Slice treating fluid and method for preserving antigen activity in slice

Abstract

The invention discloses a slice treating fluid and method for preserving antigen activity in a slice, and belongs to the technical field of immunodetection. Wherein the section is obtained by utilizing a pathological section preparation technology, and the section treatment liquid comprises a pentacyclic triterpenoid compound. By utilizing the section treating fluid disclosed by the invention, the antigen activity of the paraffin-embedded tissue section can be kept for at least 9 months when the paraffin-embedded tissue section is stored at normal temperature, and the storage life of the antigen activity in the cell slide at normal temperature can be effectively prolonged.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunohistochemical detection technology, and particularly relates to a slice processing solution and method for preserving antigen activity in a slice. BACKGROUND

[0002] Immunohistochemistry (IHC) technology locates target proteins in tissues through antigen-antibody specific reaction, and the reliability of the results thereof depends on the integrity and recognizability of epitopes in tissue slices. The traditional process requires that a specimen be fixed, embedded in paraffin and sliced as soon as possible after being taken off the body, and be stored in a dry state at low temperature (4℃) or room temperature (15-25℃), and then be dyed within 1-2 weeks. However, with the deepening of precision medicine and retrospective cohort studies, clinical and scientific research laboratories often need to perform batch dyeing, inter-laboratory quality evaluation, multi-center verification, and even re-detection after several months or years for the same batch of tissue microarrays (TMAs) or large cohort slices to supplement new markers. Thus, the demand for “batch slicing, normal-temperature storage, and on-demand dyeing” arises, but the existing preservation strategies are difficult to meet the requirement of long-term preservation of antigen activity.

[0003] After being stored at room temperature, in a dry state and in the dark for 3-4 months, the staining intensity of most antibodies decreases significantly; after being stored for 6-12 months, the positive signals of some phosphorylated proteins, transcription factors and low-abundance antigens can be reduced by 30-80%, or even completely lost. The degradation mechanism involves the following aspects: oxidative stress and protein cross-linking, which mask or change the epitope conformation; continuous aldehyde group reaction, which causes protein-protein and protein-nucleic acid cross-linking, and reduces the recognizability of antigens; room temperature fluctuations cause water adsorption on the surface of the tissue, forming a “micro-wet-micro-heat” cycle, which accelerates protein denaturation and non-specific aggregation; the distribution of static electricity on the surface of the long-stored slide changes, and during the subsequent processes of deparaffinization, hydration and heat-induced epitope retrieval (HIER), the slide is prone to be detached, the edges are prone to be dried, and the background is prone to be increased, which indirectly leads to a decrease in the signal-to-noise ratio.

[0004] The progressive loss of antigen activity not only causes the results of staining to be unable to be repeated, but also significantly increases the risk of false negatives. To avoid this problem, laboratories usually take the following remedial measures: ① placing the slice in a sealed freezer at -20℃ / -80℃, which can delay oxidation, but frequent freezing and thawing can easily cause the slice to be detached and damaged by ice crystals; ② storing in a nitrogen-filled dryer, which is too high in equipment and operation cost; ③ storing at 4℃ after being covered with a film, which is complicated to operate. The above strategies are all developed around “solid-phase low temperature / inert environment”, and cannot completely get rid of the dependence on the cold chain, and are also not conducive to remote transportation and application in resource-poor areas.

[0005] Therefore, developing a “processing solution and processing method” that can long-term preserve the antigen activity at room temperature has become the key to breaking through the bottleneck of the existing technology. SUMMARY

[0006] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0007] The first aspect of this application provides the use of pentacyclic triterpenoids in the preparation of a slide preparation solution for preserving the antigenic activity in slides obtained using pathological slide preparation techniques.

[0008] Pentacyclic triterpenoids are a class of terpenoid compounds consisting of six isoprene units (30 carbon atoms) and five rings. Pentacyclic triterpenoids are the most numerous, widely distributed, and biologically active class of triterpenoids.

[0009] The biosynthetic precursor of pentacyclic triterpenoids is 2,3-oxidosqualene. Under the action of oxidosqualene cyclase (OSC), various pentacyclic triterpenoid core structures are formed through different folding and cyclization mechanisms.

[0010] Pentacyclic triterpenoids include oleanane type (such as oleanolic acid, glycyrrhetinic acid, and notoginsenoside R1), ursane type (such as ursolic acid), lupinane type (such as betulinol and betulinic acid), tarapinane type (such as tarapinone), hopane type (such as hopane), isohopane type (such as isohopane), fernane type (such as pterene), and taraxane type (such as taraxasterol, pseudotaraxasterol, and φ-taraxasterol).

[0011] Pathological slide preparation technology refers to the entire process of preparing thin slide specimens that can be observed under a microscope by taking samples of human or animal tissues, cells, etc., through a series of complex physical and chemical treatments.

[0012] For tissue samples, pathological slide preparation techniques typically refer to paraffin-embedded tissue sections or frozen tissue sections.

[0013] Paraffin-embedded tissue sections typically involve steps such as fixation, dehydration, embedding, dehydration again, clearing, paraffin infiltration, embedding again, and sectioning. Specifically: Fixation: Use fixatives such as formalin to prevent tissue degradation; Dehydration, clearing, and paraffin impregnation: Alcohol is used to remove moisture, xylene is used to clear the tissue, and then paraffin is used to penetrate the tissue to provide support. Embedding: The tissue block is placed in molten paraffin wax, which cools to form a hard wax block; Sectioning: Use a precision microtome to cut the wax block into thin sections of 4-6 μm and mount them onto glass slides.

[0014] Frozen tissue sections do not undergo the dehydration, clearing, and paraffin embedding processes required for paraffin embedding. Instead, fresh tissue is rapidly frozen and hardened at low temperatures (-20°C to -30°C) and then directly sectioned using a cryostat.

[0015] For cell samples, smear techniques are typically used for slide preparation.

[0016] A second aspect of this application provides a slide preparation solution for preserving antigenic activity in slides obtained using pathological slide preparation techniques, wherein the slide preparation solution comprises pentacyclic triterpenoid compounds.

[0017] In some embodiments of this application, the pentacyclic triterpenoid compound is taraxasterol.

[0018] In some preferred embodiments of this application, the concentration of taraxasterol is 0.5 μg / mL to 1.5 μg / mL.

[0019] In some embodiments of this application, antibacterial agents are also included.

[0020] In some specific embodiments of this application, the antibacterial agent is selected from at least one of 5-bromo-5-nitro-1,3-dioxane, diazolidinyl urea, parabens, or their complexes. Those skilled in the art know that parabens (chemical name: para-hydroxybenzoic acid esters) are organic compounds belonging to the broad-spectrum preservative / antibacterial agents. They appear as white crystalline powder or colorless crystals, readily soluble in alcohols, ethers, and acetone, and very slightly soluble in water. Common parabens include methylparaben, ethylparaben, propylparaben, and butylparaben. The paraben complex is composed of two or more of methylparaben, ethylparaben, propylparaben, and butylparaben. In some embodiments of this application, the paraben complex is composed of methylparaben and ethylparaben, with a mixing ratio of methylparaben to ethylparaben of 0.05:0.25 to 0.08:0.12. In some embodiments of this application, the paraben compound is composed of methylparaben and propylparaben, with a compounding ratio of methylparaben to propylparaben of 0.10:0.10 to 0.25:0.05. In some embodiments of this application, the paraben compound is composed of ethylparaben and propylparaben, with a compounding ratio of ethylparaben to propylparaben of 0.05:0.15 to 0.10:0.10. In some embodiments of this application, the paraben compound is composed of methylparaben, ethylparaben, and propylparaben, with a compounding ratio of methylparaben, ethylparaben, and ethylparaben of 0.15:0.075:0.05. In some embodiments of this application, the paraben compound is composed of methylparaben, ethylparaben, propylparaben, and butylparaben, with a compounding ratio of methylparaben, ethylparaben, propylparaben, and butylparaben of 0.15:0.03:0.04:0.03.

[0021] In some preferred embodiments of this application, the antibacterial agent comprises 0.03% to 0.1% w / v parabens or a compound thereof and 0.03% to 0.1% w / v diazolidinyl urea.

[0022] In some embodiments of this application, the slicing solution is prepared using PBS or TBS containing surfactants.

[0023] In some specific embodiments of this application, the surfactant is selected from the group consisting of Tween-20, Triton X-100 and SDS.

[0024] A third aspect of this application provides a method for preserving antigen activity in a slide, wherein the slide is obtained using pathological slide preparation techniques, and includes the following steps: Add the slice treatment solution according to any one of claims 2 to 9 to the dried slices, incubate for 5 to 20 minutes, and then dry.

[0025] Compared with the prior art, this application has the following advantages: This application creatively utilizes taraxasterol to treat pathological sections, enabling antigens in tissues or cells to be better preserved at room temperature.

[0026] Using the sectioning solution of this application, paraffin-embedded tissue sections can maintain antigen activity for at least 9 months when stored at room temperature.

[0027] The slide preparation solution of this application can also effectively extend the shelf life of antigen activity in cell slides at room temperature.

[0028] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0029] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 The results of HER2 antigen detection are shown in Example 1 of this application when breast cancer tissue sections were stored at room temperature for 1 month and 3 months using the section processing solutions of formulations #1 to #3. Figure 2 The results of CD117 antigen detection are shown in Example 1 of this application when gastrointestinal stromal tumor tissue sections were preserved at room temperature for 1 month and 3 months using the sectioning solutions of formulations #1 to #3. Figure 3 The results of HER2 antigen detection are shown in Example 2 of this application when breast cancer tissue sections were stored at room temperature for 3 to 9 months using the section processing solutions of formulations #4 to #6. Figure 4 The results of CD117 antigen detection are shown in Example 2 of this application when gastrointestinal stromal tumor tissue sections were stored at room temperature for 3 to 9 months using the sectioning solutions of formulations #4 to #6. Figure 5 The results of HER2 antigen detection are shown in Example 3 of this application when liquid cell slides were stored at room temperature for 0-6 weeks using the slide preparation solution of Formula #4.

[0030] Figure 6 The results of HER2 antigen detection in breast cancer tissue sections preserved at room temperature in the section processing solution of each comparative example of this application are shown. Detailed Implementation

[0031] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0032] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided that there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0033] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0034] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0035] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0037] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0039] Example 1: Preparation and application of slicing treatment solution 1. Preparation of the sectioning solution This embodiment provides three slicing treatment solutions #1 to #3, with specific formulations shown in the table below:

[0040] The preparation methods for taraxasterol solutions of various concentrations are as follows: (1) Dissolve 0.1 mg (0.2 mg or 0.3 mg) of taraxasterol in 2 mL of ethanol (using sonication to aid dissolution) to obtain an ethanol stock solution of 0.05 mg / mL (0.1 mg / mL or 0.15 mg / mL).

[0041] (2) Take 1 mL of 0.05 mg / mL (0.1 mg / mL or 0.15 mg / mL) of clear ethanol stock solution and add it to 9 mL of PBS buffer to obtain 5 μg / mL (10 μg / mL or 15 μg / mL) taraxasterol solution.

[0042] 2. Tissue pretreatment and section preparation Fresh tissue samples from breast cancer and gastrointestinal stromal tumors were obtained and pretreated using the following methods.

[0043] Fixation: Fix the tissue samples with 10% neutral formalin fixative for 24 hours. The volume ratio of fixative to tissue sample is 10:1.

[0044] Dehydration: The fixed tissue samples were placed in 30%, 50%, 70%, 95%, and 100% ethanol solutions in sequence, and soaked in each concentration of ethanol solution for 1 hour.

[0045] Clearing: The dehydrated tissue samples were placed in xylene for clearing treatment, soaking for 1 hour each time, for a total of 2 times.

[0046] Paraffin infiltration: The transparent tissue sample is placed in molten paraffin, with the temperature controlled below 60℃, and each infiltration lasts for 1 hour, for a total of 3 times.

[0047] Embedding: The tissue sample after being impregnated with paraffin is embedded in paraffin to form a wax block.

[0048] Sectioning: Cut the embedded paraffin block into sections with a thickness of 3-4 μm (20 sections per case).

[0049] Baking: Dry the processed slices in an oven at 62°C for 1 hour.

[0050] 3. Slicing process Incubation: Remove the dried sections from the incubator, cool to room temperature, add sectioning solution, and incubate for 10 minutes after the tissue is completely covered. Five sections from each case were incubated with each of the three treatment solutions (#1~#3), for a total of 15 sections per case. The remaining five sections that were not incubated with sectioning solution served as controls.

[0051] Drying: Dry the processed slices in an oven at 62°C for 1 hour.

[0052] Storage: Place the dried slices in a sealed container and store at room temperature.

[0053] 4. Immunohistochemical detection: Immunohistochemistry was used to detect HER2 antigen in breast cancer tissue sections at month 0 (immediately after slide preparation), month 1, and month 3 of storage, and CD117 antigen in gastrointestinal stromal tumor tissue sections. The primary antibody information used is shown in the table below.

[0054] The experiments were conducted using the LYNX480 fully automated immunohistochemical staining system from Tuling (Hangzhou) Biomedical Co., Ltd., and other reagents used, such as secondary antibodies and auxiliary reagents, were all commercial kits from Tuling (Hangzhou) Biomedical Co., Ltd.

[0055] Test results as followsFigure 1 , Figure 2 And as shown in the table below:

[0056] Therefore, it can be seen that, after one month of storage, the staining of untreated pathological sections and pathological sections treated with formulas #1 to #3 remained acceptable. After three months of storage, for gastrointestinal stromal tumor tissue sections, the staining of the untreated group was unacceptable, while the staining of the groups treated with formulas #1 to #3 was acceptable, indicating that the formulas #1 to #3 have a good preservation effect on the antigen activity in gastrointestinal stromal tumor tissue sections. For breast cancer pathological sections, the staining was unacceptable regardless of whether they were untreated or treated with formulas #1 to #3, indicating that the preservation effect of formulas #1 to #3 on the activity of different antigens was inconsistent.

[0057] Example 2 Optimization of the Slicing Treatment Solution Based on the slicing solution of Formula #2 in Example 1, further optimization was made, specifically by adding Noxaprop-P-ethyl. ® NAPA-ME99 antibacterial agent (Jingmao Biotechnology, GB01023) and / or GML-2, the specific formulation is as follows:

[0058] Similarly, fresh tissue samples from breast cancer and gastrointestinal stromal tumors were used to prepare and process pathological tissue sections. The tissue pretreatment and section preparation methods were the same as in Example 1. In the section processing steps, five sections from each case tissue were incubated with each of the three formulations of processing solutions (#4~#6), that is, a total of 15 sections were processed from each case tissue. The remaining five sections that were not incubated with the section processing solution were used as backups; the remaining steps were the same as in Example 1.

[0059] HER2 antigen in breast cancer tissue sections and CD117 antigen in gastrointestinal stromal tumor tissue sections were detected by immunohistochemistry at month 0 (i.e., immediately after slide preparation), month 1, month 3, month 6, and month 9 (if staining fails at any intermediate time point, no further testing will be performed). The immunohistochemical reagents and methods are the same as in Example 1.

[0060] Test results as follows Figure 3 , Figure 4 And as shown in the table below:

[0061] As shown in the table above, after treatment with the slide preparation solutions of Formula #5 and Formula #6, the antigens in each pathological tissue slide can be preserved for at least 3 months. After treatment with the slide preparation solution of Formula #4, the antigens in each pathological tissue slide can be preserved for at least 6 months, and even after 9 months of preservation, the staining results are still acceptable.

[0062] Example 3: Liquid Cell Processing and Detection To verify the preservation effect of the slide preparation solution in Formula #4 of Example 2 on the antigen activity of liquid cell quality control samples, the inventors automatically added HER2 3+ liquid cell quality control sample (Tuling Biotechnology, catalog number: I32342) onto glass slides using a fully automated immunohistochemical staining system (model: LNYX480 PLUS), and automatically baked the slides at 45°C for 3 minutes to obtain liquid cell slides. The slide preparation solution in Formula #4 of Example 2 was then used to treat the liquid cell slides in the same way. Untreated liquid cell slides served as controls. HER2 detection was performed at weeks 0, 1, 3, and 6, and the results are as follows. Figure 5 As shown.

[0063] Depend on Figure 5 It can be seen that the immunohistochemical test results of the control liquid cell slides were unqualified after 3 weeks of storage, while the immunohistochemical test results of the liquid cell slides treated with the slide preparation solution of Formula #4 were still qualified until 6 weeks, indicating that the liquid cell slides treated with the slide preparation solution of Formula #4 can be stored for at least 1.5 months.

[0064] Comparative Example 1 The taraxasterol solution in Formula #4 was replaced with vitamin C, and the breast cancer tissue sections from Example 1 were processed in the same way. The HER2 antigen was detected one month later.

[0065] Comparative Example 2 The antibacterial agent NAPA-ME99 in formulation #4 was replaced with Proclin 950, and the breast cancer tissue sections from Example 1 were treated in the same way. HER2 antigen was detected one month later.

[0066] Comparative Example 3 Replace the GML-2 solution in Formula #4 with PC 300, and process the breast cancer tissue sections from Example 1 in the same way. Detect HER2 antigen after 1 month.

[0067] The results of the above comparative tests are as follows: Figure 6 As shown, replacing the components in the slicing solution of Formula #4 significantly reduced the preservation effect of antigen activity in breast cancer tissue slices.

[0068] Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The application of pentacyclic triterpenoids in the preparation of slide preparation solutions for preserving antigenic activity in slides, characterized in that, The slides were obtained using pathological slide preparation techniques.

2. A slide preparation solution for preserving antigen activity in slides, characterized in that, The slides were obtained using pathological slide preparation techniques, and the slide treatment solution included pentacyclic triterpenoids.

3. The slicing treatment solution according to claim 2, characterized in that, The pentacyclic triterpenoid compound is taraxasterol.

4. The slicing treatment solution according to claim 3, characterized in that, The concentration of taraxasterol is 0.5 μg / mL to 1.5 μg / mL.

5. The slicing treatment solution according to claim 2, characterized in that, It also includes antibacterial agents.

6. The slicing treatment solution according to claim 5, characterized in that, The antibacterial agent is selected from at least one of 5-bromo-5-nitro-1,3-dioxane, diazolidinyl urea, and parabens and their compounds.

7. The slicing treatment solution according to claim 6, characterized in that, The antibacterial agent includes 0.03%~0.1% w / v parabens or their compounds and 0.03%~0.1% w / v diazolidinyl urea.

8. The slicing treatment solution according to any one of claims 2 to 7, characterized in that, The slicing solution is prepared using PBS or TBS containing surfactants.

9. The slicing treatment solution according to claim 8, characterized in that, The surfactant is selected from the group consisting of Tween-20, Triton X-100 and SDS.

10. A method for preserving antigen activity in tissue sections, characterized in that, The slides were obtained using pathological slide preparation techniques, including the following steps: Add the slice treatment solution according to any one of claims 2 to 9 to the dried slices, incubate for 5 to 20 minutes, and then dry.

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