Integrated buffer for deparaffinization, hydration and antigen retrieval for IHC detection and application thereof

Abstract

The application discloses a kind of dewaxing, hydration and antigen repair integrated buffer for immunohistochemical detection, kit and application thereof, belong to immunodetection technical field.The dewaxing, hydration and antigen repair integrated buffer includes Tris Base, EDTA, nonylphenol polyoxyethylene ether, preservative, proteinase K and polyethylene glycol 2000.The dewaxing, hydration and antigen repair integrated buffer of the application is used to carry out paraffin section processing, can significantly shorten the time of immunohistochemical detection, so that antigen staining is more efficient.Immunohistochemical detection is carried out using the application, without using xylene and alcohol and other organic solvents in the process, more environmentally friendly, and, staining intensity is more obvious, more easily observed, has clinical auxiliary diagnosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunodetection technology, and particularly relates to a dewaxing, hydrating and antigen repairing integrated buffer for IHC and application thereof. BACKGROUND

[0002] Immunotherapy has shown effectiveness and safety in the treatment of various tumors and has been widely used, becoming a new milestone in the treatment of malignant tumors. However, not all patients can benefit from immunotherapy, so specific biomarkers are needed to screen suitable populations, and PD-L1 expression is the most widely used immunotherapy biomarker. A series of clinical research results have proved that the PD-L1 expression level of tumor cells and / or tumor-related immune cells is closely related to the efficacy of immunotherapy and the prognosis of patients.

[0003] At present, the detection of PD-L1 is mainly carried out by the method of immunohistochemistry (IHC), and the basic principle is that after the paraffin section is dewaxed and hydrated, the antigen is repaired with an antigen repair solution to fully expose the antigen epitopes of the tissue that are masked during the formalin fixation, and then the specific PD-L1 antibody is combined with the antigen in the tissue to form an antigen-antibody complex, and then the enzyme-labeled secondary antibody reacts with the first antibody to form an antigen-antibody-secondary antibody complex. The enzyme labeled in the secondary antibody catalyzes the substrate to produce insoluble precipitates in situ in the cell to serve as a tracer for the antigen. The basic steps of detection include baking, dewaxing, hydration, antigen repair, blocking, staining, color development, re-staining, dehydration, transparency, and mounting. The antigen repair of the PD-L1 paraffin section will directly affect the positive rate of PD-L1 and the interpretation result. Among them, dewaxing, hydration and antigen repair play an extremely important role in the whole process of immunohistochemical detection of PD-L1.

[0004] However, the pre-section processing process such as baking, dewaxing, hydration and antigen repair takes about 1.5 hours, which makes the whole process of immunohistochemical detection of PD-L1 very long, thereby affecting the detection efficiency of PD-L1. In addition, the conventional dewaxing of paraffin sections requires the use of organic solvent xylene, which has certain harmfulness to the environment and operators. SUMMARY

[0005] In order to solve at least one of the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] The first aspect of the present application provides a dewaxing, hydrating and antigen repairing integrated buffer for immunohistochemical detection, wherein the dewaxing, hydrating and antigen repairing integrated buffer comprises Tris Base, EDTA, nonylphenol polyoxyethylene ether, a preservative, proteinase K and polyethylene glycol 2000.

[0007] In some embodiments of the present application, the preservative is ProClin TM 950.

[0008] In some embodiments of the present application, the pH value of the dewaxing, hydrating and antigen-repairing integrated buffer is 9.0.

[0009] In some embodiments of the present application, the dewaxing, hydrating and antigen-repairing integrated buffer comprises Tris Base at a molar concentration of 5-20 mM, EDTA at a molar concentration of 0.5-1.5 mM, nonylphenol polyoxyethylene ether at a mass fraction of 0.25%-2%, preservative at a volume fraction of 0.3%-0.4%, proteinase K at a mass-volume ratio of 80-120 μg / mL, and polyethylene glycol 2000 at a volume fraction of 3%-6%.

[0010] In some specific embodiments of the present application, the dewaxing, hydrating and antigen-repairing integrated buffer comprises Tris Base at a molar concentration of 10 mM, EDTA at a molar concentration of 1 mM, nonylphenol polyoxyethylene ether at a mass fraction of 0.25%-2%, ProClin TM 950 at a volume fraction of 0.35%, proteinase K at a mass-volume ratio of 100 μg / mL, and polyethylene glycol 2000 at a volume fraction of 5%, with a pH value of 9.0.

[0011] The second aspect of the present application provides a preparation method of the dewaxing, hydrating and antigen-repairing integrated buffer according to the first aspect of the present application, comprising the following steps:

[0012] Tris Base, EDTA, nonylphenol polyoxyethylene ether, preservative, proteinase K, and polyethylene glycol 2000 are weighed or measured in a clean container, 70%-90% of purified water is added, and the mixture is stirred and mixed well, and then the volume is adjusted to the total volume.

[0013] Further, before adjusting the volume to the total volume, the step of adjusting the pH value to 9.0 is further included.

[0014] The third aspect of the present application provides an immunohistochemical detection kit, comprising the dewaxing, hydrating and antigen-repairing integrated buffer according to any one of the first aspect of the present application.

[0015] Further, the kit further comprises a blocking solution, a secondary antibody developing system, and / or hematoxylin. The hematoxylin here can be hematoxylin powder without being formulated, or can be a formulated hematoxylin dye solution.

[0016] In some embodiments of the present application, the immunohistochemical detection kit is used to detect whether a biological sample has PD-L1 expression, and the kit further comprises a PD-L1 antibody. Of course, the kit of the present application is a universal kit that can be used for detection of different antigens by matching different primary antibodies and secondary antibodies.

[0017] The fourth aspect of the present application provides an immunohistochemical detection method, comprising the following steps:

[0018] S1, obtaining a paraffin-embedded biological sample and slicing;

[0019] S2, adding the obtained slice to the deparaffinization, hydration and antigen retrieval integrated buffer of claim 1 at 98-100℃, and incubating for 8-10min to complete the deparaffinization, hydration and antigen retrieval of the slice;

[0020] S3, performing blocking, primary antibody incubation, secondary antibody incubation, color development and hematoxylin re-staining on the slice.

[0021] In some embodiments of the present application, before step S3, further comprising the step of cooling the deparaffinization, hydration and antigen retrieval integrated buffer to room temperature.

[0022] In some embodiments of the present application, the immunohistochemical detection method is a method for non-diagnostic and therapeutic purposes.

[0023] In some specific embodiments of the present application, the immunohistochemical detection method is used for auxiliary diagnosis.

[0024] Advantages of the present application

[0025] Compared with the prior art, the present application has the following advantages:

[0026] Using the deparaffinization, hydration and antigen retrieval integrated buffer of the present application for paraffin section processing can significantly shorten the time of immunohistochemical detection, and make antigen staining more efficient.

[0027] Using the method of the present application for immunohistochemical detection, no organic solvents such as xylene and alcohol are used in the process, which is more environmentally friendly.

[0028] Using the buffer or kit of the present application in combination with the method of the present application for detection, the staining intensity is more obvious and easier to observe, and it is useful for clinical auxiliary diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1Staining results after treatment with different buffers formulated according to Example 1 of the present application are shown. A: staining results of sections after treatment with buffer #1; B: staining results of sections after treatment with buffer #2; C: staining results of sections after treatment with buffer #3.

[0030] Figure 2 Staining results after treatment with different buffers formulated according to Example 2 of the present application are shown. A: staining results of sections after treatment with buffer #4; B: staining results of sections after treatment with buffer #5; C: staining results of sections after treatment with buffer #6; D: staining results of sections after treatment with buffer #7; E: staining results of sections after treatment with ordinary Tris-EDTA antigen retrieval solution. DETAILED DESCRIPTION

[0031] Unless otherwise indicated, all parts and percentages expressed herein are on a weight basis, and all tests and procedures are conducted in accordance with the standards and criteria synchronous with the filing date of this application. To the extent that any patent, patent application, or publication is cited in this application, the contents of all such citations are hereby incorporated by reference into this application, and their equivalents, in the event of inconsistencies between the terminology of this application and the terminology used in the above cited references, the terminology used in this application shall prevail. If a specific term used in the prior art is defined differently from any definition provided herein, the term shall be given the definition provided herein.

[0032] Numerical ranges expressed in the present application as comprising between about and / or between from about and are approximate ranges and thus include numerical values which are within the range. Numerical ranges if expressed herein encompass the range if modified to be a few off by 1 unit, for example from about 1 to about 5 or from about 6 to about 10, where the numerical limits are not shown. To the extent that numerical ranges and parameters setting forth the broadest scope of any numerical limitations recited herein are inconsistent with any definition of terms in the specification or otherwise provided herein, the inconsistency is expressly disclaimed. Except in the Examples, or where otherwise explicitly indicated, all numerical quantities in this description are by weight, unless otherwise indicated. Numerical ranges are approximations, and thus the

[0033] The terms "comprising," "including," "containing," and "having," and their derivatives, are not intended to exclude any component, step or procedure not specified, and are used to allow for the presence of additional components, steps or procedures, even though such are not necessarily present. For the avoidance of doubt, unless clearly specified otherwise, the use of the term "comprising" in this application is not intended to exclude any additional additives, adjuvants or compounds, unless clearly specified otherwise. In contrast, the term "consisting essentially of is intended to exclude any component, step or procedure not specified, other than an impurity that is incidental to the manufacture of the specified components, steps or procedures. The term "consisting of is intended to exclude any component, step or procedure not specified. The term "or" as used in this application refers to any or all possible combinations of one or more of the associated listed items, and is not intended to exclude additional additives, adjuvants or compounds.

[0034] In order to make the technical problems solved by the present application, the technical solutions and the beneficial effects clearer, the present application will be further explained in details in combination with the embodiments.

[0035] Embodiments

[0036] The following examples are presented to demonstrate preferred embodiments of the present application. Those skilled in the art will appreciate that the technology disclosed in the following examples represents the best attempts of the inventors to use the technology discovered by the inventors to practice the present application, and therefore can be considered preferred modes of practicing the present application. However, those skilled in the art will appreciate, in light of the present specification, that the particular examples disclosed herein can be modified in many ways without departing from the spirit or scope of the present application.

[0037] 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 belongs. The materials disclosed herein are cited for their disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior application or admission.

[0038] Those skilled in the art will appreciate that many of the specific embodiments of the present application described herein can be made without departing from the spirit or scope of the present application. Such equivalents are intended to be encompassed by the claims.

[0039] The experimental methods of the following examples are all routine methods unless otherwise specified. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are all purchased from routine biochemical reagent stores unless otherwise specified.

[0040] Example 1 Effect of different surfactants on immunohistochemical detection

[0041] 1. Preparation of buffers containing different surfactants

[0042] The inventors prepared buffers for immunohistochemical detection with different components in this example, and unexpectedly found that when a specific surfactant, nonylphenol polyoxyethylene ether, was selected, dewaxing, hydration and antigen repair integration treatment could be achieved. In order to verify the unexpected technical effect of nonylphenol polyoxyethylene ether, the inventors respectively used nonylphenol polyoxyethylene ether, Tween-20 and TrionX-100 as surfactants (1% v / v) to prepare buffers according to the proportions in Table 1.

[0043] Table 1 Buffer formulation with different surfactants

[0044]

[0045] The preparation method of buffers #1-#3 is as follows: accurately weigh or measure Tris Base, EDTA, nonylphenol polyoxyethylene ether or Tween-20 or TrionX-100, ProClin TM950, proteinase K, polyethylene glycol 2000 in a clean glass container, add 800 mL purified water, stir well, adjust pH to 9.0 with dilute hydrochloric acid, and then dilute to 1000 mL with purified water.

[0046] 2. Immunohistochemical detection

[0047] Immunohistochemical detection of PD-L1 was performed using the above prepared buffers #1-#3, and the other reagents required are shown in Table 2:

[0048] Table 2 Reagents for immunohistochemical detection after dewaxing, hydration and antigen repair integration treatment

[0049]

[0050] Among them, the preparation of PBST cleaning solution (1L): measure 800 mL of purified water and pour into the preparation container; weigh 8.0 g of sodium chloride, 0.2 g of potassium chloride, 2.87 g of sodium dodecyl phosphate and 0.2 g of potassium phosphate, respectively, and adjust the pH value to 7.2-7.4 after complete dissolution, then add Tween-20 1 mL and dilute to 1 L.

[0051] Take FFPE (Formalin-fixed paraffin-embedding) tissue samples, after slicing, use the above buffers #1-#3 for dewaxing, hydration and antigen repair integration operation, then add blocking solution to quench the activity of endogenous peroxidase; incubate the slice with PD-L1 rabbit monoclonal antibody for 30 minutes at room temperature; add secondary antibody color developing system and incubate according to the instructions; add hematoxylin staining solution, incubate at room temperature for 7 minutes to stop color development, and finally return to blue with PBST cleaning solution for 10 seconds.

[0052] The staining results of immunohistochemical detection using buffer #1-#3 are shown in Figure 1 As shown in Table 3, it can be seen that the staining intensity of buffer #1 containing 1% nonylphenol polyoxyethylene ether is the strongest (“+++”), while the staining intensity of buffer #2 containing 1% Tween-20 and buffer #3 containing 1% TrionX-100 is weak (“+”), and the effect is very unsatisfactory. It can be seen that using nonylphenol polyoxyethylene ether as a surfactant for dewaxing, hydration and antigen repair integrated treatment can achieve unexpected technical effects.

[0053] Table 3 Immunohistochemical detection results using buffers containing different surfactants

[0054] #1 #2 #3 Results +++ + +

[0055] Example 2 Preparation of dewaxing, hydration and antigen repair integrated buffer containing different proportions of nonylphenol polyoxyethylene ether

[0056] 1. Preparation of buffers containing different proportions of nonylphenol polyoxyethylene ether

[0057] This example provides four buffers #4-#7, all of which include Tris-Base, EDTA, nonylphenol polyoxyethylene ether, ProClin TM 950, proteinase K, polyethylene glycol 2000, and the difference is that the amount of nonylphenol polyoxyethylene ether added is different, and the specific formula is shown in Table 4:

[0058] Table 4 Formulas of buffers #4-#7 containing different proportions of nonylphenol polyoxyethylene ether

[0059]

[0060] The preparation method of buffers #4-#7 is as follows: accurately weigh or measure Tris Base, EDTA, nonylphenol polyoxyethylene ether, ProClin TM950, proteinase K, and polyethylene glycol 2000 in a clean glass container, add 800 mL of purified water, stir well, adjust the pH to 9.0 with dilute hydrochloric acid, and then dilute to 1000 mL with purified water.

[0061] From the above table, it can be seen that buffer #6 has the same formula as buffer #1 in Example 1, and in order to show the staining effects of different batches, different numbers are used to distinguish them.

[0062] 2. Preparation of ordinary Tris-EDTA antigen repair solution

[0063] As a comparison, this example also provides the preparation of an ordinary Tris-EDTA antigen repair solution, and the formula is shown in Table 5.

[0064] Table 5 Formulation of common Tris-EDTA antigen retrieval solution

[0065] Components Content (total volume of formulation 1000 mL) Tris-Base (g) 1.212 EDTA (g) 0.37 ProClin TM 950 (mL)] 3.5

[0066] The common Tris-EDTA antigen retrieval solution is prepared as follows:

[0067] Tris Base, EDTA and ProClin are accurately weighed or measured TM In a clean glass container, 800 mL of purified water is added and stirred well. The pH is adjusted to 9.0 with dilute hydrochloric acid, and then diluted to 1000 mL with purified water.

[0068] 3. Immunohistochemical detection

[0069] The immunohistochemical detection of PD-L1 is performed using the above prepared buffer solutions #4-#7 and the common Tris-EDTA antigen retrieval solution. In addition to the reagents shown in Table 2, the reagents shown in Table 6 are also included in the detection using the common Tris-EDTA antigen retrieval solution:

[0070] Table 6 Additional reagents required for immunohistochemical detection using common Tris-EDTA antigen retrieval solution

[0071] Reagent name Catalogue number Specification Supplier Dewaxing solution / 3800 mL / bottle Guangzhou Kaixiu Trade Co., Ltd. Anhydrous ethanol / 500 mL / bottle Shanghai Lingfeng Chemical Reagent Co., Ltd. 95% ethanol / 500 mL / bottle Shanghai Lingfeng Chemical Reagent Co., Ltd. 75% ethanol / / Self-made

[0072] The operation steps and time are shown in Table 7:

[0073] Table 7 Steps and time for immunohistochemical detection

[0074]

[0075]

[0076] As shown in Table 7, the total experimental time for PD-L1 detection using the deparaffinization, hydration, and antigen retrieval integrated buffer solutions #4-#7 is 102 minutes (not including the cleaning process); while the total experimental time for PD-L1 detection using the common Tris-EDTA antigen retrieval solution through the conventional method is 182 minutes (not including the cleaning process), which is shortened by 1 hour.

[0077] The results of immunohistochemical staining are shown in Figure 2 and Table 8, it can be seen that the immunohistochemical detection using the deparaffinization, hydration, and antigen retrieval integrated buffer solutions #4-#7 has relatively strong staining intensity (“+++”); while the immunohistochemical detection using the common Tris-EDTA antigen retrieval solution has relatively weak staining intensity (“++”).

[0078] Table 8 Results of immunohistochemical detection with each buffer

[0079] #4 #5 #6 #7 Routine staining Results +++ +++ +++ +++ ++

[0080] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding benefits disclosed herein.

Claims

1. An immunohistochemical detection method, characterized by, The method comprises the following steps: S1, obtaining a paraffin-embedded biological sample and slicing; S2, adding the obtained slice to a dewaxing, hydrating and antigen repairing integrated buffer at 98-100 DEG C, and incubating for 8-10 min to complete the dewaxing, hydrating and antigen repairing of the slice; S3, performing blocking, first antibody incubation, second antibody incubation, color development and hematoxylin re-staining on the slice, The solute of the dewaxing, hydrating and antigen repairing integrated buffer is composed of Tris Base, EDTA, nonylphenol polyoxyethylene ether, preservative, proteinase K and polyethylene glycol 2000, wherein the molar concentration of Tris Base is 5-20 mM, the molar concentration of EDTA is 0.5-1.5 mM, the mass fraction of nonylphenol polyoxyethylene ether is 0.25%-2%, the volume fraction of preservative is 0.3%-0.4%, the mass-volume ratio of proteinase K is 80-120 mu g / mL, the volume fraction of polyethylene glycol 2000 is 3%-6%, and the pH value of the dewaxing, hydrating and antigen repairing integrated buffer is 9.

0.

2. The immunohistochemical detection method according to claim 1, characterized in that, The preservative is ProClin TM 950.

3. The immunohistochemical detection method according to claim 1, characterized in that, In the dewaxing, hydrating and antigen repairing integrated buffer, the molar concentration of Tris Base is 10 mM, the molar concentration of EDTA is 1 mM, the mass fraction of nonylphenol polyoxyethylene ether is 0.25%-2%, the volume fraction of preservative is 0.35%, the mass-volume ratio of proteinase K is 100 mu g / mL, and the volume fraction of polyethylene glycol 2000 is 5%.

4. The immunohistochemical detection method according to any one of claims 1 to 3, characterized in that, The immunohistochemical detection method is used for detecting whether the biological sample has PD-L1 expression, and the first antibody is a PD-L1 antibody.

5. The immunohistochemical detection method according to any one of claims 1 to 3, wherein Before step S3, the method further comprises the step of cooling the dewaxing, hydrating and antigen repairing integrated buffer to room temperature.

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