commercially available enzyme-labeled secondary antibodies (Leica or Dako), the enzyme-labeled secondary antibody polymer (Baisheng) prepared by the present invention is used for KI-67 staining, and the staining intensity is stronger, the non-specific staining is cleaner, and it has high specificity and sensitivity.

[0168] (2) Comparison of CK (pan) staining results is shown in Table 2. Figure 3 and Figure 4

[0169] Table 2 Results of CK (pan) staining using different secondary antibodies

[0170]

[0171] From Table 2, Figure 3 and Figure 4 It can be seen that compared with commercially available enzyme-labeled secondary antibodies (Leica or Dako), the enzyme-labeled secondary antibody polymer (Baisheng) prepared by the present invention is used for CK (pan) staining, which has stronger staining intensity, cleaner nonspecific staining, and high specificity and sensitivity.

[0172] (3) Comparison of CD3 staining results is shown in Table 3. Figure 5 and Figure 6

[0173] Table 3 CD3 staining results using different secondary antibodies

[0174]

[0175] From Table 3, Figure 5 and Figure 6 It can be seen that compared with commercially available enzyme-labeled secondary antibodies (Leica or Dako), the enzyme-labeled secondary antibody polymer (Baisheng) prepared by the present invention is used for CD3 staining, and the staining intensity is stronger, the non-specific staining is cleaner, and it has high specificity and sensitivity.

[0176] Application Example 2

[0177] The effects of enzyme-labeled secondary antibody polymers prepared with different polymer backbones (poly(2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), polyethylene glycol diacrylate) on the sensitivity of immunohistochemical KI-67 staining were compared. The preparation steps of the enzyme-labeled secondary antibody polymers refer to Example 1. The KI-67 staining steps refer to Application Example 1.

[0178] Results Figure 8 .Depend on Figure 8 It can be seen that compared with poly (N-isopropylacrylamide) and polyethylene glycol diacrylate, the enzyme-labeled secondary antibody polymer prepared using poly methacrylate-2-hydroxyethyl ester as the polymer backbone has the highest staining intensity and the best detection sensitivity.

[0179] Application Example 3

[0180] The effects of enzyme-labeled secondary antibody polymers prepared with poly(2-hydroxyethyl methacrylate) of different molecular weights on the sensitivity of immunohistochemical KI-67 staining were compared. Fig. 9 .Depend on Fig. 9 It can be seen that poly(2-hydroxyethyl methacrylate) has better detection sensitivity when the molecular weight is 2500-7500 kDa, and the sensitivity is best when the molecular weight is 5000 kDa.

[0181] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field should fall within the protection scope determined by the claims of the present invention.

Claims

1. An enzyme-labeled secondary antibody polymer, which is formed by combining a secondary antibody with a polymer backbone, characterized in that: The enzyme-labeled secondary antibody polymer is labeled with a labeling enzyme, the polymer backbone is poly(2-hydroxyethyl methacrylate), the labeling enzyme is connected to the poly(2-hydroxyethyl methacrylate) via a coupling agent; the molecular weight of the poly(2-hydroxyethyl methacrylate) is 2500-7500 kD; the labeling enzyme is connected to the end of the poly(2-hydroxyethyl methacrylate) branch chain.

2. The enzyme-labeled secondary antibody polymer according to claim 1, characterized in that: The secondary antibody is selected from the secondary antibody Fab' fragment.

3. The enzyme-labeled secondary antibody polymer according to claim 2, characterized in that: The secondary antibody Fab' fragment is a secondary antibody Fab' fragment with a molecular weight of 50-75 kDa, which is obtained by hydrolyzing the secondary antibody with pepsin to obtain a F(ab')2 fragment and then cleaving it with 2-mercaptoethylamine.

4. The enzyme-labeled secondary antibody polymer according to claim 3, characterized in that: The secondary antibody Fab' fragment is selected from one or more of horse anti-mouse IgG-Fab' fragment, horse anti-rabbit IgG-Fab' fragment, mouse anti-goat IgG-Fab' fragment, rabbit anti-goat IgG-Fab' fragment, horse anti-goat IgG-Fab' fragment, rabbit anti-mouse IgG-Fab' fragment, goat anti-mouse IgG-Fab' fragment, goat anti-rabbit IgG-Fab' fragment, donkey anti-mouse IgG-Fab' fragment, donkey anti-rabbit IgG-Fab' fragment, mouse anti-donkey IgG-Fab' fragment, rabbit anti-donkey IgG-Fab' fragment, horse anti-donkey IgG-Fab' fragment, donkey anti-horse IgG-Fab' fragment, camel anti-goat IgG-Fab' fragment or camel anti-donkey IgG-Fab' fragment.

5. The enzyme-labeled secondary antibody polymer according to claim 1, characterized in that: The labeling enzyme is selected from one or more of horseradish peroxidase, alkaline phosphatase, glucosidase or β-galactosidase.

6. The enzyme-labeled secondary antibody polymer according to claim 1, characterized in that: The coupling agent is selected from one or more of N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt, succinimidyl 3-(2-pyridyldisulfide)-propionate or N-succinimidyl-S-acetylthioacetate.

7. A method for preparing an enzyme-labeled secondary antibody polymer according to any one of claims 1 to 6, characterized in that: The following steps are involved: Preparing an enzyme complex: connecting the labeled enzyme via a first coupling agent to form an enzyme complex; Activating the polymer skeleton: activating poly(2-hydroxyethyl methacrylate) with a second coupling agent to obtain an activated polymer skeleton; Preparation of product by coupling reaction: the enzyme complex, activated polymer backbone and secondary antibody are subjected to coupling reaction to obtain enzyme-labeled secondary antibody polymer.

8. The preparation method according to claim 7, characterized in that: The molar ratio of the enzyme complex, the activated polymer backbone and the secondary antibody is (1-15): (1-10):1.

Images