Immunohistochemical antibody diluent and preparation method thereof

Abstract

The invention discloses an immunohistochemical antibody diluent and a preparation method thereof, and belongs to the technical field of immunization, and the immunohistochemical antibody diluent is prepared by the following steps: weighing 7.8-8.1 parts by weight of sodium chloride, 0.15-0.25 part by weight of potassium chloride, 3.5-3.6 parts by weight of sodium dihydrogen phosphate and 0.25-0.3 part by weight of dipotassium phosphate, adding into 950-1050 parts by weight of deionized water, stirring at room temperature to fully dissolve, filtering, and drying to obtain the immunohistochemical antibody diluent. Then 9.5-10.5 parts of bovine serum albumin, 4.5-5.5 parts of goat serum and 0.9-1.1 parts of ProCin 300 are added, the pH is adjusted to 7.5, the color of the solution is adjusted, and an immunohistochemical antibody diluent is obtained after filtration with a 0.22-micron filter membrane; the prepared antibody diluent can improve the storage stability of the immunohistochemical antibody, is stable in performance, safe and reliable, can reduce background signals, and remarkably improves the signal-to-noise ratio of various tissue dyeing results.

Description

Technical Field

[0001] The invention belongs to the field of immunotechnology, and in particular relates to an immunohistochemical antibody diluent and a preparation method thereof. Background Art

[0002] Immunohistochemistry is the abbreviation of immunohistochemistry. It applies the basic principle of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody. Through chemical reaction, the color developer of the labeled antibody is used to determine the antigen in the tissue cell, and to conduct localization, qualitative and quantitative research on it. Immunohistochemistry antibody diluent is the antibody diluent used in immunohistochemistry experiments. Commonly used antibody reagents have a high titer and need to be diluted to a suitable concentration before they can be used in the reaction of detecting antigens. The components of the antibody diluent play a key role in the strength of immunohistochemistry detection and the stability of antibody products.

[0003] Currently, there are various formulas of antibody diluents on the market: there are general-purpose phosphate buffered saline (PBS) or triethanolamine buffered saline (TBS) as antibody diluents, which reduce the impact of pH changes on experimental results through their buffering effect. However, such antibody diluents have problems such as poor storage stability of antibodies, short storage time and low detection intensity of antigens. Some antibody diluents add sodium azide preservative to enhance the storage stability of antibodies, but sodium azide affects the enzyme activity of horseradish peroxidase (HRP) and cannot be used for dilution of immunohistochemistry secondary antibodies. In addition, it is highly toxic and not conducive to experimental safety management. Summary of the invention

[0004] The object of the present invention is to provide an immunohistochemical antibody diluent and a preparation method thereof to solve the problems in the background technology.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] An immunohistochemical antibody diluent comprises the following components in parts by weight:

[0007] 7.8-8.1 parts of sodium chloride, 0.15-0.25 parts of potassium chloride, 3.5-3.6 parts of sodium dihydrogen phosphate, 0.25-0.3 parts of dipotassium hydrogen phosphate, 9.5-10.5 parts of bovine serum albumin (BSA), 4.5-5.5 parts of goat serum, 0.9-1.1 parts of ProClin 300 and 950-1050 parts of deionized water.

[0008] Furthermore, the immunohistochemical antibody diluent comprises the following components in parts by weight:

[0009] 8 parts of sodium chloride, 0.2 parts of potassium chloride, 3.58 parts of sodium dihydrogen phosphate, 0.27 parts of dipotassium hydrogen phosphate, 10 parts of BSA, 5 parts of goat serum, 1 part of ProClin 300 and 1000 parts of deionized water.

[0010] Furthermore, the pH value of the immunohistochemistry antibody diluent is 7.5.

[0011] A method for preparing an immunohistochemical antibody diluent comprises the following steps:

[0012] Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate and dipotassium hydrogen phosphate, add them into deionized water, stir at room temperature to fully dissolve, then add BSA, goat serum and ProClin 300, adjust the pH to 7.5, adjust the color of the solution, filter through a 0.22 um filter membrane, and obtain an immunohistochemical antibody diluent.

[0013] Beneficial effects:

[0014] The antibody diluent prepared by the present invention can be used for the production of immunohistochemical antibodies, can improve the storage stability of immunohistochemical antibodies, can be stored at 2-8°C for at least 24 months, and can be used directly by users without any configuration operation, which is convenient and quick;

[0015] The antibody diluent of the present invention uses ProClin 300 as a preservative, has a broad-spectrum antibacterial ability, is effective against bacteria, fungi and yeast, and has stable performance, safety and reliability. It can not only be used for the dilution of immunohistochemical antibodies, but also can be used for the dilution of HRP secondary antibodies because it does not contain sodium azide.

[0016] The antibody diluent of the invention can improve the specificity of immunohistochemistry, and the bovine serum albumin and goat serum contained therein can be directly used as immunohistochemical blocking agents to reduce background signals and significantly improve the signal-to-noise ratio of various tissue staining results. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below in conjunction with the accompanying drawings.

[0018] Figure 1 It is a comparison diagram of the staining results of pancreatic tissue by the diluted primary antibody (CgA) prepared in Example 4 and Comparative Example 1;

[0019] Figure 2 It is a comparison diagram of the staining results of appendix tissue by the diluted primary antibody (CK20) prepared in Example 5 and Comparative Example 2;

[0020] Figure 3 It is a comparison chart of the staining results of the diluted primary antibody (CD31) prepared in Example 6 and Comparative Example 3 on placental tissue. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1

[0023] This embodiment provides an immunohistochemical antibody dilution solution, which is prepared by the following steps:

[0024] Weigh 7.8 g of sodium chloride, 0.15 g of potassium chloride, 3.5 g of sodium dihydrogen phosphate and 0.25 g of potassium dihydrogen phosphate, add them to 950 g of deionized water, stir at room temperature to fully dissolve, then add 9.5 g of BSA, 4.5 g of goat serum and 0.9 g of ProC lin 300, adjust the pH to 7.5 with 1 M NaOH or 1 M HCl, finally add green pigment to adjust the color of the solution, filter with 0.22 um filter membrane to obtain immunohistochemical antibody diluent.

[0025] Example 2

[0026] This embodiment provides an immunohistochemical antibody dilution solution, which is prepared by the following steps:

[0027] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 3.58 g of sodium dihydrogen phosphate and 0.27 g of potassium dihydrogen phosphate, add them into 1000 g of deionized water, stir at room temperature to fully dissolve, then add 10 g of BSA, 5 g of goat serum and 1 g of ProC lin 300, adjust the pH to 7.5 with 1 M NaOH or 1 M HCl, finally add green pigment to adjust the color of the solution, filter with 0.22 um filter membrane to obtain immunohistochemical antibody diluent.

[0028] Example 3

[0029] This embodiment provides an immunohistochemical antibody dilution solution, which is prepared by the following steps:

[0030] Weigh 8.1 g of sodium chloride, 0.25 g of potassium chloride, 3.6 g of sodium dihydrogen phosphate and 0.3 g of potassium dihydrogen phosphate, add them into 1050 g of deionized water, stir at room temperature to fully dissolve, then add 10.5 g of BSA, 5.5 g of goat serum and 1.1 g of ProC lin 300, adjust the pH to 7.5 with 1 M NaOH or 1 M HCl, finally add green pigment to adjust the color of the solution, filter with 0.22 um filter membrane to obtain immunohistochemistry antibody diluent.

[0031] Example 4

[0032] The immunohistochemical antibody diluent prepared in Example 2 was diluted to a working concentration of chromogranin A (CgA, Catalog No. AF20027) at 1:200 to obtain a diluted primary antibody.

[0033] Example 5

[0034] The immunohistochemical antibody diluent prepared in Example 2 was diluted to a working concentration of cytokeratin 20 (CK20, catalog number AF20037) at 1:200 to obtain a diluted primary antibody.

[0035] Example 6

[0036] The immunohistochemical antibody diluent prepared in Example 2 was diluted to a working concentration of platelet-endothelial cell adhesion molecule (CD31, Catalog No. AF20112) at 1:200 to obtain a diluted primary antibody.

[0037] Comparative Example 1

[0038] The difference between this comparative example and Example 4 is that the antibody is diluted with a commercially available antibody diluent, and the other raw materials and steps are the same.

[0039] Comparative Example 2

[0040] The difference between this comparative example and Example 5 is that the antibody is diluted with a commercially available antibody diluent, and the other raw materials and steps are the same.

[0041] Comparative Example 3

[0042] The difference between this comparative example and Example 6 is that the antibody is diluted with a commercially available antibody diluent, and the other raw materials and steps are the same.

[0043] According to the following experimental steps, the diluted primary antibodies prepared in Examples 4 to 6 and Comparative Examples 1 to 3 were used to stain pancreatic tissue, appendix tissue and placental tissue respectively:

[0044] Step 1: Dewax paraffin sections to water:

[0045] The paraffin sections were placed in xylene I, xylene II and xylene III for 15 min each, then placed in anhydrous ethanol I and anhydrous ethanol II for 5 min each, 85% alcohol for 5 min, 75% alcohol for 5 min, and washed with distilled water to obtain tissue sections;

[0046] Step 2: Antigen retrieval:

[0047] Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH = 9.0), perform antigen repair in a microwave oven, boil for 10 minutes on medium heat, stop the heat for 5 minutes to keep warm, switch to medium-low heat for 5 minutes, stop the heat for 2 minutes to keep warm, and then continue to heat for 5 minutes on medium-low heat. During this process, excessive evaporation of the buffer should be prevented, and the sections should not be dried. Take out and place at room temperature to cool naturally. Take out the tissue sections, place them in PBS buffer (pH = 7.4), and wash them 3 times on a decolorizing shaker, each time for 5 minutes;

[0048] Step 3: Block endogenous peroxidase:

[0049] The tissue sections were placed in a 3% hydrogen peroxide solution, incubated at room temperature in the dark for 15 minutes, and then placed in a PBS buffer (pH=7.4) and washed three times on a decolorizing shaker, each time for 5 minutes.

[0050] Step 4, serum blocking:

[0051] Add 3% BSA in the tissue circle to evenly cover the tissue and seal at room temperature for 30 minutes;

[0052] Step 5, add primary antibody:

[0053] The blocking solution was gently shaken off, and the diluted primary antibodies prepared in Examples 4 to 6 and Comparative Examples 1 to 3 were respectively added dropwise to the slices, and the slices were placed flat in a humidified box (a small amount of water was added to the humidified box to prevent the antibody from evaporating), and incubated at 4° C. overnight.

[0054] Step 6, add secondary antibody:

[0055] The slides were placed in PBS buffer (pH = 7.4) and washed on a decolorizing shaker for 3 times, 5 min each time; after being slightly dried, the secondary antibody of the same species as the primary antibody was added dropwise into the tissue circle: rabbit antibody (HRP labeled) to cover the tissue and incubate at room temperature for 50 min;

[0056] Step 7, DAB color development:

[0057] The slides were placed in PBS buffer (pH = 7.4) and washed on a decolorizing shaker for 3 times, 5 min each time; after being slightly dried, freshly prepared DAB color developing solution was added dropwise into the tissue circle, the color development time was controlled under a microscope, and the slides were rinsed with tap water to terminate the color development;

[0058] Step 8. Counterstaining of cell nuclei:

[0059] Hematoxylin counterstaining for about 3 minutes, washing with tap water, differentiation with hematoxylin differentiation solution for a few seconds, washing with tap water, blueing with hematoxylin blueing solution, washing with running water;

[0060] Step 9: Dehydration and sealing:

[0061] Put the slices into 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, n-butanol for 5 minutes, and xylene for 5 minutes to dehydrate and make them transparent. Take them out and dry them slightly, and seal them with neutral gum.

[0062] Step 10: Microscope inspection, image acquisition and analysis.

[0063] The results are as follows Figure 1-Figure 3 As shown, hematoxylin stains the cell nucleus in blue, and the positive expression shown by DAB is brown-yellow. Compared with the commercially available antibody diluent, the antibody diluent prepared by the present invention can not only significantly improve the specific staining of immunohistochemistry, but also reduce the background signal, and significantly improve the signal-to-noise ratio of various tissue staining results through the combination of ProClin300, bovine serum albumin and goat serum.

[0064] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0065] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An immunohistochemical antibody dilution, characterized in that: The composition comprises the following components in parts by weight: Sodium chloride 7.8-8.1 parts, potassium chloride 0.15-0.25 parts, sodium dihydrogen phosphate 3.5-3.6 parts, dipotassium hydrogen phosphate 0.25-0.3 parts, bovine serum albumin 9.5-10.5 parts, goat serum 4.5-5.5 parts, ProClin 300 0.9-1.1 parts and deionized water 950-1050 parts.

2. An immunohistochemical antibody dilution according to claim 1, characterized in that: The composition comprises the following components in parts by weight: 8 parts of sodium chloride, 0.2 parts of potassium chloride, 3.58 parts of sodium dihydrogen phosphate, 0.27 parts of dipotassium hydrogen phosphate, 10 parts of bovine serum albumin, 5 parts of goat serum, 1 part of ProClin 300 and 1000 parts of deionized water.

3. An immunohistochemical antibody dilution according to claim 1, characterized in that: The pH value of the antibody diluent is 7.

5.

4. The method for preparing an immunohistochemical antibody diluent according to claim 1, characterized in that: The following steps are involved: Weigh sodium chloride, potassium chloride, sodium dihydrogen phosphate and dipotassium hydrogen phosphate, add to deionized water, stir at room temperature to fully dissolve, then add bovine serum albumin, goat serum and ProClin 300, adjust the pH to 7.5, adjust the color of the solution, filter through a 0.22 um filter membrane, and obtain an immunohistochemical antibody diluent.

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