A polymerized enzyme-labeled antibody composition and its application

Abstract

The present invention provides a composition containing polymerase-labeled polyantibodies and polymerase-labeled anti-nanoantibodies, wherein polymerase-labeled antibody compositions of different sizes are matched to different localized antigens, thereby improving the penetration of secondary antibodies into cell nuclear membranes and overcoming the problems of unevenness and low universality exhibited by different types of antigen detection; and polyethylene glycol-connected polyethyleneimine compounds are selected as skeleton materials, and by optimizing the selection of skeleton materials, the use of highly toxic raw materials is avoided, the safety of product production is improved, the hydrophilicity of polymerase-labeled antibodies is improved, background staining is effectively reduced, and the volume of immunohistochemical secondary antibodies is reduced, thereby further improving their molecular mobility and penetration capabilities.

Description

Technical Field

[0001] The present invention relates to the technical field of immunohistochemical detection, and in particular to a polymerase-labeled antibody composition and application thereof. Technical Background

[0002] As the "gold standard" for tumor diagnosis, pathological diagnosis can study the etiology, pathogenesis, morphological structure, function and metabolic changes of the disease through a series of treatments and observations of human tissues or cells, reveal the occurrence and development of the disease, and thus clarify the nature of the disease. Histopathology and cytopathology are two basic methods of pathological diagnosis, which mainly conduct morphological observations. Combined with immune diagnosis or molecular diagnosis, pathological diagnosis has formed two other important branches: immunohistochemical pathology and molecular pathology, which deepen pathological diagnosis from morphological observations at the tissue and cell levels to protein and molecular levels. Among them, immunohistochemistry technology has the advantages of fast detection speed, low cost, and the ability to analyze paraffin-embedded samples and frozen sections, and is the most widely used in clinical practice. Immunohistochemistry can provide objective evidence at the protein expression level, and is widely used in judging the benign and malignant nature of tumors, determining the source of tumor cells, differentially diagnosing tumor types or subtypes, tumor differentiation direction, prognosis judgment, targeted therapy, and the discovery and determination of 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 requirements of detection sensitivity, a variety of secondary antibody detection systems have been developed and applied. For example, the early enzyme-labeled streptavidin-biotin staining method (SABC method) has a detection principle such as Figure 1 As shown in Figure B, this method is based on the principle of "high affinity between biotin and streptavidin". Through the combination of "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 biotin that is 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, i.e., the enzyme-labeled polymer staining method, is widely used ( Figure 1C). This method is to label the enzyme on the main chain with inert glucan as the skeleton to form a glucanase composition. Multiple enzyme molecules and antibody molecules can be connected to each glucan main chain. The system can simultaneously connect secondary antibodies with different resistances, thereby specifically binding to primary antibodies from rabbit or mouse. It effectively avoids nonspecific staining caused by endogenous biotin, and its sensitivity is comparable to that of enzyme-labeled streptavidin-biotin staining, so this method is the mainstream method for clinical application at present. However, the molecular weight of the glucan skeleton used in this type of secondary antibody is relatively large. Although multiple enzyme and antibody molecules are connected at the same time, its structure is loose and the hydrodynamic volume is relatively large, thus forming a large steric hindrance, which makes the polymer have low penetration of the cell nuclear membrane and poor nuclear staining effect.

[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, and has better nuclear staining effect. A polymer with too small a volume means faster molecular movement and worse sensitivity. For immunohistochemical staining such as cell staining and membrane staining, the effective binding rate of a polymer with too small a volume and 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, using compact dendritic polymers as the skeleton, overcoming the main defects of the current polymer enzyme labeling method using chain polymers (such as dextran or polypeptide) 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 many 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 new type of compact polymerase-labeled antibody composition, preparation method and application thereof. The polymerase-labeled antibody composition of the present invention adopts the form of a combination of polyantibodies and nanoantibodies, and matches polymerase-labeled antibody compositions of different sizes for different localization antigens, thereby improving the penetration of the secondary antibody for the cell nuclear membrane, while overcoming the unevenness and universality problems shown by different types of antigen detection. The present invention further avoids the use of highly toxic raw materials and improves the safety of product production by optimizing the selection of skeleton materials. Furthermore, the skeleton of the polymerase-labeled antibody of 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 composition containing a compact polymerase-labeled antibody, the composition comprising:

[0007] A first polymerase-labeled antibody, wherein the first polymerase-labeled antibody comprises a polyclonal antibody, a first polymer skeleton and a first enzyme;

[0008] A second polymer enzyme-labeled antibody, wherein the second polymer enzyme-labeled antibody comprises a nanobody, a second polymer backbone, and a second enzyme;

[0009] Wherein, the first enzyme and the second enzyme are selected from horseradish peroxidase, alkaline phosphatase or β-glycosidase respectively.

[0010] Furthermore, the polyantibody 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, F(ab')2 fragments of the above corresponding IgGs; 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 be targeted, has high affinity, strong specificity, and has no cross reaction with human IgG.

[0011] Furthermore, for the needs of nuclear staining of some samples, it is considered beneficial that the mass ratio of the first polymerase-labeled antibody to the second polymerase-labeled antibody in the composition is less than 13:1, preferably less than 5:1. Based on the 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 is 1:5-13:1, preferably 1:2-5:1.

[0012] Further, the first enzyme and the second enzyme are directly connected to the first polymer skeleton and the second polymer skeleton respectively. Preferably, the connection method is through the reaction of the active functional group on the enzyme with the active functional group on the skeleton or through 1-3 small molecules, such as alcohols, aldehydes and fatty acids, etc., to avoid the presence of indirect connection structure will increase the volume of polymerase-labeled antibodies, thereby weakening the penetration of polymerase-labeled antibodies. The polymer skeleton is a polymer compound in which polyethylene glycol or its derivatives are connected to polyethyleneimine and / or its derivatives. This type of skeleton material has the effect of improving the hydrophilicity of compact polymerase-labeled antibodies. Compared with other skeleton materials, it can greatly reduce the volume of immunohistochemical secondary antibodies, improve their molecular mobility and penetration ability, and reduce background staining.

[0013] The polyethylene glycol derivative has a polyethylene glycol skeleton or main chain, and the skeleton or main chain has branches. Depending on whether the end groups are modified, the polyethylene glycol structure includes but is not limited to unmodified end groups and modified end groups. The end group modification groups include but are not limited to methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, etc.

[0014] The polyethyleneimine derivative is a polymer having a polyethyleneimine skeleton or main chain and having branches in the skeleton or main chain; preferably, the polyethyleneimine derivative is a type having branches and amino groups.

[0015] Furthermore, by connecting polyethyleneimine and / or its derivatives only at one end of polyethylene glycol or its derivatives, it is possible to avoid connecting enzymes and / or antibodies at both ends of the backbone material, thereby increasing the volume of the polymerase-labeled antibody.

[0016] 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).

[0017]

[0018]

[0019] in,

[0020] R1 = hydrogen, methyl, ethyl, isopropyl, tert-butyl, cyclopropyl;

[0021] n = 0-6;

[0022] q≥2;

[0023] X is a linking moiety containing a carbon atom and / or an oxygen atom, such as a carbonyl group;

[0024] m=22-900, preferably m=25-500;

[0025] j+p=2-500; preferably 2≤j+p≥250;

[0026] k=0-50, preferably 1≤k≤25.

[0027] The "linear type" refers to the situation where there is no branch in the polyethyleneimine part of the skeleton; the "polyheaded type" refers to the situation where there are multiple polyethyleneimine and / or polyethyleneimine derivative parts (such as shown in Formula 3) in the skeleton, or there is a polyethyleneimine part with an amino branch.

[0028] Optionally, the molecular weight of polyethylene glycol or its derivatives in the polymer skeleton is 1000-40000, preferably 2000-30000; the molecular weight of polyethyleneimine and / or its derivatives is 100-30000. Due to the large steric hindrance of antibodies and enzymes, in order to facilitate the connection of appropriate amounts of antibodies and enzymes to the polyethyleneimine and / or its derivatives in the skeleton material, the molecular weight of the polyethyleneimine and / or its derivatives in the skeleton material is preferably greater than 500, more preferably greater than 1000. If the molecular weight of the skeleton material is too large, the volume of the polymerized enzyme-labeled antibody complex will be larger, resulting in decreased penetration. Therefore, preferably, the molecular weight of the skeleton material is less than 45000, more preferably less than 35000.

[0029] 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-head skeleton is preferred.

[0030] The connection modes of polyethylene glycol and polyethyleneimine include: carbon-nitrogen bond, amide bond, and carbon-amide bond.

[0031] Another aspect of the present invention is the use of any of the aforementioned compositions in the preparation of a marker capture or marker binding product, for example, in the preparation of an immunohistochemical detection product; preferably, the product is a kit.

[0032] "Marker" 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 organisms. Based on their natural properties, they can be divided into nucleic acids (such as DNA and RNA), proteins, sugars and related derivatives or inorganic substances.

[0033] 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 the preparation method thereof provided by the present invention have the following advantages:

[0034] (1) The present invention simultaneously uses a mixture of polyantibodies and nanoantibodies as raw materials for preparing enzyme-labeled secondary antibodies, and matches polymer enzyme-labeled antibodies of different sizes to different localized antigens. On the one hand, the sensitivity of polyantibodies is fully utilized, and on the other hand, the volume of immunohistochemical secondary antibodies is reduced by taking advantage of the small size of nanoantibodies, thereby improving their molecular mobility and penetration ability, 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.

[0035] (2) 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 size of the polymerase and improves the penetration of the polymerase-labeled antibody and the stability of the polymerase.

[0036] (3) 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 staining background caused by hydrophobic effects during tissue section staining.

[0037] (4) Based on the optimized selection of the backbone material, the preparation conditions of the polymerase-labeled antibody of the present invention are mild and the use of highly toxic raw materials is avoided, making the preparation process safer and more environmentally friendly.

[0038] Instruction Manual

[0039] Figure 1 Schematic diagram of commonly used enzyme-labeled secondary antibody detection systems for immunohistochemistry; Figure A is the traditional enzyme-labeled antibody, Figure B is the enzyme-labeled streptavidin-biotin staining method (SABC method), and Figure C is the polymer-enzyme-labeled secondary antibody method.

[0040] Figure 2 Schematic diagram of the structure of the polymerase-labeled antibody of the present invention.

[0041] Figure 3 Comparison of immunohistochemical staining results of polymerase-labeled antibody composition and polymerase-labeled nanoantibody; wherein, Figure A is the staining result of polymerase-labeled antibody composition 1a, and Figure B is the staining result containing only polymerase-labeled nanoantibody.

[0042] Figure 4 Comparison of immunohistochemical staining results of polymerase-labeled antibody compositions with different compositions; wherein, 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.

[0043] Figure 5 Comparison of immunohistochemical staining results of polymerase-labeled antibody compositions of different skeleton materials; wherein, Figure A is the staining result of polymerase-labeled antibody composition 1a, and Figure B is the staining result of polymerase-labeled antibody composition 2a.

[0044] Figure 6 Comparison of immunohistochemical staining results of polymerase-labeled antibody compositions of different skeleton materials; wherein, Figure A is the staining result of polymerase-labeled antibody composition 5, Figure B is the staining result of polymerase-labeled antibody composition 6, and Figure C is the staining result of polymerase-labeled antibody composition 7.

[0045] Figure 7Comparative pictures of the staining results of the polymerase-labeled antibody composition and the commercially available product Novolink RE7200-K in different tissue samples; wherein, the upper picture is the staining result of the polymerase-labeled antibody composition of the present invention, and the lower picture is the staining result of the commercially available product. DETAILED DESCRIPTION

[0046] The present invention is further explained in conjunction with specific examples. If no specific techniques or conditions are specified in this example, the operation is carried out according to the conventional technical methods and instrument instructions in the art. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased from the market or obtained through self-development.

[0047] Preparation Example 1

[0048] The preparation of the polymerase-labeled antibody composition comprises the following steps:

[0049] 1. Preparation of multi-enzyme polymer:

[0050] (1) Weigh 10 mg of horseradish peroxidase, add 2 mL of pure water, mix well to fully dissolve. Then weigh 10 mg of sodium periodate and add it to the above reaction solution. React at 25°C in the dark for 1 hour.

[0051] (2) 400 μL of ethylene glycol solution was added to the reaction solution, and the solution was placed at 4°C in the dark for 1 h. The reaction solution was then dialyzed against 2 mM sodium acetate buffer (pH 4.1) overnight.

[0052] (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.

[0053] (4) Weigh 2 mg of sodium borohydride and add it to the reaction system, and place it at 4°C for 1 hour to terminate the reaction. Then dialyze it overnight using 10 mM PBS (pH 7.4) to obtain a multi-enzyme polymer, which is then placed at 4°C for later use.

[0054] 2. Multi-enzyme polymer activation:

[0055] (1) Weigh 1 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (sulfo-SMCC) and add 1 mL of pure water to dissolve. Add 500 μL of sulfo-SMCC solution to the enzyme polymer solution prepared in step 1, mix well, and place at 25°C for 1 h.

[0056] (2) The activated multi-enzyme polymer solution is passed through a PD-10 desalting column to obtain a maleimide-modified multi-enzyme polymer.

[0057] 3. Activation of labeled antibodies:

[0058] (1) Take 150uL of polyclonal IgG (antibody content 10mg / mL) and add a certain volume of 100mM PBS (containing 5mM EDTA, pH 8.0) solution to make the final antibody concentration 3mg / mL.

[0059] (2) Weigh 0.1 mg of 2-iminothiolane hydrochloride and add it to the above polyclonal antibody reaction solution, and place it at 25°C for 1 hour. Then, the reaction solution is passed through a PD-10 desalting column to obtain thiol-modified polyclonal antibody IgG.

[0060] Referring to the above steps, the polyantibody was replaced with the nanobody to prepare the thiol-modified nanobody.

[0061] 4. Multi-enzyme polymer labeled antibodies:

[0062] (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, and place them at 25°C for 1 hour. The reaction solution is purified by Sephacryl S-200 protein purification column, and the equilibrium buffer is 10mM PBS (pH7.4). The first main peak is collected to obtain the first multi-enzyme-labeled antibody (multi-enzyme-labeled multi-antibody IgG).

[0063] (2) Take 5 mg of maleimide-modified multi-enzyme polymer and mix it evenly with the thiol-modified nano-antibody prepared in step 3, and place it at 25°C for 1 hour. The reaction solution is purified by Sephacryl S-200 protein purification column, and the equilibrium buffer is 10mM PBS (pH 7.4). The first main peak is collected to obtain the second polymerase-labeled antibody (polymerase-labeled nano-antibody).

[0064] 5. Multi-enzyme polymer labeled antibody composition:

[0065] (1) Take appropriate amounts of polymerase-labeled antibody IgG and polymerase-labeled nanoantibody, respectively, 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.

[0066] 6. Multi-enzyme polymer labeled antibody composition:

[0067] (1) Take appropriate amounts of polymerase-labeled antibody IgG and polymerase-labeled nanoantibody, respectively, 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.

[0068] 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.

[0069] Table 1 Composition information of polymerase-labeled polyclonal antibody composition

[0070]

[0071]

[0072] 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.

[0073] 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".

[0074] Boltorn™ H40 was purchased from Polymer Factory.

[0075] Both polyclonal antibodies and nanoantibodies are independently developed by Chongqing Aisens Bioengineering Co., Ltd.

[0076] Preparation Example 2

[0077] The specific steps of the standard operating procedure for manual immunohistochemistry are as follows:

[0078] (1) Slice baking: Select the corresponding paraffin-embedded tissue slices and bake them at 65°C for 30 min on a slide baking machine.

[0079] (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.

[0080] (3) Antigen repair: Add pH 9.0 EDTA repair solution to a stainless steel pot, immerse the above sections in it, heat the water on the induction cooker to boiling, and maintain for 20 minutes. Turn off the heating, transfer the stainless steel pot to the sink, rinse with running water, and cool to room temperature.

[0081] (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.

[0082] (5) Primary antibody incubation: Shake off the PBS buffer, add 0.1 mL of the corresponding primary antibody to the tissue, make sure the primary antibody solution covers the entire tissue, and incubate at room temperature for 1 h. Then rinse the sections with PBS buffer.

[0083] (6) Primary antibody post-incubation: Shake off the PBS buffer, add 0.1 mL of the corresponding secondary antibody A reagent to the tissue, ensuring that the primary antibody solution covers the entire tissue, and incubate at room temperature for 15 minutes. Then rinse the sections with PBS buffer.

[0084] (7) Secondary antibody incubation: Shake off the PBS buffer, add 0.1 mL of the corresponding secondary antibody polymer reagent to the tissue, ensure that the secondary antibody solution covers the entire tissue, and incubate at room temperature for 15 min. Then rinse the sections with PBS buffer.

[0085] (8) DAB color development: Shake off the PBS buffer, add 0.1 mL of DAB color development solution onto the tissue, making sure that the DAB color development solution covers the entire tissue, and incubate at room temperature for 5 min. Then rinse the sections with deionized water.

[0086] (9) Staining: Shake off the deionized water. Add 0.1 mL of dye solution to the tissue, making sure the dye solution covers the entire tissue, and incubate at room temperature for 5 minutes. Then rinse the sections with deionized water.

[0087] (10) Dehydration, transparency, and sealing: Soak the slices in 75% ethanol for 3 min; soak the slices in 95% ethanol for 3 min; soak the slices in anhydrous ethanol for 3 min, replace anhydrous ethanol and soak for another 3 min; soak the slices in xylene for 5 min, replace xylene and soak for another 5 min. Remove the slices from the solvent, add a drop of neutral resin on the tissue, cover with a coverslip, and place in a fume hood to air dry overnight.

[0088] (11) Observe the tissue morphology under a microscope and determine the staining results.

[0089] Effect verification example

[0090] In order 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 the polymerase-labeled antibody and composition prepared in Example 1 and commercially available products were subjected to immunohistochemical testing and effect comparison according to the experimental steps described in Example 2.

[0091] Effect Example 1

[0092] The polymerized enzyme-labeled antibody composition 1a prepared in Example 1 and the polymerized 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 index to be tested. The staining results are shown in FIG. 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.

[0093] Effect Example 2

[0094] In order to demonstrate the effect of the composition of the polymerase-labeled antibody composition on the staining of tissue samples, 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 index to be tested. The staining results are shown in Figure 2. Figure 4 As shown in the figure, due to the high density of the cell nucleus, it is difficult for macromolecular substances to enter, so the staining effect of the protein expressed in the cell nucleus gradually weakens as the content of the polymerase-labeled nanoantibody 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 equivalent to the staining effect of the working solution containing only polymerase-labeled polysomes ( Figure 4 B), based on the consideration of staining effectiveness, the mass ratio of polymerase-labeled polyantibody to polymerase-labeled nanoantibody in the polymerase-labeled antibody composition is less than 13:1, preferably less than 5:1.

[0095] Effect Example 3

[0096] In order to demonstrate 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 polyethyleneimine part in the framework of composition 1a and composition 2a is a multi-head type, which can connect more enzymes and secondary antibodies, so the staining effect is better than that of the polymerase-labeled antibody composition 6 containing a linear framework. Composition 3 can still stain tissue samples and achieve effective resolution, but the molecular weight of the polymerase-labeled antibody framework in composition 4 is too large and there are too many amino-coupled enzymes and secondary antibodies in the polyethyleneimine part, resulting in a weakened staining effect.

[0097] Therefore, the present invention uses polyethylene glycol to connect 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-head 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.

[0098] Effect Example 4

[0099] To further demonstrate the beneficial effects of the skeleton material selection of the present invention, the polymerase-labeled antibody composition 5, composition 6 and composition 7 prepared in Example 1 were used as working solutions and compared on the basis of the same solid content. In this experiment, a human lung squamous cell carcinoma tissue sample fixed with formalin and embedded in paraffin was used, and the nuclear expressed protein P63 was selected as the primary antibody index to be tested. The staining results are shown in Figure 2. Figure 6 As shown, the staining effect of the polymerase-labeled antibody composition containing 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.

[0100] Effect Example 5

[0101] In order to evaluate the specificity and sensitivity of the prepared polymerized enzyme-labeled antibody composition in immunohistochemistry experiments. We used the polymerized 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 were used, including human tonsil tissue and human breast tissue, 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 staining intensity of the polymerase-labeled antibody composition prepared by the present invention for CD5 and CK8&18 primary antibodies is significantly stronger than that of the control sample, and both have comparable effects on P53 primary antibody staining. This further indicates that the polymerase-labeled antibody composition of the present invention has good universality and better overall effects than similar products.

[0102] Through the above experiments and verifications, it can be known 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 polymerase-labeled multi-antibody and the polymerase-labeled nano-antibody are used in combination, which improves the staining effect and also improves the universality of the product.

[0103] It should also be noted that the exemplary embodiments mentioned in the present invention describe some methods or systems based on a series of steps or devices. However, the present invention is not limited to the order of the above steps, that is, the steps can be performed in the order mentioned in the embodiments, or in a different order from the embodiments, or several steps can be performed simultaneously.

[0104] 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.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the embodiments of the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition containing a compact polymerase-labeled antibody, characterized in that: The composition comprises: A first polymerase-labeled antibody, wherein the first polymerase-labeled antibody comprises a polyclonal antibody, a first polymer skeleton and a first enzyme; A second polymer enzyme-labeled antibody, wherein the second polymer enzyme-labeled antibody comprises a nanobody, a second polymer backbone, and a second enzyme; wherein the first enzyme and the second enzyme are selected from horseradish peroxidase, alkaline phosphatase or β-glycosidase, respectively; The first polymer skeleton and the second polymer skeleton are polymer compounds of polyethylene glycol connected with polyethylene imine; the molecular weights of the first polymer skeleton and the second polymer skeleton are lower 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 composition according to claim 1, characterized in that The polyclonal antibody is selected from goat anti-mouse IgG, rabbit anti-mouse IgG, horse anti-mouse IgG, goat anti-rabbit IgG, horse anti-rabbit IgG, mouse anti-rabbit IgG, donkey anti-goat IgG, horse anti-goat IgG, mouse anti-goat IgG, rabbit anti-goat IgG and one or more of the Fab fragments, Fab' fragments and F(ab')2 fragments of the corresponding IgGs above.

3. The composition according to claim 2, characterized in that The nanobody is obtained by phage display technology.

4. A combination according to any one of claims 1 to 3, characterized in that The nanobody is selected from one or more of those targeting mouse, rabbit or goat IgG.

5. The composition 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.

6. The composition according to claim 4, 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.

7. The composition according to claim 5, characterized in that In the polymer compound in which polyethylene glycol is connected to polyethylene imine, polyethylene imine is connected only at one end of the polyethylene glycol.

8. The composition 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.

9. The composition 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 4 enzymes.

10. The composition according to claim 5, characterized in that The first polymer skeleton and the second polymer skeleton each contain at least two enzymes.

11. The composition according to claim 5, characterized in that The first polymer skeleton and the second polymer skeleton each contain at least 4 enzymes.

12. The composition according to any one of claims 1 to 3, characterized in that The molecular weight of the polyethylene glycol in the first polymer skeleton and / or the second polymer skeleton is 1000-40000; the molecular weight of the polyethylene imine is 100-30000.

13. The composition according to claim 8, characterized in that The molecular weight of the polyethylene glycol in the first polymer skeleton and / or the second polymer skeleton is 1000-40000; the molecular weight of the polyethylene imine is 100-30000.

14. Use of the composition according to any one of claims 1 to 13 in preparing a marker capture or marker binding product.

15. The use according to claim 14, characterized in that: The product is a test kit.

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