Secondary antibody diluent suitable for full-automatic immunohistochemical staining instrument

Abstract

The invention discloses a secondary antibody diluent suitable for a full-automatic immunohistochemical stainer. The secondary antibody diluent comprises the following components in percentage by mass: 0.1%-1% of Tris, 0.1%-1% of sodium chloride, 0.01%-0.1% of a stabilizer, 0.01%-0.05% of Tween-20, 0.01%-0.05% of a bacteriostatic agent, 1%-5% of trehalose, 0.5%-1.5% of a protein protective agent A, 0.01%-0.05% of a protein protective agent B, 0.01%-0.05% of a tackifying aqueous solvent and the balance of pure water. The secondary antibody diluent disclosed by the invention is non-toxic, harmless, difficult to volatilize, good in stability and excellent in slide preparation effect when being matched with a full-automatic immunohistochemical staining instrument, and can meet the requirements of clinical departments of hospitals on large-batch and high-quality pathological slide preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to a secondary antibody diluent suitable for a full-automatic immunohistochemical staining instrument. Background Art

[0002] At present, the initial diagnosis of benign and malignant tumors in clinical practice mainly relies on the staining results of pathological tissue sections. The doctor will make sections or smears of the suspected lesions, observe them under a microscope after staining, think about the causes, mechanisms, and occurrence processes of the lesions, and finally make a pathological diagnosis. In general, the process of pathological tissue preparation includes sampling, fixation, dehydration, transparency, wax immersion, embedding, slicing, dewaxing, staining, re-dehydration, re-transparency, and sealing. In the above steps, the secondary antibody incubation process plays a vital role, and the influence of choosing a suitable secondary antibody diluent on the final result is obvious. Therefore, it is necessary to study a secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument. Summary of the invention

[0003] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument, which is non-toxic, harmless, non-volatile, has good stability and excellent preparation effect when used with a fully automatic immunohistochemical staining instrument, and can meet the needs of hospital clinical departments for large-scale, high-quality pathology preparation.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: A secondary antibody diluent suitable for a full-automatic immunohistochemical staining instrument comprises the following components by mass fraction: 0.1%-1% Tris, 0.1%-1% sodium chloride, 0.01%-0.1% stabilizer, 0.01%-0.05% Tween-20, 0.01%-0.05% antibacterial agent, 1%-5% trehalose, 0.5%-1.5% protein protective agent A, 0.01%-0.05% protein protective agent B, 0.01%-0.05% viscosity-enhancing aqueous solvent, and the balance is pure water.

[0005] Preferably, the stabilizer is magnesium sulfate.

[0006] Preferably, the antibacterial agent is PC300 antibacterial agent.

[0007] Preferably, the protein protective agent A is bovine serum albumin (BSA).

[0008] Preferably, the protein protective agent B is casein.

[0009] Preferably, the viscosity-increasing aqueous solvent is polyethylene oxide (PEO).

[0010] The present invention is beneficial in that: (1) The secondary antibody diluent of the present invention is non-toxic, non-volatile, stable, and has excellent slide preparation effect when used with a fully automatic immunohistochemical staining instrument, which can meet the needs of clinical departments in hospitals for large-scale, high-quality pathology slide preparation; (2) The magnesium ions in magnesium sulfate can form multiple points of contact with proteins, increase the rigidity of proteins, and improve the stability of proteins in responding to temperature changes; trehalose can interfere with the structural overlap of enzyme molecules. At the same time, the multi-structure in trehalose can combine with the molecules on the surface of the enzyme through hydrogen bonds, thereby promoting the stability of the enzyme molecules; BSA and casein are both protein protectants that can increase the concentration of protein in the solution, protect the enzyme, prevent enzyme decomposition and nonspecific adsorption, and reduce enzyme or protein denaturation caused by harsh environments; PEO can increase the viscosity of the solution, thereby slowing down the flow of water molecules and better promoting the specific binding of antigens and antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a staining effect diagram of the paraffin-embedded prostate tissue section sample (Calponin) in Example 1; Figure 2 is a staining effect diagram of the paraffin-embedded tonsil tissue section sample (Ki67) in Example 2; Figure 3 is a staining effect diagram of the paraffin-embedded colon cancer tissue section sample (CEA) in Example 3; Figure 4 is a picture of the staining effect of the paraffin-embedded prostate tissue section sample (Calponin) in Example 4 (using the diluent that has been placed for 6 months); Figure 5 This is a staining effect diagram of the paraffin-embedded tonsil tissue section sample (Ki67) in Example 5 (using the diluent that has been stored for 6 months); Figure 6 This is a staining effect diagram of the paraffin-embedded colon cancer tissue section sample (CEA) in Example 6 (using a diluent that has been stored for 6 months); Figure 7 is a staining effect diagram of the paraffin-embedded prostate tissue section sample (Calponin) in Comparative Example 1; Figure 8 This is a staining effect diagram of the paraffin-embedded tonsil tissue section sample (Ki67) in Comparative Example 2; Fig. 9 This is a staining effect diagram of the paraffin-embedded colon cancer tissue section sample (CEA) in Comparative Example 3. DETAILED DESCRIPTION

[0012] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0013] A secondary antibody diluent suitable for a full-automatic immunohistochemical staining instrument comprises the following components by mass fraction: 0.1%-1% Tris, 0.1%-1% sodium chloride, 0.01%-0.1% magnesium sulfate, 0.01%-0.05% Tween-20, 0.01%-0.05% PC300 antibacterial agent, 1%-5% trehalose, 0.5%-1.5% BSA, 0.01%-0.05% casein, 0.01%-0.05% PEO, and the balance is pure water.

[0014] As a biological buffer reagent, Tris-sodium chloride buffer has a high solubility in water and is inert to many enzyme reactions. It can be used as a buffer system for enzyme-linked secondary antibodies. The appropriate sodium ion concentration can stabilize the activity of the enzyme well, and at the same time, as an electrolyte, it is beneficial to enhance the specific binding reaction of antigen and antibody. The magnesium ions in magnesium sulfate can form multiple points of contact with the protein, increase the rigidity of the protein, and enhance the stability of the protein in response to temperature changes. Trehalose is a non-reducing disaccharide composed of two glucose molecules, often existing in the form of a dihydrate. It is a typical stress metabolite that can form a unique protective film under harsh environments such as high temperature, high cold, high osmotic pressure, and drying and dehydration, effectively protecting the structure of biological molecules from being destroyed. Trehalose has certain commonalities in the mechanism of its protective effect on different cells, such as inhibiting the synthesis of proteins to inhibit some cell functions or activating certain cell functions to protect itself or other cells. Therefore, adding this substance to the antibody diluent can interfere with the structural overlap of enzyme molecules. At the same time, the multi-structure in trehalose can bind to the molecules on the surface of the enzyme through hydrogen bonds to promote the stability of the enzyme molecules. PC300 is a broad-spectrum antibacterial agent. At a certain concentration, it can inhibit the growth and reproduction of microorganisms such as bacteria and fungi for a long time, and will not affect the activity of enzymes such as HRP. It has no interference with the binding reaction of antibodies and is an ideal biological preservative. BSA and casein are both protein protectants that can increase the concentration of protein in the solution, protect the enzyme, prevent enzyme decomposition and non-specific adsorption, and reduce enzyme or protein denaturation caused by harsh environments. At the same time, casein is a type of biological polymer that can be used as a blocking agent to avoid antibody adsorption to a certain extent and act as a stabilizer in the diluent. PEO is a type of polyethylene oxide with good water solubility and rheological properties. It can increase the viscosity of the solution, thereby slowing down the flow rate of water molecules and better promoting the specific binding of antigens and antibodies.

[0015] Example 1. A secondary antibody dilution solution suitable for a fully automatic immunohistochemical staining instrument, comprising the following components by mass fraction: 0.25% Tris, 0.4% sodium chloride, 0.05% stabilizer, 0.02% Tween-20, 0.01% antibacterial agent, 2.5% trehalose, 0.75% protein protective agent A, 0.03% protein protective agent B, 0.02% thickening aqueous solvent, and the balance is pure water.

[0016] The steps of using the repair solution with the fully automatic immunohistochemical instrument for on-machine testing and preparation are: dewaxing, repairing, blocking, primary / secondary antibody incubation, DAB color development, and hematoxylin blueing. The secondary antibody incubation process includes the following specific steps: set the temperature to 20~40 ℃ in the on-machine program, add 150 μL of secondary antibody diluent, and react for 20~50 min. Use paraffin-embedded prostate tissue slice samples (Calponin), and the instrument runs automatically. The staining results are as follows: Figure 1 shown.

[0017] Example 2: The specific difference between this example and Example 1 is that: paraffin-embedded tonsil tissue slice samples (Ki67) are used, and the staining results are as follows: Figure 2 shown.

[0018] Example 3: The specific difference between this example and Example 1 is that: paraffin-embedded colon cancer tissue slice samples (CEA) are used, and the staining results are as follows: Figure 3 shown.

[0019] Example 4: The tissue section samples of this example are the same as those of Example 1, except that the prepared dilution solution is placed for 6 months before the staining test. The staining results are as follows: Figure 4 shown.

[0020] Example 5: The tissue section samples of this example are the same as those of Example 2, except that the prepared dilution solution is placed for 6 months before the staining test. The staining results are as follows: Figure 5 shown.

[0021] Example 6: The tissue section samples of this example are the same as those of Example 3, except that the prepared dilution solution is placed for 6 months before the staining test. The staining results are as follows: Figure 6 shown.

[0022] Comparative Example 1: The specific difference between this comparative example and Example 1 is that conventional antibody diluent is used, and the staining result is as follows: Figure 7 shown.

[0023] Comparative Example 2: The specific difference between this comparative example and Example 2 is that conventional antibody diluent is used, and the staining result is as follows: Figure 8 shown.

[0024] Comparative Example 3: The specific difference between this comparative example and Example 2 is that conventional antibody diluent is used, and the staining result is as follows: Fig. 9 shown.

[0025] pass Figures 1 to 9It can be seen that, compared with Examples 1 to 6 and Comparative Examples 7 to 9, there is no difference in the intensity of the film-making effect between the secondary antibody diluent of the present invention and the conventional antibody diluent on the market, and the background is cleaner, and the slightly yellow non-specific binding area is significantly reduced. Figures 4 to 6 and Figures 1 to 3 By comparison, there is no significant change in the intensity of the film-making effect and the background level between the diluent that has been placed for 6 months and the diluent that has just been obtained, which indicates that the antibody diluent of the present invention can maintain excellent stability.

[0026] 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 secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument, characterized in that: The invention comprises the following components by mass fraction: 0.1%~1% Tris, 0.1%~1% sodium chloride, 0.01%~0.1% stabilizer, 0.01%~0.05% Tween-20, 0.01%~0.05% antibacterial agent, 1%~5% trehalose, 0.5%~1.5% protein protective agent A, 0.01%~0.05% protein protective agent B, 0.01%~0.05% thickening aqueous solvent, and the balance is pure water.

2. A secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument according to claim 1, characterized in that: The stabilizer is magnesium sulfate.

3. The secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument according to claim 1, characterized in that: The antibacterial agent is PC300 antibacterial agent.

4. The secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument according to claim 1, characterized in that: The protein protective agent A is bovine serum albumin.

5. The secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument according to claim 1, characterized in that: The protein protective agent B is casein.

6. The secondary antibody diluent suitable for a fully automatic immunohistochemical staining instrument according to claim 1, characterized in that: The viscosity-increasing aqueous solvent is polyethylene oxide.

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