Glucan-based HRP-goat anti-mouse IgG labeled antibody and preparation method thereof

Abstract

The invention discloses a dextran-based HRP-goat anti-mouse IgG labeled antibody and a preparation method thereof, and the preparation method is characterized by comprising the following steps: S1, oxidizing dextran to obtain aldehyde group-containing dextran; s2, carrying out polymerization on the dextran containing the aldehyde group and horse radish peroxidase, so as to obtain a horse radish peroxidase polymer; s3, performing desalination and ultrafiltration on the horse radish peroxidase polymer to obtain an ultrafiltration product I; s4, connecting a goat anti-mouse IgG antibody to the ultrafiltration product I to obtain oxidized glucan coupled with goat anti-mouse IgG and HRP enzyme; and S5, purifying the oxidized dextran of the product coupled with the goat anti-mouse IgG and the HRP enzyme, so as to obtain the dextran coupled HRP-goat anti-mouse IgG labeled antibody. The dextran product obtained by the invention is wide in molecular weight coverage, different molecular weights can be selected to design polymer substances with different sizes, the number of aldehyde groups can be controlled to control coupling of different numbers of protein molecules under the same molecular weight, and the signal intensity is easy to control.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a dextran-based HRP-sheep anti-mouse IgG labeled antibody and a preparation method thereof. Background Art

[0002] Immunohistochemistry is an immunohistochemical technique that uses the characteristics of antigen-antibody specific recognition to detect tumor-specific proteins or antigens in pathological specimens. This technique can not only detect the nature and quantity of characteristic substances, but also because it can detect characteristic proteins or antigens at their original locations and on the basis of maintaining the original morphology of the tissue, it has become the most effective method for pathological etiology analysis (especially for tumor pathology analysis) and has become one of the most commonly used clinical methods for tumor diagnosis, identification, differentiation and efficacy prediction.

[0003] The experimental operation steps and principles of immunohistochemistry are as follows: (1) Obtain pathological specimens (tumor tissue or other lesion tissue) through surgical operation, fix with formalin, dehydrate with alcohol, embed in paraffin, slice into 3-5 micron thick tissue slices, and adhere to glass slides; (2) Dewax with organic solvent, wash with alcohol and hydrate, and then cook at high temperature to restore the characteristic substances on the tissue (such as tumor characteristic proteins or antigens, or pathogen proteins) to their original shape; (3) Use specific antibodies (antibodies with the ability to bind to characteristic substances or specific antigens, called "primary antibodies") to identify characteristic proteins; (4) Use marker enzymes to label The antibody against the primary antibody (called the "secondary antibody") is incubated to allow the enzyme-labeled secondary antibody to bind to the primary antibody and determine the location of the specific characteristic substance (protein); (5) A color developer is used to react chemically under the catalysis of the labeling enzyme to form an insoluble colored compound in situ, thereby displaying the location of the enzyme-labeled secondary antibody (that is, the location of the characteristic protein or antigen); (6) Hematoxylin is used to stain the entire tissue morphology to facilitate the identification of the tissue morphology; (7) After such a series of operation processes, the location and amount of the tumor characteristic protein or pathogen protein are displayed through the location and depth of the color, which plays a role in identification and differentiation and guides clinical treatment.

[0004] The entire immunohistochemistry experimental operation process requires nearly thirty steps, including: dewaxing, hydration, endogenous enzyme blocking, washing, high temperature heat repair, cooling, washing, primary antibody incubation, washing, secondary antibody incubation, washing, enhancer incubation, washing, color development, washing, back staining, washing, dehydration, and sealing. In addition to the standardized tissue sampling and pretreatment required in these steps, the key factors affecting the processing effect of the slice specimens are the specificity and affinity of the primary antibody and the signal amplification effect of the secondary antibody. The amplification ability of the secondary antibody depends on the specificity and affinity of the secondary antibody and the number of labeled enzymes labeled on the secondary antibody. The principles and shortcomings of the existing technology: The working principles of the secondary antibodies labeled with labeled enzymes commonly used in the market are the following two methods: (a) using biotin-avidin amplification method ( Figure 1 ), the principle of this method is: use biotin labeling on the secondary antibody, and then label the marker enzyme with avidin. In the process of processing the slice specimen, add the primary antibody to the specimen, then add the biotin-labeled secondary antibody, and then add the avidin labeled with the marker enzyme. The specific high affinity between avidin and biotin is used to locate the marker enzyme labeled with avidin at the location of the characteristic substance. The advantage of this method is that biotin is a small organic molecule. Because of its small size, it can be labeled more on the secondary antibody without affecting the specificity and affinity of the secondary antibody. The more biotin labeled on the secondary antibody, the more likely it is to combine with more avidin labeled with the marker enzyme, which can improve the effect of signal amplification and detection sensitivity. However, this method has a serious disadvantage: biotin is one of the vitamins present in organisms, especially in small intestinal tissue, kidney tissue, brain tissue and liver tissue. The use of biotin-avidin amplification method will appear in the above tissues. Positive signals unrelated to the detection itself, that is, false positive misdiagnosis may occur. Another disadvantage of this method is that it requires an extra step. (b) Direct enzyme-secondary antibody labeling method ( Figure 2 ), this method is to directly label the marker enzyme on the secondary antibody through an organic small molecule coupling reagent. Since the marker enzyme itself has a large molecular weight (the commonly used molecular weight of peroxidase is 40,000Da, the molecular weight of alkaline phosphatase is 140,000Da, and the molecular weight of urease is 200,000Da), and the volume is large, only 1-2 enzyme molecules can be labeled on the secondary antibody molecule. Too many labels will reduce the specificity of the secondary antibody itself (reduced specificity will cause high background and non-specific signals) and reduce the affinity of the secondary antibody, thereby reducing the sensitivity of its detection. Summary of the invention

[0005] To address the deficiencies of the prior art, the present invention provides a dextran-based HRP-sheep anti-mouse IgG labeled antibody and a preparation method thereof. The obtained dextran product has a wide range of molecular weights, and different molecular weights can be selected to design polymers of different sizes. The number of aldehyde groups can also be controlled to control the coupling of different numbers of protein molecules at the same molecular weight.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody comprises the following steps: S1, oxidizing dextran to obtain dextran containing aldehyde groups; S2, polymerizing the aldehyde-containing dextran with horseradish peroxidase to obtain a horseradish peroxidase polymer; S3, performing desalting and ultrafiltration on the horseradish peroxidase polymer to obtain an ultrafiltration product 1; S4, connecting the goat anti-mouse IgG antibody to the ultrafiltration product 1 to obtain oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme; S5. Purify the oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme to obtain dextran-coupled HRP-goat anti-mouse IgG labeled antibody.

[0007] Preferably, the specific method of the aforementioned step S1 is: dissolving dextran in 10-100 mM carbonate buffer with a pH of 9-10, and then adding sodium periodate to oxidize the dextran, wherein the mass ratio of the dextran to sodium periodate is 1:2-1:10.

[0008] Preferably, the specific method of the aforementioned step S2 is: adding the aldehyde-containing dextran to the horseradish peroxidase, fully mixing, then adding sodium triacetoxyborohydride, and rotating the mixture at 2-8° C. to obtain the horseradish peroxidase polymer.

[0009] Preferably, in the aforementioned step S2, the mass ratio of the aldehyde-containing dextran, horseradish peroxidase and sodium triacetoxyborohydride is 1:5:5-1:5:10.

[0010] Preferably, in the aforementioned step S3, the molecular retention capacity of the ultrafiltration tube is 30-100 Kd, and the ultrafiltration is performed at room temperature for 3-10 min.

[0011] Preferably, the specific method of the aforementioned step S4 is: adding goat anti-mouse IgG antibody to the ultrafiltration product, adding sodium triacetoxyborohydride after sufficient mixing, and rotating the reaction for 12-24 hours.

[0012] Preferably, in the aforementioned step S4, the mass ratio of the ultrafiltration product, the goat anti-mouse IgG antibody and sodium triacetoxyborohydride is 1:0.01:5 to 1:0.1:10.

[0013] Preferably, the specific method of the aforementioned step S5 is: (1) Desalting and ultrafiltration of oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme; (2) Using dextran G50 as a molecular sieve filler, a mixed solution containing oxidized dextran coupled to goat anti-mouse IgG and HRP enzyme was purified by column chromatography, and the product flowing out from the bottom of the chromatography column was detected using a nucleic acid protein detector, and the product was collected in the order of peak elution with an automatic fraction collector; (3) Use a spectrophotometer to test the protein concentration of the ultrafiltration product, add antibody diluent to control the concentration to 0.025~1 mg / mL, and store at -15~25°C to obtain a dextran-based HRP-sheep anti-mouse IgG labeled antibody.

[0014] A dextran-based HRP-goat anti-mouse IgG labeled antibody.

[0015] A dextran-based HRP-goat anti-mouse IgG labeled antibody for use in immunohistochemistry.

[0016] The present invention is beneficial in that: (1) The present invention uses dextran as a skeleton, couples antibody molecules and enzymes to the dextran skeleton, oxidizes the ortho-hydroxyl groups on the dextran by the sodium periodate method to form aldehyde groups, and then connects the oxidized dextran skeleton to the amino groups on HRP through the aldehyde groups to form a dextran-HRP polymer. Finally, goat anti-mouse IgG is connected to dextran-HRP, and after purification, ultrafiltration and dilution treatment, a dextran-based HRP-goat anti-mouse IgG labeled antibody is obtained. The dextran product obtained by the present invention has a wide range of molecular weights, and different molecular weights can be selected to design polymers of different sizes. The number of aldehyde groups can also be controlled to control the number of protein molecules coupled at the same molecular weight, which is easy to control the signal intensity. (2) The preparation steps of the present invention are simple, the obtained product has good application effect and higher detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the biotin-avidin enzyme-labeled secondary antibody immunohistochemistry method; Figure 2 This is a schematic diagram of the direct enzyme-labeled secondary antibody immunohistochemistry method; Figure 3 It is a schematic diagram of the chain polymerase enzyme-labeled secondary antibody immunohistochemistry method; Figure 4 To detect Ki67 protein in tonsil by immunohistochemistry using the multimeric enzyme-labeled goat anti-mouse secondary antibody produced by the method of the present invention (produced in Example 2, 1:20); Figure 5 Immunohistochemistry was used to detect Ki67 protein in tonsils using polymerase goat anti-rabbit secondary antibody (Dako Catalog No.: K4001). DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Polymer enzyme-secondary antibody labeling method is a new labeling enzyme and secondary antibody labeling method ( Figure 3 ). In order to reduce the damage of the marker enzyme to the specificity and affinity of the secondary antibody, the marker enzyme is not directly linked to the secondary antibody molecule, but first linked to a chain organic polymer (such as dextran, poly-lysine polypeptide, etc.), and then the polymerized polymer enzyme is linked to the secondary antibody molecule, which not only increases the number of marker enzymes on each secondary antibody molecule, but also allows a certain spatial distance between the marker enzyme and the secondary antibody, reducing the adverse effects on the specificity and affinity of the secondary antibody. Compared with the previous two labeling methods, the polymer enzyme labeling method eliminates the possibility of interference from endogenous biotin, simplifies the operation steps (reducing one step, changing two steps: adding biotin-labeled secondary antibody and adding enzyme-labeled biotin to one step: adding polymer enzyme-labeled secondary antibody) and has higher detection sensitivity.

[0020] Example 1: A method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody, comprising the following steps: S1. Prepare oxidized dextran containing aldehyde groups, select dextran with a molecular weight of 3500-50000D, prepare a dextran aqueous solution with a mass concentration of 10 mg / mL, adjust the pH value of the dextran solution to 9 with 100 mM sodium carbonate solution, then add sodium periodate to oxidize the dextran, the mass ratio of dextran to sodium periodate is 1:2, react at room temperature for 7 h, and after the reaction is completed, load it into an ultrafiltration tube with a molecular weight cutoff of 30 Kd, centrifuge at 4000 rpm for 5 minutes to obtain oxidized glucose containing aldehyde groups; S2, adding HRP enzyme and sodium triacetoxyborohydride to aldehyde-containing dextran, the mass ratio of aldehyde-containing dextran, horseradish peroxidase and sodium triacetoxyborohydride is 1:5:5, and rotating reaction is carried out at 2-8 ℃ for 12 h to obtain horseradish peroxidase polymer; S3, the obtained horseradish peroxidase polymer is concentrated by ultrafiltration at room temperature for 5 minutes using an ultrafiltration tube with a molecular cutoff of 100 Kd to obtain an ultrafiltration product; S4, adding goat anti-mouse IgG antibody and sodium triacetoxyborohydride to the ultrafiltration product, the mass ratio of ultrafiltration product, goat anti-mouse IgG antibody and sodium triacetoxyborohydride is 1:0.01:5, rotating reaction at 2-8 ℃ for 24 h, and obtaining a mixed solution of oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme; S5, the obtained mixed solution of oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme is concentrated by ultrafiltration using an ultrafiltration tube with a molecular cutoff of 100 Kd, and ultrafiltration is performed at room temperature for 5 minutes to obtain an ultrafiltration product; Dextran G50 was used as a molecular sieve filler, and the concentrated solution of oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme was purified by column chromatography. The elution solution was PBS (PBS was 10 mM sodium phosphate, 140 mM sodium chloride, pH=8.5). The product flowing out from the bottom of the chromatography column was detected by a nucleic acid protein detector, and the product was collected in the order of peaks with an automatic fraction collector. The collected product was loaded into an ultrafiltration tube with a molecular weight cutoff of 100 Kd and centrifuged at 4000 rpm for 10 minutes to obtain an ultrafiltration product; The protein concentration of the ultrafiltration product was tested using a spectrophotometer, and the concentration was controlled to 0.025 mg / mL by adding antibody diluent and stored at -15°C to obtain a dextran-based HRP-sheep anti-mouse IgG labeled antibody.

[0021] Example 2: The preparation steps of this example are the same as those of Example 1, with the specific difference being that in step S1, the pH value of the dextran solution after adjustment is 9.5.

[0022] Example 3: The preparation steps of this example are the same as those of Example 1, with the specific difference being that in step S1, the pH value of the dextran solution after adjustment is 10.5.

[0023] Performance testing The dextran-based HRP-sheep anti-mouse IgG labeled antibody prepared from the earliest product in Example 2 was used as the experimental group, and the polymer enzyme-labeled secondary antibody (ready-to-use) purchased from Dako Company, USA, catalog number K4000 and K4001 was used as the control group, which was used for immunohistochemical staining of pathological specimens.

[0024] The specific staining steps include: (1) Dewaxing: Take the corresponding paraffin-embedded tissue sections and immerse them in xylene for 10 minutes each, three times.

[0025] (2) Hydration: Transfer the above sections to 100% alcohol and soak for 5 minutes each, once; soak in 95% alcohol for 5 minutes, once; soak in 75% alcohol for 5 minutes, once; soak in purified water for 3 minutes, once.

[0026] (3) Thermal repair: Soak the above sections in EDTA antigen repair solution and heat them in a pressure cooker. Maintain the temperature at 120°C for 3.5 minutes. Stop heating and cool naturally for 30 minutes. Take out the sections and soak them in PBS buffer for 3 minutes.

[0027] (4) Primary antibody incubation: Remove the sections, spin dry the PBS, add 0.1 mL of the corresponding primary antibody to each section to cover the tissue, incubate at room temperature for 60 minutes, spin dry the primary antibody, and soak the sections in PBS buffer for 3 minutes three times.

[0028] (5) Secondary antibody incubation: Remove the sections, spin dry the PBS, add 0.1 mL of the corresponding secondary antibody to each section to cover the tissue, incubate at room temperature for 20 minutes, spin dry the secondary antibody, and soak the sections in PBS for 3 minutes three times.

[0029] (6) Color development: Remove the slices, spin off PBS, add 0.1 mL of DAB to each slice to cover the tissue on the slice, incubate at room temperature for 5 minutes, spin off DAB, and soak the slices in purified water for 1 minute.

[0030] (7) Back staining: Take out the above sections, shake off the water, add 0.1 mL of hematoxylin on each section to cover the tissue on the section, incubate at room temperature for 3 minutes, shake off the hematoxylin, place the section on a section rack and rinse under tap water for 3 minutes.

[0031] (8) Dehydration: Take out the above slices and soak them in the following liquids for 3 minutes respectively: 95% alcohol, 100% alcohol, 100% alcohol, and xylene.

[0032] (9) Sealing: Take out the above sections and blow dry them in a fume hood. Add one drop (about 0.02 mL) of resin mounting medium on each section, cover with a coverslip, and blow dry in a fume hood (about 5 minutes).

[0033] (10) Film review: Observe the staining effect under a microscope.

[0034] The application example shows that under the same operation steps, the reagent obtained by using the globular polymer enzyme secondary antibody reagent (Example 2) produced by the present invention (1:20 dilution) is used to perform immunohistochemistry to detect the staining intensity of Ki67 antigen protein (Ki67) in human tonsil tissue ( Figure 4 ) was significantly stronger than the staining intensity of Ki67 antigen protein (Ki67) detected by immunohistochemistry using DAKO polymer enzyme-linked secondary antibody reagent in the same tonsil tissue ( Figure 5 ), it can be seen that the polymer enzyme-labeled secondary antibody reagent produced by the present invention has a higher sensitivity for detecting nuclear antigens.

[0035] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for preparing a HRP-goat anti-mouse IgG labeled antibody based on dextran, characterized in that: The steps include: S1, oxidizing dextran to obtain dextran containing aldehyde groups; S2, polymerizing the aldehyde-containing dextran with horseradish peroxidase to obtain a horseradish peroxidase polymer; S3, performing desalting and ultrafiltration on the horseradish peroxidase polymer to obtain an ultrafiltration product 1; S4, connecting the goat anti-mouse IgG antibody to the ultrafiltration product 1 to obtain oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme; S5. Purify the oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme to obtain dextran-coupled HRP-goat anti-mouse IgG labeled antibody.

2. The method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody according to claim 1, characterized in that: The specific method of step S1 is: dissolving dextran in 10-100 mM carbonate buffer with a pH of 9-10, and then adding sodium periodate to oxidize the dextran, wherein the mass ratio of the dextran to sodium periodate is 1:2-1:

10.

3. The method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody according to claim 1, characterized in that: The specific method of step S2 is: adding aldehyde-containing dextran to horseradish peroxidase, fully mixing, then adding sodium triacetoxyborohydride, and rotating at 2-8° C. to react to obtain horseradish peroxidase polymer.

4. The method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody according to claim 1, characterized in that: In the step S2, the mass ratio of the aldehyde-containing dextran, horseradish peroxidase and sodium triacetoxyborohydride is 1:5:5-1:5:

10.

5. The method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody according to claim 1, characterized in that: In step S3, the molecular cutoff of the ultrafiltration tube is 30-100 Kd, and ultrafiltration is performed at room temperature for 3-10 min.

6. The method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody according to claim 1, characterized in that: The specific method of step S4 is: adding goat anti-mouse IgG antibody to the ultrafiltration product, adding sodium triacetoxyborohydride after sufficient mixing, and rotating the reaction for 12-24 hours.

7. The method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody according to claim 1, characterized in that: In step S4, the mass ratio of the ultrafiltration product, the goat anti-mouse IgG antibody and sodium triacetoxyborohydride is 1:0.01:5-1:0.1:

10.

8. The method for preparing a dextran-based HRP-sheep anti-mouse IgG labeled antibody according to claim 1, characterized in that: The specific method of step S5 is: (1) Desalting and ultrafiltration of oxidized dextran coupled with goat anti-mouse IgG and HRP enzyme; (2) Using dextran G50 as a molecular sieve filler, a mixed solution containing oxidized dextran coupled to goat anti-mouse IgG and HRP enzyme was purified by column chromatography, and the product flowing out from the bottom of the chromatography column was detected using a nucleic acid protein detector, and the product was collected in the order of peak elution with an automatic fraction collector; (3) Use a spectrophotometer to test the protein concentration of the ultrafiltration product, add antibody diluent to control the concentration to 0.025~1 mg / mL, and store at -15~25°C to obtain a dextran-based HRP-sheep anti-mouse IgG labeled antibody.

9. The dextran-based HRP-sheep anti-mouse IgG labeled antibody obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the dextran-based HRP-goat anti-mouse IgG labeled antibody according to claim 9 in immunohistochemistry.

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