A method for preparing a polymerase-labeled antibody composition

Abstract

The present invention avoids the use of highly toxic raw materials in the production and preparation of polymerase-labeled antibodies by optimizing the types of skeleton materials, coupling agents and preparation methods, thereby improving the safety of product production. The composition containing the polymerase-labeled polyantibody and polymerase-labeled nanoantibody prepared by the present invention improves the penetration of the secondary antibody into the cell nuclear membrane, while overcoming the problems of uneven performance and low universality of different types of antigen detection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunohistochemistry detection, and in particular to a method for preparing a polymerase-labeled antibody composition. Technical Background

[0002] Pathological diagnosis, the "gold standard" for tumor diagnosis, uses a series of processes and observations on human tissues or cells to study the etiology, pathogenesis, morphological structure, function, and metabolism of the disease, revealing the patterns of disease development and ultimately elucidating the underlying nature of the disease. Histopathology and cytopathology are the two basic methods of pathological diagnosis, primarily focusing on morphological observation. Integrating immunodiagnosis with molecular diagnosis has led to two other important branches of pathological diagnosis: immunohistochemical pathology and molecular pathology. These advance pathological diagnosis from morphological observation at the tissue and cellular level to the protein and molecular levels. Immunohistochemistry is the most widely used technique in clinical practice due to its rapid testing, low cost, and ability to analyze paraffin-embedded specimens and frozen sections. Immunohistochemistry provides objective evidence at the protein expression level and is widely used in determining whether a tumor is benign or malignant, determining the origin of tumor cells, differentiating tumor types and subtypes, determining tumor differentiation, assessing prognosis, promoting targeted therapy, and detecting and identifying micrometastases.

[0003] In immunohistochemical detection technology, the combination of antibodies and enzymes (secondary antibodies) plays an extremely important role in the specificity and sensitivity of immunoassays. The traditional methods of enzyme-labeled antibodies are sodium periodate oxidation and glutaraldehyde oxidation. The enzyme-labeled products obtained by these methods only carry a small number of enzyme molecules ( Figure 1 A), cannot meet the sensitivity requirements and is difficult to apply in pathological immunohistochemistry detection. In order to meet the detection sensitivity requirements, a variety of secondary antibody detection systems have been developed and applied. For example, the early enzyme-labeled streptavidin-biotin staining method (SABC method), the detection principle is as follows Figure 1 As shown in B, this method is based on the principle of "high affinity between biotin and streptavidin". By combining "biotin-labeled secondary antibody" and "streptavidin-biotin-enzyme composition", a polymer is formed, which greatly improves the sensitivity of detection. The main drawback of this method is that the core ABC composition will bind to the biotin commonly present in cell tissues, thereby producing serious non-specific staining, which greatly interferes with the interpretation of the results. Therefore, this method has been gradually eliminated in clinical immunoassays. At present, the polymer-enzyme-labeled secondary antibody method, that is, the enzyme-labeled polymer staining method, is more widely used ( Figure 1C) This method involves labeling the enzyme onto a main chain with an inert glucan backbone, forming a glucanase composition. Multiple enzyme and antibody molecules can be attached to each glucan backbone. This system can simultaneously attach secondary antibodies of varying resistance, allowing for specific binding to rabbit or mouse primary antibodies. This method effectively avoids nonspecific staining caused by endogenous biotin, and its sensitivity is comparable to that of enzyme-labeled streptavidin-biotin staining, making it the current mainstream method in clinical practice. However, the glucan backbone used in this type of secondary antibody has a relatively large molecular weight. Although multiple enzyme and antibody molecules are attached simultaneously, its structure is loose and its hydrodynamic volume is large, resulting in significant steric hindrance. This reduces the polymer's ability to penetrate the nuclear membrane, resulting in poor nuclear staining.

[0004] To improve such problems, Chinese patents CN112305222A and CN113391059A respectively use smaller polymer enzyme-antibody fragments and polymer enzyme-labeled nanoantibodies. Compared with ordinary polymer enzyme-labeled antibody products, this type of product has improved molecular mobility and penetration ability, and has better nuclear staining effect. A polymer with too small a volume means that the molecule moves faster and has poorer sensitivity. For immunohistochemical staining situations such as cell staining and membrane staining, the effective binding rate of a polymer with too small a volume to the target (primary antibody) decreases, resulting in poor universality of the product and uneven effects on different types of antigen detection. In addition, Chinese invention patent CN105566499A optimizes the skeleton material and uses compact dendrimers as the skeleton to overcome the main defects of the current polymer enzyme-labeled method using chain polymers (such as dextran or polypeptides) as carriers, reducing the volume of the polymer enzyme and increasing the number of enzymes connected to the secondary antibody per unit volume and the molecular penetration. However, this technology uses highly toxic divinyl sulfone (DVS) as a coupling reagent multiple times during the preparation process, which is harmful to the environment and human health. Summary of the Invention

[0005] Based on the existing technical problems and difficulties, the present invention proposes a novel method for preparing a compact polymerase-labeled antibody. The present invention avoids the use of highly toxic raw materials in the production and preparation process by optimizing the types of skeleton materials, coupling agents and preparation methods, thereby improving the safety of product production; the polymerase-labeled antibody composition prepared by the present invention adopts the form of a combination of polyantibodies and nanoantibodies, and matches polymerase-labeled antibody compositions of different sizes for different positioning antigens, thereby improving the penetration of the secondary antibody for the cell nuclear membrane, and at the same time overcoming the unevenness and universality problems shown by different types of antigen detection. Furthermore, the skeleton of the polymerase-labeled antibody described in the present invention improves the hydrophilicity of the polymerase-labeled antibody and effectively reduces background staining; at the same time, it reduces the volume of the immunohistochemistry secondary antibody, further improving its molecular mobility and penetration ability.

[0006] Therefore, one aspect of the present invention is to provide a method for preparing a polymerase-labeled antibody composition, comprising the following steps:

[0007] (1) preparing a first polymerase-labeled antibody and a second polymerase-labeled antibody respectively;

[0008] (2) mixing the first polymerase-labeled antibody and the second polymerase-labeled antibody to obtain a polymerase composition;

[0009] Wherein, the first polymerase-labeled antibody comprises a polyclonal antibody, a first polymer backbone and a first enzyme;

[0010] The second multi-enzyme-labeled antibody comprises a nanobody, a second polymer skeleton and a second enzyme.

[0011] Furthermore, the preparation method of the first polymerase-labeled antibody and the second polymerase-labeled antibody comprises the following steps:

[0012] a. Preparation of multi-enzyme polymers;

[0013] b. Multi-enzyme polymer activation;

[0014] c. Activation of labeled antibodies;

[0015] d. Multi-enzyme polymer labeled antibodies.

[0016] Furthermore, the preparation steps of the multi-enzyme polymer include:

[0017] a1 preparing an activated enzyme with an active functional group; optionally, preparing the activated enzyme by glutaraldehyde cross-linking method or sodium periodate oxidation method;

[0018] a2. Adding the polymer skeleton compound to a solution containing an activated enzyme for reaction; wherein the reaction pH is 9-10, the reaction temperature is 20-30° C., and the reaction time is 2-6 hours;

[0019] a3. Add an appropriate amount of reducing agent to the reaction system, then adjust the pH with a buffer solution and dialyze overnight to obtain a multi-enzyme polymer solution;

[0020] Preferably, the reducing agent is sodium borohydride, and the buffer solution is a buffer solution containing PBS.

[0021] Furthermore, the preparation steps of the multi-enzyme polymer activation include:

[0022] b1 Weigh an appropriate amount of coupling agent, dissolve it, and add it to the enzyme polymer solution prepared in step a3, mix well, and react; wherein the reaction temperature is 20-30°C and the reaction time is 0.5-2h;

[0023] Optionally, the coupling agent is a heterobifunctional coupling agent, preferably having a molecular weight of less than 1000, such as sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sodium salt (sulfo-SMCC);

[0024] b2 Pass the activated multi-enzyme polymer solution through a desalting column to obtain the modified multi-enzyme polymer.

[0025] Furthermore, the preparation step of activating the labeled antibody includes:

[0026] c1 dissolve the antibody in buffer solution;

[0027] c2. Adding the protein modifying agent to the polyclonal antibody reaction solution, reacting for a period of time, and then passing the reaction solution through a desalting column to obtain the modified antibody;

[0028] Preferably, the protein modifying agent is a heterobifunctional coupling agent, such as 2-iminothiolane hydrochloride; and the buffer is a buffer containing PBS.

[0029] Furthermore, the preparation steps of the multi-enzyme polymer labeled antibody include:

[0030] The modified multi-enzyme polymer and the modified antibody are evenly mixed and reacted for a period of time. The reaction solution is purified by a protein purification column, and the first main peak is collected to obtain a polymerized enzyme-labeled antibody. When the antibodies are polyclonal antibodies and nanoantibodies, a first polymerized enzyme-labeled antibody (polymerized enzyme-labeled polyclonal antibody IgG) and a second polymerized enzyme-labeled antibody (polymerized enzyme-labeled nanoantibody) are prepared respectively.

[0031] Without limitation to the examples, those skilled in the art are capable of selecting desalting columns and protein purification columns from commercially available products or preparing them on their own. The desalting column can be a commercially available product, such as a PD-10 desalting column with G-25M filler; and the protein purification column can be Sephacryl S-200.

[0032] The first enzyme and the second enzyme are respectively selected from horseradish peroxidase, alkaline phosphatase or β-glucosidase.

[0033] Furthermore, the polyclonal antibody is selected from one or more of goat anti-mouse IgG, rabbit anti-mouse IgG, horse anti-mouse IgG, goat anti-rabbit IgG, horse anti-rat IgG, mouse anti-rabbit IgG, donkey anti-goat IgG, horse anti-goat IgG, mouse anti-goat IgG, rabbit anti-goat IgG and the Fab fragments, Fab' fragments and F(ab')2 fragments of the corresponding IgGs above; the nanoantibody is obtained by phage display technology; preferably, the nanoantibody is selected from one or more of mouse, rabbit or goat IgG that can target, has high affinity, strong specificity, and has no cross reaction with human IgG.

[0034] For nuclear staining of some tissue samples, a mass ratio of the first polymerase-labeled antibody to the second polymerase-labeled antibody in the composition of less than 13:1 is considered beneficial, and preferably less than 5:1. Based on the need for universality of the polymerase-labeled antibody composition for staining different samples, the mass ratio of the first polymerase-labeled antibody to the second polymerase-labeled antibody in the composition of the composition is 1:5-13:1, preferably 1:2-5:1.

[0035] Furthermore, the first enzyme and the second enzyme are directly connected to the first polymer backbone and the second polymer backbone, respectively. Preferably, the connection is through the reaction of the active functional groups on the enzyme with the active functional groups on the backbone or through 1-3 small molecules, such as alcohols, aldehydes and fatty acids, etc., to avoid the indirect connection structure being too long, resulting in an increase in the volume of the polymerase-labeled antibody, thereby weakening the penetration of the polymerase-labeled antibody. The polymer backbone is a polymer compound of polyethylene glycol or its derivatives connected to polyethyleneimine and / or its derivatives. This type of backbone material has the effect of improving the hydrophilicity of compact polymerase-labeled antibodies. Compared with other backbone materials, it can greatly reduce the volume of the immunohistochemical secondary antibody, improve its molecular mobility and penetration ability, and reduce background staining.

[0036] The polyethylene glycol derivative has a backbone or main chain of polyethylene glycol, and the backbone or main chain has branches; according to whether the end groups are modified, the polyethylene glycol structure includes but is not limited to unmodified end groups and modified end groups, and the end group modification groups include but are not limited to methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, etc.

[0037] The polyethyleneimine derivative is a polymer having a polyethyleneimine skeleton or main chain and branched chains. Preferably, the polyethyleneimine derivative is a polymer having branched chains and amino groups.

[0038] Furthermore, by connecting polyethyleneimine and / or its derivatives only to one end of polyethylene glycol or its derivatives, it is possible to avoid connecting enzymes and / or antibodies to both ends of the backbone material, thereby increasing the volume of the polymerase-labeled antibody.

[0039] For example, the polyethylene glycol-linked polyethyleneimine compound includes one of a linear type (such as shown in Formula 1) and a multi-head type (such as shown in Formulas 2 and 3).

[0040]

[0041]

[0042] in,

[0043] R1 = hydrogen, methyl, ethyl, isopropyl, tert-butyl, cyclopropyl;

[0044] n = 0-6;

[0045] q≥2;

[0046] X is a linking moiety containing a carbon atom and / or an oxygen atom, such as a carbonyl group;

[0047] m=22-900, preferably m=25-500;

[0048] j+p=2-500; preferably 2≤j+p≥250;

[0049] k=0-50, preferably 1≤k≤25.

[0050] The "linear type" refers to the case where there is no branch in the polyethyleneimine part of the skeleton (for example, as shown in Formula 1); the "multi-headed type" refers to the case where there are multiple polyethyleneimine and / or polyethyleneimine derivative parts in the skeleton (for example, as shown in Formula 3), or there is a polyethyleneimine part with an amino branch.

[0051] Optionally, the molecular weight of polyethylene glycol or its derivatives in the polymer backbone is 1,000-40,000, preferably 2,000-30,000; the molecular weight of polyethyleneimine and / or its derivatives is 100-30,000. Due to the significant steric hindrance of antibodies and enzymes, to facilitate the attachment of an appropriate amount of antibodies and enzymes to the polyethyleneimine and / or its derivatives in the backbone material, the molecular weight of the polyethyleneimine and / or its derivatives in the backbone material is preferably greater than 500, more preferably greater than 1,000. Excessively high molecular weights of the backbone material will increase the volume of the polymerized enzyme-labeled antibody complex and result in decreased penetration. Therefore, preferably, the molecular weight of the backbone material is less than 45,000, more preferably less than 35,000.

[0052] Based on the need to enhance the dyeing effect, the first polymer skeleton and the second polymer skeleton respectively contain at least 2 enzymes; preferably, the first polymer skeleton and the second polymer skeleton respectively contain at least 4 enzymes, so a multi-headed skeleton is preferred.

[0053] The connection modes between polyethylene glycol and polyethyleneimine include: carbon-nitrogen bond, amide bond, and carbon-amide bond.

[0054] Furthermore, the first polymer skeleton material and the second polymer skeleton material are of different types.

[0055] Another aspect of the present invention is the use of any of the aforementioned compositions in preparing a marker capture or marker binding product, for example, in preparing an immunohistochemical detection product; preferably, the product is a kit.

[0056] "Marker" has the commonly known meaning in the art, and refers to a biochemical substance that can mark changes or possible changes in the structure or function of systems, organs, tissues, cells and subcellular structures, and has the function of indicating the physiological state of an organism. Based on its natural properties, it can be divided into nucleic acids (such as DNA and RNA), proteins, sugars and related derivatives or inorganic substances, etc.

[0057] The applicant believes that, based on the current state of the art and the content verified in this application, the polymerase-labeled antibody composition and preparation method provided by the present invention have the following advantages:

[0058] (1) Based on the optimization of the types of backbone materials, coupling agents, and preparation methods, the polymerase-labeled antibody of the present invention is prepared under mild conditions and avoids the use of highly toxic raw materials, making the preparation process safer and more environmentally friendly.

[0059] (2) The present invention simultaneously uses a mix of polyantibodies and nanoantibodies as raw materials for the preparation of enzyme-labeled secondary antibodies, and matches polymer enzyme-labeled antibodies of different sizes to different positioning antigens. On the one hand, it fully utilizes the sensitivity of polyantibodies, and on the other hand, it takes advantage of the small size of nanoantibodies to reduce the volume of immunohistochemical secondary antibodies, thereby improving their molecular mobility and penetration ability, improving staining intensity and sensitivity, and improving the penetration of secondary antibodies for the cell nuclear membrane. Therefore, the composition of the present invention can also have a significant staining and marking effect on nuclear staining areas and non-nuclear staining areas, overcoming the problem that polymer enzyme-labeled antibodies show uneven performance when detecting different types of antigens, thereby improving the universality of the product.

[0060] (3) The polymerase composition provided by the present invention uses a polymer compound of polyethylene glycol linked to polyethyleneimine as a skeleton, so that the enzyme molecules are tightly connected to form a compact polymerase composition structure, which greatly reduces the volume of the polymerase, improves the penetration of the polymerase-labeled antibody and the stability of the polymerase.

[0061] (4) Hydrophilic polyethylene glycol is used as a backbone component to modify the polymerase-labeled antibody complex to improve its water solubility, thereby reducing polymerase aggregation and the staining background caused by hydrophobic effects during tissue section staining.

[0062] Figures in the specification

[0063] Figure 1 Schematic diagram of commonly used enzyme-labeled secondary antibody detection systems in immunohistochemistry; Figure A shows traditional enzyme-labeled antibodies, Figure B shows the enzyme-labeled streptavidin-biotin staining method (SABC method), and Figure C shows the polymer-enzyme-labeled secondary antibody method.

[0064] Figure 2 Schematic diagram of the structure of the polymerase-labeled antibody of the present invention.

[0065] Figure 3 Comparison of immunohistochemical staining results of the polymerase-labeled antibody composition and the polymerase-labeled nanoantibody; Figure A is the staining result of the polymerase-labeled antibody composition 1a, and Figure B is the staining result containing only the polymerase-labeled nanoantibody.

[0066] Figure 4 Comparison of immunohistochemical staining results of polymerase-labeled antibody compositions with different compositions; Figure A is the staining result of the polymerase-labeled antibody composition 2a, and Figure B is the staining result containing only the polymerase-labeled multi-antibody reagent.

[0067] Figure 5 Comparison of immunohistochemical staining results of polymerase-labeled antibody compositions made of different skeleton materials; Figure A shows the staining results of the polymerase-labeled antibody composition 1a, and Figure B shows the staining results of the polymerase-labeled antibody composition 2a.

[0068] Figure 6 Comparison of immunohistochemical staining results of polymeric enzyme-labeled antibody compositions with different skeleton materials; Figure A is the staining result of polymeric enzyme-labeled antibody composition 5, Figure B is the staining result of polymeric enzyme-labeled antibody composition 6, and Figure C is the staining result of polymeric enzyme-labeled antibody composition 7.

[0069] Figure 7 Comparative images of immunohistochemical staining results of the polymerase-labeled antibody composition and the commercially available product Novolink RE7200-K in different tissue samples; the upper image shows the staining results of the polymerase-labeled antibody composition described in the present invention, and the lower image shows the staining results of the commercially available product.

[0070] Figure 8 Comparison of immunohistochemical staining results of polymerase-labeled antibody compositions prepared with different coupling agents; Figure A shows the staining results of polymerase-labeled antibody composition 1a, and Figure B shows the staining results of polymerase-labeled antibody composition 8. DETAILED DESCRIPTION

[0071] The present invention will be further explained with reference to specific examples. Where specific techniques or conditions are not specified in the examples, the operations were performed in accordance with conventional techniques and instrument specifications in the art. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially or developed independently.

[0072] Preparation Example 1

[0073] The preparation of the polymerase-labeled antibody composition comprises the following steps:

[0074] 1. Preparation of multi-enzyme polymer:

[0075] (1) Weigh 10 mg of horseradish peroxidase, add 2 mL of pure water, and mix thoroughly to dissolve. Then weigh 10 mg of sodium periodate and add it to the reaction solution. Incubate at 25°C in the dark for 1 hour.

[0076] (2) Add 400 μL of ethylene glycol solution to the reaction solution and incubate at 4°C in the dark for 1 h. Then dialyze the reaction solution against 2 mM sodium acetate buffer (pH 4.1) overnight.

[0077] (3) Weigh 10 mg of the skeleton material and add it to the activated enzyme solution after dialysis. Then, use sodium carbonate solution to adjust the pH value of the reaction solution to 9.5, and place the reaction solution at 25°C for 4 hours.

[0078] (4) Weigh 2 mg of sodium borohydride and add it to the reaction system. Incubate at 4°C for 1 h to terminate the reaction. Then, dialyze overnight with 10 mM PBS (pH 7.4) to obtain the multi-enzyme polymer, which is then stored at 4°C until use.

[0079] 2. Multi-enzyme polymer activation:

[0080] (1) Weigh 1 mg of sulfo-SMCC and dissolve it in 1 mL of pure water. Add 500 μL of the sulfo-SMCC solution to the enzyme-polymer solution prepared in step 1, mix well, and incubate at 25°C for 1 h.

[0081] (2) The activated multi-enzyme polymer solution is passed through a PD-10 desalting column to obtain a maleimide-modified multi-enzyme polymer.

[0082] 3. Activation of labeled antibodies:

[0083] (1) Take 150 μL of polyclonal IgG (antibody content 10 mg / mL) and add a certain volume of 100 mM PBS (containing 5 mM EDTA, pH 8.0) solution to make the final antibody concentration 3 mg / mL.

[0084] (2) Weigh 0.1 mg of 2-iminothiolane hydrochloride and add it to the above polyclonal antibody reaction solution. Incubate at 25°C for 1 hour. Then, pass the reaction solution through a PD-10 desalting column to obtain the thiol-modified polyclonal antibody IgG.

[0085] Referring to the above steps, the polyclonal antibody was replaced with the nanobody to prepare the thiol-modified nanobody.

[0086] 4. Multi-enzyme polymer labeled antibodies:

[0087] (1) Take 5 mg of maleimide-modified multi-enzyme polymer and the thiol-modified multi-antibody IgG prepared in step 3 and mix them evenly. Incubate at 25°C for 1 hour. Purify the reaction solution through a Sephacryl S-200 protein purification column. Use 10 mM PBS (pH 7.4) as the equilibration buffer. Collect the first main peak to obtain the first multi-enzyme-labeled antibody (multi-enzyme-labeled multi-antibody IgG).

[0088] (2) 5 mg of maleimide-modified multi-enzyme polymer was mixed evenly with the thiol-modified nanobody prepared in step 3 and allowed to react at 25°C for 1 h. The reaction solution was purified by Sephacryl S-200 protein purification column using 10 mM PBS (pH 7.4) as the equilibration buffer. The first main peak was collected to obtain the second multi-enzyme-labeled antibody (multi-enzyme-labeled nanobody).

[0089] 5. Multi-enzyme polymer labeled antibody composition:

[0090] (1) Take appropriate amounts of polymerase-labeled antibody IgG and polymerase-labeled nanobody, and prepare them with PBS buffer (containing 1% BSA and 0.05% Proclin 300, pH 7.4) to prepare a liquid with a polymerase-labeled antibody solid content of 1 μg / ml. Mix as needed and store at 2-8°C for later use.

[0091] Among them, the selection of framework materials, polyclonal antibodies, nano-monoclonal antibodies and the composition information of their combinations in steps 1-5 are shown in Table 1.

[0092] Table 1 Composition information of polymerase-labeled polyclonal antibody composition

[0093]

[0094]

[0095] Among them, mPEG2k-g-PEI2k (PEG molecular weight 2k, PEI molecular weight 2k), mPEG10k-g-PEI1k (PEG molecular weight 10k, PEI molecular weight 1k), mPEG15k-g-PEI30k (PEG molecular weight 15k, PEI molecular weight 30k) and mPEG10k-g-PEI25k (PEG molecular weight 10k, PEI molecular weight 25k) are multi-head skeletons purchased from "Carbohydrate Technology" company.

[0096] PPE-104 (PEG molecular weight 5k, PEI molecular weight 4k) and PPE-101 (PEG molecular weight 2k, PEI molecular weight 4k) are linear backbones purchased from Creative PEGWorks.

[0097] Boltorn™ H40 was purchased from Polymer Factory.

[0098] Both polyclonal antibodies and nanoantibodies are independently developed by Chongqing Aisens Bioengineering Co., Ltd.

[0099] Preparation Example 2

[0100] Composition 8 was prepared according to the preparation method of composition 1a, except that bisaminopolyethylene glycol (NH2-PEG-NH2) with a polymerization degree of 24 was used instead of sulfo-SMCC as the coupling agent for the multi-enzyme polymer and the antibody.

[0101] Preparation Example 3

[0102] The specific steps of the standard operating procedure for manual immunohistochemistry are as follows:

[0103] (1) Slide baking: Select the corresponding paraffin-embedded tissue slices and bake them on a slide baking machine at 65°C for 30 minutes.

[0104] (2) Dewaxing and rehydration: Soak the sections in xylene for 10 min, then replace xylene and soak for another 10 min; soak the sections in anhydrous ethanol for 5 min, then replace anhydrous ethanol and soak for another 5 min; soak the sections in 95% ethanol for 5 min; soak the sections in 85% ethanol for 5 min; soak the sections in 75% ethanol for 5 min; soak in ultrapure water for 5 min.

[0105] (3) Antigen retrieval: Add pH 9.0 EDTA retrieval solution to a stainless steel pot and immerse the sections in it. Heat the water on an induction cooker until boiling and maintain for 20 min. Turn off the heat, transfer the stainless steel pot to a sink, rinse with running water, and cool to room temperature.

[0106] (4) Blocking: Rinse the sections with PBS buffer and spin dry the PBS buffer. Add 0.1 mL of endogenous peroxidase blocking agent to the tissue and incubate at room temperature for 10 min. Then rinse the sections with PBS buffer.

[0107] (5) Primary antibody incubation: Shake off the PBS buffer and add 0.1 mL of the corresponding primary antibody to the tissue, ensuring that the primary antibody solution covers the entire tissue. Incubate at room temperature for 1 hour. Then rinse the sections with PBS buffer.

[0108] (6) Post-primary antibody incubation: Shake off the PBS buffer and add 0.1 mL of the corresponding secondary antibody A reagent to the tissue, ensuring that the primary antibody solution covers the entire tissue. Incubate at room temperature for 15 minutes. Then rinse the sections with PBS buffer.

[0109] (7) Secondary antibody incubation: Shake off the PBS buffer and add 0.1 mL of the corresponding secondary antibody polymer reagent to the tissue, ensuring that the secondary antibody solution covers the entire tissue. Incubate at room temperature for 15 minutes. Then rinse the sections with PBS buffer.

[0110] (8) DAB color development: Shake off the PBS buffer and add 0.1 mL of DAB color development solution to the tissue, ensuring that the DAB color development solution covers the entire tissue. Incubate at room temperature for 5 minutes. Then rinse the sections with deionized water.

[0111] (9) Backstaining: Shake off the deionized water. Add 0.1 mL of dye solution to the tissue, ensuring that the dye solution covers the entire tissue. Incubate at room temperature for 5 minutes. Then rinse the sections with deionized water.

[0112] (10) Dehydration, transparency, and mounting: Soak the sections in 75% ethanol for 3 minutes; soak the sections in 95% ethanol for 3 minutes; soak the sections in anhydrous ethanol for 3 minutes, then replace the anhydrous ethanol and soak for another 3 minutes; soak the sections in xylene for 5 minutes, then replace the xylene and soak for another 5 minutes. Remove the sections from the solvent, add a drop of neutral resin to the tissue, cover with a coverslip, and place in a fume hood to air-dry overnight.

[0113] (11) Observe the tissue morphology under a microscope and determine the staining results.

[0114] Effect verification example

[0115] To verify the effect of the polymerase-labeled antibody composition of the present invention, an appropriate primary antibody was selected according to the type of secondary antibody, and immunohistochemistry tests and effect comparisons were performed on the polymerase-labeled antibodies and compositions prepared in Examples 1 and 2 and commercially available products according to the experimental steps described in Example 3.

[0116] Effect Example 1

[0117] The polymer enzyme-labeled antibody composition 1a prepared in Example 1 and the polymer enzyme-labeled nanoantibody corresponding to the composition 1a were used as working solutions and compared on the basis of the same solid content. In this experiment, formalin-fixed and paraffin-embedded human intestinal cancer tissue was used, and the cytoplasmic expressed protein DOG1 was selected as the primary antibody indicator to be tested. The staining results are shown in Figure 2. Figure 3 As shown, since the polymerase-labeled antibody composition of the present invention contains both a polymerase-labeled antibody structure and a small-volume polymerase-labeled nanoantibody, the staining uniformity and staining depth are significantly better than those of the polymerase-labeled nanoantibody used alone, indicating that the polymerase-labeled antibody composition of the present invention has a better staining effect on some tissue samples and is more universal.

[0118] Effect Example 2

[0119] To demonstrate the effect of the composition of the polymerase-labeled antibody composition on tissue sample staining, the polymerase-labeled antibody composition 2a, composition 2b, composition 2c prepared in Example 1 and the polymerase-labeled antibody corresponding to composition 2a were used as working solutions and compared on the basis of the same solid content. In this experiment, formalin-fixed and paraffin-embedded human lung squamous cell carcinoma tissue samples were used, and the nuclear expressed protein P63 was selected as the primary antibody indicator to be tested. The staining results are shown in Figure 2. Figure 4 As shown in FIG, due to the high density of the cell nucleus, it is difficult for macromolecular substances to enter, so the staining effect of the cell nucleus expressed protein gradually weakens as the content of the polymerase-labeled nanobody in the polymerase-labeled antibody composition decreases, among which the composition 2a has the best staining effect ( Figure 4 A); When the mass ratio of polymerase-labeled polyantibody to polymerase-labeled nanoantibody in the composition is greater than 15:1, the staining effect is the same as the staining effect of the working solution containing only polymerase-labeled polyantibody ( Figure 4 Based on the consideration of staining effectiveness, the mass ratio of the polymerase-labeled polyantibody to the polymerase-labeled nanobody in the polymerase-labeled antibody composition is less than 13:1, preferably less than 5:1.

[0120] Effect Example 3

[0121] In order to prove the influence of the type, molecular weight and structure of the skeleton material on the actual application effect, the polymerase-labeled antibody composition 1a, composition 2a, composition 3, composition 4 and composition 6 prepared in Example 1 were used as working solutions and compared on the basis of the same solid content. In this experiment, formalin-fixed and paraffin-embedded human lung squamous cell carcinoma tissue samples were used, and the nuclear expressed protein P63 was selected as the primary antibody index to be tested. By comparing the staining results of the tissue samples, they were ranked from best to worst according to the staining effect: composition 1a, composition 2a, composition 6, composition 3 and composition 4. Among them, the hydrophilicity of the polymerase-labeled antibody skeleton in composition 1a is higher than that of the polymerase-labeled antibody skeleton in composition 2a, the background staining is reduced, and the staining depth is further increased (such as Figure 5 (As shown). The polyethylenimine moieties in the backbones of compositions 1a and 2a are multi-headed, allowing for the attachment of more enzymes and secondary antibodies, resulting in better staining than composition 6, which contains a linear polymerase-labeled antibody. Composition 3 still stained tissue samples and achieved effective resolution, but the molecular weight of the polymerase-labeled antibody backbone in composition 4 was too large, and the polyethylenimine moiety was overloaded with amino-coupled enzymes and secondary antibodies, resulting in a weakened staining effect.

[0122] Therefore, the present invention uses polyethylene glycol-linked polyethyleneimine polymer compounds as the backbone material of polymerase-labeled antibodies to effectively reduce background staining and improve the staining effect. The use of multi-headed polyethyleneimine backbone materials can further improve the staining effect. Further considering the staining effect and the application scenarios of polymerase-labeled antibodies, the backbone molecular weight should be controlled below 45,000, preferably below 35,000.

[0123] Effect Example 4

[0124] To further demonstrate the beneficial effects of the skeleton material selection of the present invention, the polymerase-labeled antibody compositions 5, 6, and 7 prepared in Example 1 were used as working solutions and compared on the basis of the same solid content. In this experiment, formalin-fixed and paraffin-embedded human lung squamous cell carcinoma tissue samples were used, and the nuclear expressed protein P63 was selected as the primary antibody indicator to be tested. The staining results are shown in Figure 2. Figure 6 As shown, the staining effect of the polymerase-labeled antibody composition comprising the backbone of the present invention ( Figure 6 A- Figure 6 B) is significantly better than composition 7 ( Figure 6 C), further proving that the skeleton material of the present invention has a promoting effect on the tissue staining effect of the polymerase-labeled antibody composition.

[0125] Effect Example 5

[0126] In order to evaluate the specificity and sensitivity of the prepared polymer enzyme-labeled antibody composition in immunohistochemistry experiments. We used the polymer enzyme-labeled antibody composition 1a prepared in Example 1 for the experiment, and used a commercially available polymer kit containing anti-rabbit IgG polymerized HRP (Novolink RE7200-K, Cell IDx) as a control sample. In this experiment, formalin-fixed and paraffin-embedded tissue samples, including human tonsil tissue and human breast tissue, were used, and CD5, CK8&18 and P53 were selected as the primary antibodies to be tested. Figure 7 As shown, under the same experimental conditions, the polymerase-labeled antibody composition prepared by the present invention exhibited significantly stronger staining intensities for CD5 and CK8&18 primary antibodies than the control sample, and both exhibited comparable effects in staining for P53 primary antibody. This further demonstrates that the polymerase-labeled antibody composition of the present invention has good universal applicability and an overall superior effect to similar products.

[0127] Effect Example 6

[0128] From the above verification results, it can be seen that the polymerase-labeled antibody composition prepared by the method of the present invention has unexpected technical effects in terms of staining and universality, and no highly toxic coupling reagents are used in the preparation process. Composition 1a and composition 8 were further used as working solutions to evaluate the effect of coupling agent selection. In this experiment, formalin-fixed and paraffin-embedded human tissue samples were used, and the nuclear expression protein Ki-67 was selected as the primary antibody indicator to be tested. The staining results are shown in Figure 2. Figure 8 As shown in the figure, on the basis of meeting the spatial position requirements of macromolecules, sulfo-SMCC has a shorter molecular chain than bis-amino polyethylene glycol, the formed multi-enzyme-labeled antibody polymer is smaller in size, and the staining effect is more prominent.

[0129] Through the above experiments and verifications, it can be seen that the conditions used in the preparation method of the polymerase-labeled antibody composition of the present invention are mild, there are no toxic raw materials, and the production is safer; the polymerase-labeled antibody made of a special skeleton is more hydrophilic and smaller in size, and has a better staining effect; at the same time, the use of a combination of polymerase-labeled polyantibody and polymerase-labeled nanoantibody improves the staining effect and the universality of the product.

[0130] It should also be noted that the exemplary embodiments described herein describe methods or systems based on a series of steps or devices. However, the present invention is not limited to the order of the steps described above. In other words, the steps may be performed in the order described in the embodiments, or in a different order, or several steps may be performed simultaneously.

[0131] In the present invention, features described and / or illustrated for one embodiment may be used in the same or similar manner in one or more other embodiments, and / or combined with or replace features of other embodiments.

[0132] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a polymerase-labeled antibody composition, characterized in that: The following steps are involved: Polymerase-labeled antibodies Preparation: prepare the first polymerase-labeled antibody and the second polymerase-labeled antibody respectively; Mixing the first polymerase-labeled antibody, the second polymerase-labeled antibody and a buffer to obtain a polymerase composition; Wherein, the first polymerase-labeled antibody comprises a polyclonal antibody, a first polymer backbone and a first enzyme; The second multi-enzyme labeled antibody comprises a nanobody, a second polymer backbone and a second enzyme; The first polymer skeleton and the second polymer skeleton are polymer compounds of polyethylene glycol connected to polyethylene imine; the molecular weights of the first polymer skeleton and the second polymer skeleton are less than 45,000; The mass ratio of the first polymerase-labeled antibody to the second polymerase-labeled antibody in the composition is 1:5-13:

1.

2. The preparation method according to claim 1, characterized in that The preparation method of the polymerase-labeled antibody comprises: a. Preparation of multi-enzyme polymers: a1 Preparation of activated enzyme with active functional groups; a2. Adding the polymer skeleton compound to a solution containing an activated enzyme for reaction; a3. Add an appropriate amount of reducing agent to the reaction system, adjust the pH with a buffer solution, and dialyze overnight to obtain a multi-enzyme polymer solution; b. Multi-enzyme polymer activation: b1 Weigh an appropriate amount of coupling agent, dissolve it, and add it to the multi-enzyme polymer solution prepared in step a3, mix well, and react; b2 passing the activated multi-enzyme polymer solution through a desalting column to obtain the modified multi-enzyme polymer; c. Activation of labeled antibodies: c1 dissolve the polyclonal antibody in buffer solution; c2. Add the protein modifier to the polyclonal antibody reaction solution, react for a period of time, and then pass the reaction solution through a desalting column to obtain the modified polyclonal antibody; d. Multi-enzyme polymer labeled antibodies: The modified multi-enzyme polymer and the modified multi-antibody are mixed evenly and reacted for a period of time. The reaction solution is purified by a protein purification column, and the first main peak is collected to obtain the first multi-enzyme-labeled antibody; e. Referring to the method of steps ad, a second polymerase-labeled antibody containing a nanobody was prepared by replacing the polyclonal antibody with a nanobody.

3. The preparation method according to claim 2, characterized in that The activated enzyme with active functional groups is prepared by a glutaraldehyde cross-linking method or a sodium periodate oxidation method.

4. The preparation method according to claim 2 or 3, characterized in that The coupling agent is selected from heterobifunctional coupling agents.

5. The preparation method according to claim 2 or 3, characterized in that The coupling agent is sulfo-SMCC.

6. The preparation method according to any one of claims 1 to 3, characterized in that In the polyethylene glycol-connected polyethylene imine polymer compound, polyethylene imine is connected only at one end of the polyethylene glycol.

7. The preparation method according to any one of claims 1 to 3, characterized in that The first polymer skeleton and the second polymer skeleton each contain at least two enzymes.

8. The preparation method according to any one of claims 1 to 3, characterized in that The first polymer skeleton and the second polymer skeleton each contain at least four enzymes.

9. The preparation method according to any one of claims 1 to 3, characterized in that The mass ratio of the first polymerase-labeled antibody to the second polymerase-labeled antibody in the composition is 1:2-5:

1.

10. A polymerase-labeled antibody composition, characterized in that: The composition is prepared by the method according to any one of claims 1 to 9.

11. Use of the composition according to claim 10 in preparing a marker capture or marker binding product.

12. The use according to claim 11, characterized in that The product is a test kit.

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