Immune cytochemical antigen repair liquid for liquid-based cells and use method of immune cytochemical antigen repair liquid

Abstract

The invention relates to an immune cytochemical antigen repair liquid for liquid-based cells and a use method of the immune cytochemical antigen repair liquid, and belongs to the technical field of immunohistochemistry. The antigen repair liquid disclosed by the invention is prepared from tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid disodium salt, urea, boric acid, 2-methyl-4-isothiazolin-3-ketone, a nonionic surfactant, water and alkali metal hydroxide. The liquid-based cell antigen repairing liquid is good in repairing effect, repaired cell sheets are easy to dye, and the liquid-based cell antigen repairing liquid has the advantages of high immunohistochemical dyeing quality, accurate positive positioning, low non-specific background, clear background and good repeatability, safety and stability, so that the effects of improving the antigen detection rate, sterilizing and removing enzyme are achieved; therefore, the immunocytochemical staining of the liquid-based cytology sample can be adapted.

Description

Technical Field

[0001] This invention relates to an immunocytochemical antigen repair solution for liquid-based cells and its application method, belonging to the field of immunohistochemistry technology. Background Technology

[0002] Immunohistochemistry is a rapidly developing diagnostic technique in pathology in recent years. As an important auxiliary diagnostic method, it is receiving increasing attention in clinical applications, and the requirements for staining quality are also rising. Because tissue or cell slides are fixed using chemical reagents during preparation, some antigens may not be detected during subsequent staining, leading to the concept of antigen "masking or blocking." In this case, the antigen (or a specific epitope) is hidden, preventing antibodies that recognize it from binding. To address this issue and better expose the antigen, antigen retrieval is employed. Therefore, antigen retrieval plays a crucial role in the entire immunohistochemical staining process, and the quality of the retrieval conditions directly determines the quality of the final staining results.

[0003] Ethanol fixative is commonly used in cytopathology as an ideal fixative for liquid-based cytology samples (such as cervical exfoliated liquid-based cytology samples) (based on 95% ethanol). Cell samples must be fixed before subsequent processing and testing can be performed. Ethanol fixative causes protein precipitation and coagulation, i.e., protein denaturation, altering its internal molecular structure and properties. It also changes the three-dimensional conformation of antigenic proteins, distorting or burying antigenic determinants that were originally exposed on the protein surface, preventing them from binding to immunohistochemical antibodies. Therefore, an antigen retrieval step is necessary to hydrolyze and renature the proteins.

[0004] Ethanol-fixed liquid-based cell slides contain inflammatory cells, blood samples, microorganisms, bacteria, fungi, mucus, and various fermentation enzymes. If traditional antigen retrieval solutions (such as citrate antigen retrieval solution or EDTA antigen retrieval solution) for formaldehyde-fixed tissue sections are used, satisfactory immunohistochemical results will not be obtained, especially severe non-specific background staining, which will affect the accuracy of diagnosis. Summary of the Invention

[0005] The purpose of this invention is to provide an immunocytochemical antigen retrieval solution for liquid-based cells and its method of use, in order to solve the problem of non-specific background staining when using traditional antigen retrieval solutions for formaldehyde-fixed tissue sections to treat ethanol-fixed liquid-based cells.

[0006] This invention provides an immunocytochemical antigen retrieval solution for liquid-based cell immunotherapy, comprising tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, a nonionic surfactant, water, and an alkali metal hydroxide; wherein the concentration of tris(hydroxymethyl)aminomethane is 0.60~0.65 g / L, the concentration of disodium ethylenediaminetetraacetate is 0.35~0.40 g / L, the concentration of urea is 0.2~0.3 g / L, the concentration of boric acid is 0.06~0.08 g / L, and the concentration of 2-methyl-4-isothiazolin-3-one is 0.01~0.02 g / L; the nonionic surfactant is a nonionic polymer or a composition of a nonionic polymer and an alkanolamine compound, wherein the alkanolamine compound is triisopropanolamine or tetrahydroxyethylenediamine, and the chemical structure of the tetrahydroxyethylenediamine compound is as follows: .

[0007] Preferably, the nonionic polymer is Tween-20 or Triton X-100.

[0008] Preferably, the mass ratio of the nonionic polymer to the alkanolamine compound is 0.4~0.5:0.15~0.2.

[0009] Preferably, the pH value of the antigen retrieval solution is 8.5~9.0; and the alkali metal hydroxide is sodium hydroxide.

[0010] Preferably, the preparation method of the tetrahydroxyethylenediamine compound is as follows: Ethylamine-butanol and ethylene oxide are reacted at 90-95°C for 7-8 hours under sodium hydroxide catalysis. After cooling to room temperature, the mixture is filtered. The filtrate is distilled under reduced pressure to obtain a crude product. The crude product and ethanol are heated to 70°C at a mass ratio of 1:3, and stirred until the crude product is fully dissolved. The mixture is then filtered while hot, and the filtrate is cooled to 5°C. After precipitating a solid product, the filtrate is filtered. The filtered solid is dried to obtain a polyol compound. The molar ratio of ethylamine-butanol to ethylene oxide is 1:2.1, and the mass of sodium hydroxide is 0.1% of the sum of the masses of ethylamine-butanol and ethylene oxide.

[0011] Preferably, the preparation method of the immunocytochemical antigen repair solution for liquid-based cells is as follows: dissolve the prescribed amounts of tris(hydroxymethyl)aminomethane and disodium ethylenediaminetetraacetate in water, then add the prescribed amounts of urea, boric acid and 2-methyl-4-isothiazolin-3-one, mix well, adjust the pH to the set value with alkali metal hydroxide, and finally add a nonionic surfactant to obtain the immunocytochemical antigen repair solution for liquid-based cells.

[0012] The present invention also provides a method for using the immunocytochemical antigen repair solution for liquid-based cells as described above, comprising the following steps: collecting liquid-based cell samples onto a glass slide, fixing them with ethanol, then heating and repairing them with the immunocytochemical antigen repair solution for liquid-based cells, followed by washing and dehydration.

[0013] Preferably, liquid-based cell samples are collected onto a glass slide and centrifuged using gradient centrifugation or density separation and natural sedimentation.

[0014] Preferably, the ethanol fixation time is 10-15 minutes.

[0015] Preferably, the temperature for the heat repair is 95℃~100℃, and the time is 16~20min.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The liquid-based cell antigen repair solution of the present invention has good repair effect, and the cell slices after repair are easy to stain. It has the advantages of high quality of immunohistochemical staining, accurate positive localization, low non-specific background, clear background, reproducibility, safety and good stability, thereby improving the antigen detection rate, sterilization and enzyme removal, so as to adapt to the immunocytochemical staining of liquid-based cytology samples.

[0017] (2) The antigen retrieval solution of the liquid-based cell method of the present invention uses tris(hydroxymethyl)aminomethane as an alkaline buffer, and is compounded with disodium ethylenediaminetetraacetate (EDTA) and boric acid. These three basic components constitute a stable TBE (Tris-Boric acid-EDTA) antigen retrieval solution buffer system. Ethylenediaminetetraacetic acid (EDTA) is a metal ion chelating agent, which can chelate metal ions (such as Ca2+, Ca2+, and ... 2+ Mg 2+Mechanisms such as [list of mechanisms] promote the dissociation of protein cross-linked structures, thereby releasing the cross-linking bonds between proteins, restoring the proteins to their natural extended state, and re-exposing the masked antigenic determinants, enhancing the affinity between immunohistochemical antibodies and antigens, and improving antigen detection rates. EDTA is a strong chelating agent that can form stable complexes with boron ions in boric acid. Simultaneously, boric acid, as a disinfectant, can effectively remove the influence of inflammatory cells in the sample. Urea, as an auxiliary reagent for antigen retrieval, can break the chemical bonds between molecules, releasing the cross-linking between proteins, allowing the peptide chains of proteins to unfold from a tightly folded state, thereby exposing the antigenic determinants. 2-Methyl-4-isothiazolin-3-one is a broad-spectrum bactericide that can effectively remove background staining caused by bacteria, fungi, microorganisms, and enzymes in the sample. Its advantages include minimal impact on the active ingredients in the antigen retrieval solution, good thermal stability, and low toxicity. Tween-20 or Triton X-100 have a permeabilizing effect, increasing cell membrane permeability. The triisopropanolamine and tetrahydroxyethylenediamine compounds have branched structures that can increase the viscosity of the antigen retrieval solution, enhance the affinity between antibodies and antigens, and inhibit the evaporation of water molecules under high temperature and high pressure conditions, raise the boiling point, intensify the movement of liquid molecules, improve heat transfer efficiency and heat absorption for antigen retrieval, thereby improving the antigen retrieval effect.

[0018] (3) The liquid-based cell antigen repair solution of the present invention uses heating repair during use. The denatured protein is hydrolyzed and refolded at high temperature through heat treatment. Heat treatment can accelerate molecular motion, further destabilize the cross-linked structure between proteins, correct the distortion of protein peptide chains, and restore the antigen epitope to a conformation close to the natural state. This synergistic effect of physical and chemical methods can more thoroughly expose the antigen and kill bacteria and remove enzymes, significantly improving the immunohistochemical staining effect. The heat antigen retrieval process is a protein hydrolysis and renaturation process. Heat induction, through EDTA-boric acid chelation, solution pH, reaction time, and temperature, continuously disrupts protein cross-linking caused by ethanol fixation, restoring the natural spatial structure of antigenic epitopes and re-exposing intracellular antigenic determinants in tissues. This improves the antigen detection rate. At the same time, it greatly reduces interference from inflammatory cells, blood samples, microorganisms, bacteria, fungi, mucus, enzymes, and other substances, reduces non-specific background staining, enhances signal intensity, improves the accuracy of immunoassay, reduces the false negative rate, and improves the quality of liquid-based cytochemical staining. It is widely applicable to various gynecological or non-gynecological exfoliative cytology samples, as well as immunocytochemical staining slides prepared by manual, semi-automatic, or fully automatic equipment. Attached Figure Description

[0019] Figure 1 The infrared spectrum of the tetrahydroxyethylenediamine compound prepared in Example 6 of this invention; Figure 2This is a negative control staining image of a liquid-based cell sample treated with commercially available EDTA antigen retrieval solution in this invention; Figure 3 This is a negative control staining image of a liquid-based cell sample treated with the antigen retrieval solution of Example 1 in this invention; Figure 4 This is a staining image of a p16(INK4a) antibody positive control treated with the antigen retrieval solution of Example 1 in this invention. Detailed Implementation

[0020] The following examples are intended to further illustrate the content of the present invention, rather than to limit the scope of protection of the present invention.

[0021] I. Specific embodiments of the immunocytochemical antigen repair solution for liquid-based cells of the present invention are as follows: Example 1

[0022] The immunocytochemical antigen retrieval solution for liquid-based cell therapy in this embodiment is composed of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, Tween-20, deionized water, and an alkali metal hydroxide. The concentrations of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, and Tween-20 are 0.55 g / L. The alkali metal hydroxide is sodium hydroxide, and the pH of the antigen retrieval solution is 8.5.

[0023] The method for preparing the immunocytochemical antigen repair solution for liquid-based cell therapy in this embodiment includes the following steps: dissolving 0.605g of tris(hydroxymethyl)aminomethane and 0.372g of disodium ethylenediaminetetraacetate in 900mL of deionized water, stirring until the solids are fully dissolved, then adding 0.2g of urea, 0.06g of boric acid, and 0.01g of 2-methyl-4-isothiazolin-3-one, stirring until the solids are fully dissolved, adjusting the pH of the solution to 8.5 with NaOH, then adding 0.55g of Tween-20, and finally bringing the volume to 1000mL with deionized water to obtain the immunocytochemical antigen repair solution for liquid-based cell therapy.

[0024] Example 2

[0025] The immunocytochemical antigen retrieval solution for liquid-based cell therapy in this embodiment is composed of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, Tween-20, deionized water, and an alkali metal hydroxide. The concentrations of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, and Tween-20 are 0.01 g / L and 0.55 g / L, respectively. The alkali metal hydroxide is sodium hydroxide, and the pH of the antigen retrieval solution is 9.0.

[0026] The method for preparing the immunocytochemical antigen repair solution for liquid-based cell therapy in this embodiment includes the following steps: dissolving 0.605g of tris(hydroxymethyl)aminomethane and 0.372g of disodium ethylenediaminetetraacetate in 900mL of deionized water, stirring until the solids are fully dissolved, then adding 0.2g of urea, 0.06g of boric acid, and 0.01g of 2-methyl-4-isothiazolin-3-one, stirring until the solids are fully dissolved, adjusting the pH of the solution to 9.0 with NaOH, then adding 0.55g of Tween-20, and finally bringing the volume to 1000mL with deionized water to obtain the immunocytochemical antigen repair solution for liquid-based cell therapy.

[0027] Example 3

[0028] The immunocytochemical antigen retrieval solution for liquid-based cell therapy in this embodiment is composed of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, Triton X-100, deionized water, and an alkali metal hydroxide. The concentrations of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, and Triton X-100 are 0.55 g / L. The alkali metal hydroxide is sodium hydroxide, and the pH of the antigen retrieval solution is 8.5.

[0029] The method for preparing the immunocytochemical antigen repair solution for liquid-based cell therapy in this embodiment includes the following steps: dissolving 0.605g of tris(hydroxymethyl)aminomethane and 0.372g of disodium ethylenediaminetetraacetate in 900mL of deionized water, stirring until the solids are fully dissolved, then adding 0.2g of urea, 0.06g of boric acid, and 0.01g of 2-methyl-4-isothiazolin-3-one, stirring until the solids are fully dissolved, adjusting the pH of the solution to 8.5 with NaOH, then adding 0.55g of Triton X-100, and finally bringing the volume to 1000mL with deionized water to obtain the immunocytochemical antigen repair solution for liquid-based cell therapy.

[0030] Example 4

[0031] The immunocytochemical antigen retrieval solution for liquid-based cell therapy in this embodiment is composed of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, a polyol compound, Triton X-100, deionized water, and an alkali metal hydroxide. The concentrations of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, and 2-methyl-4-isothiazolin-3-one are 0.01 g / L, and Triton X-100 is 0.55 g / L. The alkali metal hydroxide is sodium hydroxide, and the pH of the antigen retrieval solution is 9.0.

[0032] The method for preparing the immunocytochemical antigen repair solution for liquid-based cell therapy in this embodiment includes the following steps: dissolving 0.605g of tris(hydroxymethyl)aminomethane and 0.372g of disodium ethylenediaminetetraacetate in 900mL of deionized water, stirring until the solids are fully dissolved, then adding 0.2g of urea, 0.06g of boric acid, and 0.01g of 2-methyl-4-isothiazolin-3-one, stirring until the solids are fully dissolved, adjusting the pH of the solution to 9.0 with NaOH, then adding 0.55g of Triton X-100, and finally bringing the volume to 1000mL with deionized water to obtain the immunocytochemical antigen repair solution for liquid-based cell therapy.

[0033] Example 5

[0034] The immunocytochemical antigen retrieval solution for liquid-based cell therapy in this embodiment is composed of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, triisopropanolamine, Tween-20, deionized water, and an alkali metal hydroxide. The concentrations of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, triisopropanolamine, and Tween-20 are 0.4 g / L. The alkali metal hydroxide is sodium hydroxide, and the pH of the antigen retrieval solution is 8.5.

[0035] The method for preparing the immunocytochemical antigen repair solution for liquid-based cell therapy in this embodiment includes the following steps: Dissolve 0.605g of tris(hydroxymethyl)aminomethane and 0.372g of disodium ethylenediaminetetraacetate in 900mL of deionized water, and stir until the solids are fully dissolved. Then add 0.2g of urea, 0.06g of boric acid, and 0.01g of 2-methyl-4-isothiazolin-3-one, and stir until the solids are fully dissolved. Adjust the pH of the solution to 8.5 with NaOH. Then add 0.15g of triisopropanolamine and 0.4g of Tween-20. Finally, bring the volume to 1000mL with deionized water to obtain the immunocytochemical antigen repair solution for liquid-based cell therapy.

[0036] Example 6

[0037] The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this embodiment and the immunocytochemical antigen repair solution for liquid-based cells in Example 5 is that triisopropanolamine is replaced with a tetrahydroxyethylenediamine compound in the immunocytochemical antigen repair solution for liquid-based cells in this embodiment. The chemical structure of the tetrahydroxyethylenediamine compound is as follows: ; The preparation method of the polyol compound is as follows: Ethylaminobutanol, sodium hydroxide, and n-butanol are added to a high-pressure reactor. Nitrogen gas is then introduced into the reactor, and stirring and heating are started. When the temperature of the material in the reactor reaches 95°C, ethylene oxide is introduced into the reactor. After the ethylene oxide is completely introduced, the reaction is maintained at this temperature for 7 hours. When the pressure in the reactor no longer changes, the mixture is cooled to room temperature, filtered, and the filtrate is distilled under reduced pressure to remove n-butanol, yielding a crude product. The crude product and ethanol are heated to 70°C at a mass ratio of 1:3, and stirred until the crude product is fully dissolved. The mixture is then filtered while hot to remove insoluble impurities. The filtrate is cooled to 5°C, and a solid product precipitates. This solid is then filtered while cold, and the solid obtained by vacuum filtration is placed in a 50°C oven and dried for 8 hours to obtain the polyol compound. The infrared spectrum of the polyol compound is shown below. Figure 1As shown (Test conditions: KBr tableting method; wavenumber range: 4000~400cm2), -1 The resolution is 4cm. -1 The molar ratio of ethylbutanol to ethylene oxide is 1:2.1, the mass of sodium hydroxide is 0.1% of the sum of the masses of ethylbutanol and ethylene oxide, and the mass of butanol is 5 times the mass of ethylbutanol.

[0038] Comparative Example 1 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 1 is that the concentration of boric acid in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example is 0.

[0039] Comparative Example 2 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 1 is that boric acid is replaced with salicylic acid in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0040] Comparative Example 3 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 1 is that boric acid is replaced with citric acid in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0041] Comparative Example 4 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 1 is that the concentration of 2-methyl-4-isothiazolin-3-one in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example is 0.

[0042] Comparative Example 5 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 1 is that 2-methyl-4-isothiazolin-3-one is replaced with 5-chloro-2-methyl-4-isothiazolin-3-one in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0043] Comparative Example 6 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 1 is that 2-methyl-4-isothiazolin-3-one is replaced with 1,2-benzisothiazolin-3-one in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0044] Comparative Example 7 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 5 is that triisopropanolamine is replaced with pentaerythritol in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0045] Comparative Example 8 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 5 is that triisopropanolamine is replaced with glycerol in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0046] Comparative Example 9 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 5 is that triisopropanolamine is replaced with triethanolamine in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0047] Comparative Example 10 The only difference between the immunocytochemical antigen repair solution for liquid-based cells in this comparative example and the immunocytochemical antigen repair solution for liquid-based cells in Example 5 is that triisopropanolamine is replaced with ethambutol in the immunocytochemical antigen repair solution for liquid-based cells in this comparative example.

[0048] II. Specific embodiments of the method of using the immunocytochemical antigen repair solution for liquid-based cells of the present invention are as follows: Liquid-based cell samples were first collected onto glass slides using density separation and natural sedimentation. After fixation with 95% ethanol for 15 minutes, the slides were placed in a layered plastic slide holder and rinsed briefly with water. 1500 mL of the immunocytochemical antigen retrieval solution from Example 1 was placed in a flat-bottomed stainless steel pot. The pot was covered and heated to boiling on an induction cooker. The slide holder, along with the cell slides, was placed in and completely submerged in the boiling retrieval solution. The induction cooker was set to minimum power to maintain a gentle boil, with the temperature controlled between 95°C and 100°C. The pot was covered and heated for another 20 minutes at minimum power. The pot was then removed from the induction cooker, the lid was opened, and the slides were allowed to cool naturally for 10 minutes. The slides were then rinsed under running water for 2 minutes, followed by dehydration with 70% ethanol for 1 minute to thoroughly remove any residual antigen retrieval solution. After rinsing with water, the slides were soaked in PBS buffer to prevent drying, awaiting subsequent immunohistochemical staining.

[0049] Experimental Example To examine the efficacy of the immunocytochemical antigen repair solutions used in the various embodiments and comparative examples for liquid-based cell immunotherapy, cervical exfoliated liquid-based cell samples were first collected onto glass slides using density separation and natural sedimentation. After fixation with 95% ethanol for 15 minutes, the slides were placed in a layered plastic slide holder and rinsed briefly with water. 1500 mL of commercially available EDTA antigen repair solution and the immunocytochemical antigen repair solutions used in the various embodiments or comparative examples for liquid-based cell immunotherapy were placed in a covered stainless steel container, sealed, and heated to boiling on an induction cooker. The slide holder, along with the cell slides, was then placed in and completely submerged in the boiling repair solution. Meanwhile, add 2000mL of water to a stainless steel pot and heat it to boiling on an induction cooker. Then, place the stainless steel container into the boiling water in the stainless steel pot, turn the induction cooker to the minimum power to keep the repair solution in a gentle boiling state, and maintain the temperature between 95℃ and 100℃. Cover the pot and continue heating at the minimum power for 20 minutes. Then, remove the stainless steel pot from the induction cooker, open the lid, and let it cool naturally for 30 minutes until the temperature of the antigen repair solution drops below 60℃. Take out the slide and rinse it with running water for 2 minutes, then dehydrate it with 70% ethanol for 1 minute to thoroughly remove the residual antigen repair solution. Rinse with water again, and then perform immunohistochemical staining.

[0050] The staining results of liquid-based cell samples corresponding to the blank control group (without antibody reagent staining) treated with commercially available EDTA antigen retrieval solution are as follows: Figure 2 As shown, the staining results of the liquid-based cell samples of the negative control sample treated with the immunocytochemical antigen retrieval solution used in Example 1 are as follows: Figure 3 As shown, the staining results of liquid-based cell samples treated with the immunocytochemical antigen retrieval solution for liquid-based cells in Example 1, which was a positive control for p16(INK4a) antibody, are as follows: Figure 4 As shown. By Figure 2 It can be seen that in the staining images obtained by treating liquid-based cell samples with traditional antigen retrieval solutions, most cells exhibit severe non-specific background staining, while the staining images of the negative control samples obtained by treating with the antigen retrieval solution of Example 1 show no non-specific background staining. Figure 4 It can be seen that the p16(INK4a) antibody positive control of liquid-based cell samples treated with the antigen retrieval solution of Example 1 was stained and located in the cell nucleus and cytoplasm or only the cytoplasm.

[0051] To further quantify and evaluate the repair effect of the antigen repair solutions in each embodiment and comparative example, the stained sections were observed under a microscope. Ten high-power fields were selected from each section, and 100 cells were counted in each high-power field. The number of positive cells in all liquid-based cells (staining brownish-yellow or brownish-red) was calculated using an image acquisition and analysis system. The percentage of positive cells to the total number of tumor cells was calculated to obtain the positive rate. The experimental results are shown in Table 1.

[0052] Table 1. Positive staining rate of liquid-based cytology cells after antigen retrieval solution. Antigen repair solution p16(INK4a) Antigen repair solution p16(INK4a) Example 1 89 Comparative Example 3 25 Example 2 86 Comparative Example 4 31 Example 3 88 Comparative Example 5 42 Example 4 87 Comparative Example 6 39 Example 5 96 Comparative Example 7 48 Example 6 99 Comparative Example 8 51 Comparative Example 1 11 Comparative Example 9 74 Comparative Example 2 20 Comparative Example 10 37 As shown in Table 1, the antigen retrieval solution of the present invention has a good retrieval effect on ethanol-fixed liquid-based cells. The positive rate of cells after retrieval staining is greater than 85%, showing good immunohistochemical staining results.

[0053] As demonstrated in Example 1 and Comparative Examples 1-6, boric acid and 2-methyl-4-isothiazolin-3-one in the antigen retrieval solution of the present invention have a significant impact on the retrieval results. Boric acid, as a disinfectant, can effectively remove the influence of inflammatory cells in the sample. Urea, as an auxiliary reagent for antigen retrieval, can break the chemical bonds between molecules, thereby breaking the cross-linking between proteins and allowing the peptide chains of proteins to unfold from a tightly folded state, thus exposing the antigenic determinants. 2-methyl-4-isothiazolin-3-one is a broad-spectrum bactericide that can effectively remove background staining caused by bacteria, fungi, microorganisms, and enzymes in the sample. Its advantages are that it has little impact on the active ingredients in the antigen retrieval solution, good thermal stability, and low toxicity. In contrast, 5-chloro-2-methyl-4-isothiazolin-3-one and 1,2-benzisisothiazolin-3-one may have a greater impact on the active ingredients in the antigen retrieval solution, resulting in poor staining effects and a low positive rate.

[0054] As shown in Example 1 and Comparative Examples 7-10, the addition of triisopropanolamine or tetrahydroxyethylenediamine compounds to the antigen retrieval solution further improves the retrieval effect on ethanol-fixed liquid-based cells. This may be because the tertiary amine groups in the triisopropanolamine or tetrahydroxyethylenediamine compounds can enhance the surface activity of the retrieval solution and increase its affinity for tissues. The branched structure can increase the viscosity of the antigen retrieval solution, enhance the affinity between antibodies and antigens, and inhibit the evaporation of water molecules under high temperature and high pressure, raise the boiling point, intensify the movement of liquid molecules, improve heat transfer efficiency and heat absorption for antigen retrieval, thereby improving the antigen retrieval effect.

Claims

1. An immunocytochemical antigen retrieval solution for liquid-based cell immunotherapy, characterized in that, It is composed of tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, urea, boric acid, 2-methyl-4-isothiazolin-3-one, a nonionic surfactant, water, and an alkali metal hydroxide; the concentration of tris(hydroxymethyl)aminomethane is 0.60~0.65 g / L, the concentration of disodium ethylenediaminetetraacetate is 0.35~0.40 g / L, the concentration of urea is 0.2~0.3 g / L, the concentration of boric acid is 0.06~0.08 g / L, and the concentration of 2-methyl-4-isothiazolin-3-one is 0.01~0.02 g / L; the nonionic surfactant is a nonionic polymer or a composition of a nonionic polymer and an alkanolamine compound, wherein the alkanolamine compound is triisopropanolamine or tetrahydroxyethylenediamine, and the chemical structure of the tetrahydroxyethylenediamine compound is as follows: 。 2. The immunocytochemical antigen repair solution for liquid-based cells as described in claim 1, characterized in that, The nonionic polymer is Tween-20 or Triton X-100.

3. The immunocytochemical antigen repair solution for liquid-based cells as described in claim 2, characterized in that, The mass ratio of the nonionic polymer to the alkanolamine compound is 0.4~0.5:0.15~0.

2.

4. The immunocytochemical antigen repair solution for liquid-based cells as described in claim 1, characterized in that, The pH value of the antigen retrieval solution is 8.5~9.0; the alkali metal hydroxide is sodium hydroxide.

5. The immunocytochemical antigen repair solution for liquid-based cells as described in claim 1 or 4, characterized in that, The preparation method of the tetrahydroxyethylenediamine compound is as follows: Ethylamine-butanol and ethylene oxide are reacted at 90-95°C for 7-8 hours under sodium hydroxide catalysis. After cooling to room temperature, the mixture is filtered, and the filtrate is distilled under reduced pressure to obtain a crude product. The crude product and ethanol are heated to 70°C at a mass ratio of 1:3 and stirred until the crude product is fully dissolved. The mixture is then filtered while hot, and the filtrate is cooled to 5°C to precipitate a solid product. The precipitated solid is then filtered, and the solid obtained is dried to obtain a polyol compound. The molar ratio of ethylamine-butanol to ethylene oxide is 1:2.1, and the mass of sodium hydroxide is 0.1% of the sum of the masses of ethylamine-butanol and ethylene oxide.

6. The immunocytochemical antigen repair solution for liquid-based cells as described in claim 1, characterized in that, The preparation method of the immunocytochemical antigen repair solution for liquid-based cells is as follows: Dissolve the prescribed amounts of tris(hydroxymethyl)aminomethane and disodium ethylenediaminetetraacetate in water, then add the prescribed amounts of urea, boric acid and 2-methyl-4-isothiazolin-3-one, mix well, adjust the pH to the set value with alkali metal hydroxide, and finally add a nonionic surfactant to obtain the immunocytochemical antigen repair solution for liquid-based cells.

7. A method of using an immunocytochemical antigen repair solution for liquid-based cell therapy as described in any one of claims 1-6, characterized in that, Includes the following steps: Liquid-based cell samples were collected onto glass slides, fixed with ethanol, and then heated and repaired using an immunocytochemical antigen retrieval solution for liquid-based cells, followed by washing and dehydration.

8. The method of using the immunocytochemical antigen repair solution for liquid-based cells as described in claim 7, characterized in that, The temperature for the heat repair is 95℃~100℃, and the time is 16~20min.

9. The method of using the immunocytochemical antigen repair solution for liquid-based cells as described in claim 7, characterized in that, The ethanol fixation time is 10-15 minutes.

10. The method of using the immunocytochemical antigen repair solution for liquid-based cells as described in claim 7, characterized in that, Liquid-based cell samples were collected onto glass slides and centrifuged using gradient centrifugation or density separation and natural sedimentation.

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