CN109839503B

CN109839503B - High-stability PD-L1 antibody reagent and application thereof

Info

Publication number: CN109839503B
Application number: CN201711208310.2A
Authority: CN
Inventors: 董艳; 许嘉森; 吴诗扬; 刘苏燕
Original assignee: Surexam Bio Tech Co Ltd
Current assignee: Surexam Bio Tech Co Ltd
Priority date: 2017-11-27
Filing date: 2017-11-27
Publication date: 2022-04-22
Anticipated expiration: 2037-11-27
Also published as: CN109839503A

Abstract

The high-stability PD-L1 antibody reagent comprises a mouse anti-human PD-L1monoclonal antibody and an antibody stabilizer, wherein the antibody stabilizer is selected from the following components: peroxidase Stabilizing Buffer, Sigma: P9209 (cat # P9209); the mouse anti-human PD-L1monoclonal antibody is selected from the group consisting of: PD-L1Monoclonal Antibody, Thermo Fisher: 14-5983-80. The high-stability PD-L1 antibody reagent provided by the invention obtains an antibody which can be stably stored at 4 ℃ through reasonable matching of an antibody stabilizer and a PD-L1monoclonal antibody, is convenient to store, avoids reduction of antibody titer caused by repeated freeze thawing, and integrates reagents required by immunohistochemical detection of PD-L1, so that immunohistochemical detection of PD-L1 is more visual, more convenient and faster.

Description

High-stability PD-L1 antibody reagent and application thereof

Technical Field

The invention relates to the field of molecular biology, and particularly relates to a PD-L1 immunohistochemical kit.

Background

Lung cancer is one of the most common malignant tumors worldwide, and its morbidity and mortality rate ends up in the tumor, and has a tendency to rise year by year. Non-small cell lung cancer (NSCLC) accounts for about 80-85% of the total lung cancer, about 75% of patients are found to be in the middle-advanced stage, the average survival period is 12.9 months, and the survival rates in 3 years and 5 years are lower, namely 19% and 11%, respectively. The current treatment modes of the non-small cell lung cancer mainly comprise surgical treatment, chemotherapy, radiotherapy and molecular targeted treatment. Surgical treatment remains the only treatment modality that may cure patients, however many non-small cell lung cancer patients often lose surgical opportunity when discovered. In recent years, some progress has been made in the treatment of NSCLC, particularly in drug therapy (such as molecular targeted therapy), but the 5-year survival rate of cured patients is not significantly improved, and the search for effective therapeutic means is urgent. In recent years, immunotherapy of tumors becomes a new hotspot after molecular targeted therapy, and with the deep research of tumor immunological mechanisms, some immunotherapy methods are introduced into the clinic, but most of the immunotherapy methods are still in an exploration stage, and the tumor therapy is still limited. Therefore, the deep research on the tumor immunity mechanism, the understanding of the tumor occurrence and development rules and the important significance for searching more effective treatment methods are realized.

Through extensive and intensive research on tumor immune tolerance and escape mechanisms, the expression abnormality of the co-stimulatory molecules involved in the immune regulation of the body plays an important role in the immune escape of tumors. The programmed death factor 1 (PD-1) and the ligand programmed death factor 1 ligand 1 (PD-L1) thereof are used as members of a CD28/B7 co-stimulatory molecule superfamily, can mediate negative co-stimulatory signals, can effectively inhibit the function and proliferation of T, B cells, and simultaneously reduce the secretion of cytokines IL-2, IL-10 and IFN-gamma, and the immune regulation involved in the inhibition pathway plays an important role in tumor immune escape and tumor microenvironment formation and is closely related to the generation and development of tumors. Blocking the PD-1/PD-L1 pathway can inhibit the formation of the tumor microenvironment and enhance endogenous anti-tumor immune responses. There are many PD-1/PD-L1 inhibitors currently in clinical trials, and the PD-1 inhibitor nivolumab was first approved by the FDA and used to treat metastatic squamous non-small cell lung cancer with disease progression during or after platinum-based chemotherapy in 3 months 2015. It has been shown that the expression of PD-L1 in tumor tissues correlates with the effectiveness of immunotherapy in NSCLC patients and also correlates well with poor prognosis and high invasiveness in NSCLC patients. Therefore, the detection of the expression of PD-L1 in NSCLC tissues can assist NSCLC patients to predict the curative effect of the PD-1/PD-L1 inhibitor.

How to accurately and rapidly detect the expression condition of the PD-L1 protein in the non-small cell lung cancer tissue is a key factor for assisting in predicting the curative effect of the PD-1/PD-L1 inhibitor. At present, methods mainly used for detecting the expression condition of PD-L1 include sequencing method, real-time quantitative PCR method, tissue immunohistochemistry method and the like. The methods have advantages and disadvantages, but the sequencing method and the real-time quantitative PCR method have the defects of complex operation, long time consumption, relatively high cost and the like, and the immunohistochemical method has simple operation, short time consumption and relatively low cost and is favorable for detecting the expression condition of PD-L1 in the non-small cell lung cancer tissue.

The tissue immunohistochemical method uses the reagent comprising mainly primary antibody, secondary antibody and color developing agent combined specifically with the protein to be detected in the tissue. Primary antibodies in conventional reagents need to be stored at higher concentrations, at temperatures of-20 ℃ or even lower, to maintain higher stability. Therefore, the primary antibody reagent or kit used in the general immunohistochemical experiment should be used immediately after dissolving and mixing the primary antibody taken out from-20 ℃ before incubating the primary antibody and diluting the primary antibody to the working concentration with PBS or similar antibody diluent. Therefore, the traditional primary antibody reagent for immunohistochemistry or other immunological experiments has the problems that the storage at the low temperature of-20 ℃ is required, a dilution step is required before the use, and the storage and the taking are inconvenient. Meanwhile, the antibody needs to be dissolved when being stored at the temperature of-20 ℃, and the stability of the antibody is deteriorated due to repeated freeze thawing, so that the titer is easily reduced.

Therefore, it is necessary to provide a PD-L1monoclonal antibody reagent which has been diluted to a working concentration, can be stored at 0 ℃ or higher, has good stability, and can be taken and used immediately.

Disclosure of Invention

Based on this, the present invention aims to provide a PD-L1monoclonal antibody reagent which has been diluted to a working concentration, can be stored at 0 ℃ or higher, has good stability, and can be taken immediately.

In order to achieve the purpose, the specific technical scheme is as follows:

a high-stability PD-L1 antibody reagent comprises a mouse anti-human PD-L1monoclonal antibody and an antibody stabilizer, wherein the antibody stabilizer is selected from the group consisting of: peroxidase Stabilizing Buffer, Sigma-P9209; the mouse anti-human PD-L1monoclonal antibody is selected from the group consisting of: PD-L1Monoclonal Antibody, Thermo Fisher: 14-5983-80.

In some of these embodiments, the concentration of the murine anti-human PD-L1monoclonal antibody in the antibody stabilizing agent is 0.4-0.8 μ g/ml.

In some of these embodiments, the murine anti-human PD-L1monoclonal antibody is present in the antibody stabilizer at a concentration of 0.4 μ g/ml.

The invention also aims to provide application of the PD-L1 antibody reagent with high stability.

In order to realize the purpose, the specific technical scheme is as follows:

the high-stability PD-L1 antibody reagent is applied to the preparation of a PD-L1 immunohistochemical kit.

The invention also aims to provide a PD-L1 immunohistochemical kit.

In order to realize the purpose, the specific technical scheme is as follows:

a PD-L1 immunohistochemical kit comprises the high-stability PD-L1 antibody reagent.

In some of these embodiments, the kit further comprises HRP-goat anti-mouse IgG conjugated to the murine anti-human PD-L1monoclonal antibody.

In some embodiments, the kit further comprises an endogenous peroxidase blocking agent and a blocking solution.

The invention also provides an application of the immunohistochemical kit. The specific scheme is as follows:

the application of the kit in preparing a detection reagent for predicting the curative effect of the PD-L1 inhibitor.

The application of the kit in preparing products for predicting the treatment effect of the non-small cell lung cancer.

Compared with the prior art, the invention has the following advantages and effects:

the invention screens and uses the Peroxidase Stabilizing Buffer, Sigma-P9209, in a plurality of antibodies and antibody stabilizers through the creative work of the inventor; as Antibody stabilizers, for dilution of PD-L1Monoclonal Antibody, Thermo Fisher: 14-5983-80 monoclonal antibody, through the reasonable selection and collocation of antibody stabilizer and PD-L1monoclonal antibody, the antibody can be preserved at 4 ℃ and has high stability after being diluted to corresponding working concentration by Peroxidase Stabilizing Buffer and Sigma-P9209, thereby realizing instant use after taking, achieving the purpose of convenient storage and taking, and simultaneously avoiding the reduction of antibody titer caused by repeated freeze thawing during storage at-20 ℃.

On the other hand, the invention discovers that when the antibody is diluted to different concentrations, the storage stability of the antibody is different, and the concentration of 0.4-0.8 mu g/ml is selected as the working concentration, the antibody stability is high at the working concentration, and the staining effect of the immunohistochemical test is good. Meanwhile, the antibody reagent with specific concentration and ready to use is taken and is not required to be prepared temporarily before each use, so that the problem that the antibody is inconvenient to take in the method for preparing the antibody on site in an immunohistochemical experiment can be solved, and errors caused by concentration difference in each preparation can be avoided.

In addition, the kit provided by the invention uses the high-stability antibody, on one hand, the contained monoclonal antibody can specifically recognize PD-L1, the kit is convenient to store, and on the other hand, the required reagents for immunohistochemical detection of PD-L1 are integrated, so that the immunohistochemical detection of PD-L1 is more visual, more convenient and faster.

Drawings

FIG. 1 is the result of staining diseased lung tissue for NSCLC in example 1;

FIG. 2 is the result of staining of normal lung tissue in example 1;

FIG. 3 shows the staining results of immunohistochemical experiments using antibodies diluted with Peroxidase Stabilizing Buffer (P9209) from Sigma;

FIG. 4 shows staining results of immunohistochemical experiments using antibodies diluted in Antibody Stabilizer PBS (131500) from CANDOR Bioscience;

FIG. 5 shows the use

Staining results of immunohistochemical experiments with HRP-Stab (270500) diluted antibodies;

FIG. 6 shows the staining results of immunohistochemical experiments performed when antibodies were now formulated;

FIG. 7 shows staining results of immunohistochemical experiments using PBS-diluted antibodies;

FIG. 8 shows staining results of an anti-PD-L1 antibody immunohistochemical experiment purchased from R & D;

FIG. 9 shows the staining results of immunohistochemical experiments performed after the antibody of the present invention was diluted at a ratio of 1: 5000;

FIG. 10 shows the staining results of immunohistochemical experiments performed after the antibody of the present invention was diluted at a ratio of 1: 8000;

FIG. 11 shows the staining results of immunohistochemical experiments performed after the antibody of the present invention was diluted at a ratio of 1: 10000;

FIG. 12 shows the staining results of immunohistochemical experiments performed after dilution of the antibody of the present invention at a ratio of 1: 15000;

FIG. 13 shows the effect of the incubation time of PD-L1 primary antibody on the immunohistochemical detection in example 4;

FIG. 14 shows the effect of the incubation time of the secondary antibody on the immunohistochemical detection effect in example 5.

Detailed Description

The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and do not limit the scope of the present invention.

Example 1: PD-L1 immunohistochemical kit for detecting expression of PD-L1 in NSCLC tissue sections

The embodiment provides an immunohistochemical kit for detecting PD-L1 and application thereof, wherein the kit comprises the following reagents:

endogenous peroxidase blocker, 3% (v/v) H₂O₂A solution;

high stability antibody reagent: murine anti-human PD-L1 Antibody PD-L1Monoclonal Antibody, (Thermo Fisher: 14-5983-80), has been diluted to 0.5. mu.g/ml by Peroxidase Stabilizing Buffer;

HRP-goat anti-mouse IgG purchased from New Biotechnology development, Inc., Kit-0014;

5% (w/v) Bovine Serum Albumin (BSA) solution prepared by using phosphate buffer PBS as a solvent;

DAB color developing solution purchased from Mi New Biotechnology development Inc.

The detection principle of the kit is as follows: the immunological antigen and the antibody can be specifically combined based on the structural complementarity and high affinity between the two molecules, and the enzyme color developing agent for marking the antibody is developed through oxidation-reduction reaction to determine the antigen in the tissue cell, and the antigen is positioned and determined qualitatively.

Firstly, connecting an anti-human PD-L1 antibody and a PD-L1 antigen on tissues;

secondly, the HRP-goat anti-mouse IgG polymer recognizes the connected anti-human PD-L1 antibody;

thirdly, a chromogenic substrate is added, and horseradish peroxidase on the polymer can catalyze H in the DAB chromogenic solution₂O₂Decomposing to oxidize benzidine into benzidine, thereby leading the antigen sites in the tissue slices to show yellow or brown yellow coloration;

and finally, counterstaining and mounting the sample.

The presence and condition of PD-L1 on the tissue section were inferred by observing the color development under a microscope.

Two formaldehyde fixed paraffin embedded tissue sections are obtained from a pathology department of a hospital, and after pathological diagnosis, one is a normal lung tissue, and the other is a NSCLC tissue which is diagnosed and used for determining the expression of PD-L1 protein. Tissue sections were first deparaffinized and hydrated: the slices were dewaxed three times with xylene for 10 minutes each, dehydrated with gradient alcohol twice with absolute ethanol for 5 minutes each, 5min in 95% ethanol, 5min in 75% ethanol, 5min in PBS solution. Immunohistochemical staining procedure was as follows:

1. antigen retrieval

The tissue slices were placed in a boiled 1mM EDTA antigen retrieval solution (pH 9.0), heated in a microwave oven over a slow fire for 10min, and allowed to cool to room temperature.

The amount of the repairing liquid must be ensured to be always soaked in the liquid when the antigen is repaired, the section must be always kept in the liquid when the section is cooled to the room temperature after being heated, and the section cannot be taken out to be cooled in the air.

2. Blocking endogenous peroxidase

1) Washing the tissue section subjected to antigen retrieval with PBS for 3 times, and each time for 5 minutes;

2) taking out the section, throwing away and wiping off liquid around the tissue, and fixing the area to be detected on the slide by an immunohistochemical pen ring;

3) dropwise adding 3% of H in the area to be detected₂O₂And standing at room temperature for 15min to extinguish endogenous horseradish peroxidase.

4) The PBS solution was washed 3 times for 5 minutes each.

3. Sealing of

And dropwise adding confining liquid on the cell slide until the cell slide covers the area to be detected, standing at room temperature for 30min, and then throwing off the redundant liquid.

4. PD-L1 antibody incubation

1) Removing the PBS solution, adding PD-L1 antibody reagents diluted according to different dilution times, and incubating for 60 minutes at room temperature;

2) the PBS solution was washed 3 times for 5 minutes each.

5. Enzyme-labeled Polymer incubation

1) Removing the PBS solution, adding 100 mul of HRP-labeled HRP-goat anti-mouse IgG working solution, and incubating for 30 minutes at room temperature;

2) the PBS solution was washed 3 times for 5 minutes each.

6. DAB color development

And (3) sequentially dripping 1ml of stable DAB buffer solution, 50-100 mul of stable DAB substrate and 50-100 mul of stable DAB chromogen into the prepared small test tube to prepare DAB chromogenic solution, uniformly mixing and keeping out of the sun. Mixing, and adding into slices. Developing at room temperature, and controlling the developing time under a mirror, wherein the developing time is generally 5-10 minutes. The reaction was terminated by washing with distilled water.

If necessary, hematoxylin counterstaining can be performed: adding hematoxylin staining solution dropwise onto the slices, incubating for 1-2min, and washing with distilled water.

Note that: depending on the intensity of the hematoxylin staining solution acted and the length of incubation time: counterstaining results cause the nucleus to appear light blue to dark blue, and either over-staining or under-staining may compromise the determination of the correct result.

7. Dewatering, transparent and sealing sheet

Respectively soaking in 75% ethanol, 95% ethanol, anhydrous ethanol and xylene for 5 min;

and (4) dropwise adding neutral resin, and covering with a glass slide for mounting.

8. Biological microscope slide reading and result judgment

The staining results are shown in FIGS. 1 and 2. Wherein, FIG. 1 shows the staining result of lung tissue with NSCLC, PD-L1 is colored as brown part, and blue part is cell nucleus; FIG. 2 shows the result of staining normal lung tissue, with only the nucleus portion stained blue. In FIG. 2, no brown coloration exists in the normal lung tissue, and no PD-L1 expression exists in the normal lung tissue, so that the experiment system has no problem and can effectively avoid nonspecific background coloration, namely false positive. In FIG. 13, the brown portion is a positive stain for PD-L1, PD-L1 is mainly expressed in the cell membrane of tumor cells in tumor tissues, and it can be seen that the brown stain is mostly distributed around the blue nucleus. While the normal immune cells have no PD-L1 brown coloration, and are normal negative results, and false positive and false negative also do not occur. The PD-L1 shows that the PD-L1 is specifically distributed in the cell membrane of the NSCLC tissue tumor cell and shows strong positive coloring distribution, and the kit can effectively detect the PD-L1 in the NSCLC tissue.

Example 2 study of the Effect of antibody stabilizers on antibody stability

The use of Peroxidase Stabilizing Buffer (cat # P9209), Antibody Stabilizer PBS from CANDOR Bioscience (cat # 131500) and Sigma

HRP-Stab (cat # 270500) was used as an Antibody stabilizer to dilute mouse anti-human PD-L1Monoclonal Antibody, (Thermo Fisher: 14-5983-80) to 0.4. mu.g/ml, and each Antibody stabilizer was tested for its ability to stabilize the potency of the Monoclonal Antibody using an accelerated stability assay. The diluted primary antibody is stored at 37 ℃, and immunohistochemical detection and contrast staining effects are respectively carried out on B-CPAP and MCF-7 cells by using a Mixin DAB detection kit respectively at 0 day, 4 days, 7 days and 9 days. Mouse anti-human PD-L1Monoclonal Antibody (Thermo Fisher: 14-5983-80) diluted with PBS was stored at 37 ℃ for 0, 4, 7, and 9 days, respectively, and on the same day, diluted with PBS and prepared on-siteAnti-human PD-L1Monoclonal Antibody, (Thermo Fisher: 14-5983-80) was used as a control. All the above were diluted to 0.4. mu.g/ml. The immunohistochemical assay procedure was as in example 1.

It was subjected to semi-quantitative analysis, and the staining marker immune features were classified into three stages, weak (1+), medium (2+), and strong (3 +). The results are shown in FIGS. 8-11, and the corresponding semi-quantitative analysis results are shown in Table 1.

TABLE 1

As shown in FIGS. 3 to 7, FIG. 3 shows the results of immunohistochemical analysis of B-CPAP cells and MCF-7 cells using an antibody diluted with Peroxidase Stabilizing Buffer (cat # P9209) after 0, 4, 7 and 9 days at 37 ℃; FIG. 4 shows the results of immunohistochemical assays performed on B-CPAP cells and MCF-7 cells after 0, 4, 7, and 9 days at 37 ℃ using Antibody Stabilizer PBS (cat # 131500) from CANDOR Bioscience; FIG. 5 shows the use

The results of immunohistochemical experiments on B-CPAP cells and MCF-7 cells were carried out after HRP-Stab (cat # 270500) had been left at 37 ℃ for 0, 4, 7, and 9 days; FIG. 6 shows the results of immunohistochemical assays performed on B-CPAP cells and MCF-7 cells on days 0, 4, 7, and 9, after undiluted antibody was removed from the day at-20 ℃ and diluted 8000-fold with PBS; FIG. 7 is a graph showing the results of immunohistochemistry tests on B-CPAP cells and MCF-7 cells after 0, 4, 7, and 9 days of incubation at 37 ℃ using PBS diluted antibody; wherein A represents B-CPAP cells, B represents MCF-7 cells, and 1-4 correspond to the experimental results of 0, 4, 7 and 9 days respectively.

The staining results show that: since PD-L1 is highly expressed in B-CPAP cells but not in MCF-7 cells, none of the MCF-7 cells in FIGS. 3-8 had significant brown staining, effectively eliminating false positive results. In the results of B-CPAP, the staining effect can reach 2-3+ by using Peroxidase Stabilizing Buffer (Cat No. P9209) from Sigma; before use, the PD-L1 antibody is taken out from a refrigerator at the temperature of-20 ℃ and diluted to be 0.4 mu g/ml, and is used for an immunohistochemical experiment, and the staining effect is also 2-3 +; as can be seen, the effects of the two are not obviously different on

days

0, 4, 7 and 9. Therefore, the PD-L1 antibody obtained by dilution of the Peroxidase Stabilizing Buffer has stability.

Example 3: effect of antibody concentration on immunohistochemical assay Effect

Diluting according to different concentrations:

the PD-L1 antibody stock solution described in example 1 was diluted with Peroxidase Stabilizing Buffer (cat. No. P9209) at a concentration of 0.5mg/ml to a concentration of: 0.8. mu.g/ml, 0.6. mu.g/ml, 0.4. mu.g/ml and 0.2. mu.g/ml. The 4 concentrations of the antibody solutions and the anti-human PD-L1 antibody (product number MAB1561) purchased from R & D were used as controls in immunohistochemical experiments at 0, 4, 7, and 9 days at room temperature to determine the effects.

In this example, immunohistochemistry was performed using a michigan DAB kit using cultured thyroid cancer cell line B-CPAP and human breast cancer cell line MCF-7 formaldehyde-fixed paraffin-embedded tissue sections as samples to test the titer of PD-L1monoclonal antibody, and the optimal working concentration of antibody for immunohistochemistry was found. Immunohistochemistry was performed as in example 1.

The staining effect of the sections was observed under a microscope and semi-quantitatively analyzed to classify the staining marker immune features into three classes, weak (1+), medium (2+), and strong (3 +). The corresponding semi-quantitative analysis results are shown in Table 2.

TABLE 2

As in fig. 8-12, fig. 8 is PD-L1 antibody purchased from R & D, cat # s: MAB1561 immunohistochemical tests were performed using a primary antibody solution at a concentration of 15ug/ml, recommended by the trade company for immunohistochemical experiments, and used as a control group. FIG. 9 is a graph showing the results of immunohistochemistry experiments using 0.8. mu.g/ml of the PD-L1 antibody of the present invention, 0.6. mu.g/ml of the PD-L1 antibody of the present invention, 0.4. mu.g/ml of the PD-L1 antibody of the present invention, 0.4. mu.g/ml of the PD-L1 antibody of the present invention, and 0.2. mu.g/ml of the PD-L1 antibody as a primary antibody. Wherein, A is the staining result of B-CPAP cells, B is the staining result of MCF-7 cells, A1, A2, A3, A4 or B1, B2, B3 and B4 are the experimental result graphs of PD-L1 antibody as a primary antibody when the antibody is placed at room temperature for 0, 4, 7 and 9 days.

The PD-L1monoclonal antibody and the antibody stabilizer are respectively diluted into the following concentrations: 0.8. mu.g/ml, 0.6. mu.g/ml, 0.4. mu.g/ml and 0.2. mu.g/ml as primary antibody solutions, and the results of immunohistochemical tests with the antibody purchased from R & D as a control group, revealed that: four concentrations and the antibody purchased from R & D were equivalent in the immunohistochemical test on day 0 after dilution (i.e., when used as prepared), while the antibody at a concentration of 0.2 μ g/ml was less colored in the positive cells B-CPAP when used in the immunohistochemical test on day 4 after dilution, and therefore, the stability of the antibody was low at this concentration. Therefore, under the concentration of 0.4-0.8 mug/ml, the antibody stability is high, the staining effect of an immunohistochemical experiment is good, and the effect is obviously superior to that of the antibody purchased by R & D, which shows that the PD-L1monoclonal antibody can be stored under the working concentration of 0.4 mug/ml-0.8 mug/ml, and the stability is high.

Example 4: effect of PD-L1 Primary antibody incubation time on immunohistochemical detection Effect

This example uses cultured thyroid cancer cell line B-CPAP and human breast cancer cell line MCF-7 formaldehyde-fixed paraffin-embedded tissue sections, divided into 3 groups, and incubated at room temperature for different periods of primary antibody incubation, wherein B-CPAP cells were used to detect PDL1 protein expression, and MCF-7 cells were used as a negative control because they do not express PD-L1. PD-L1 was tested as in the immunohistochemical procedure of example 1, in which 3 groups were incubated for 0.5, 1, and 2 hours at room temperature for the primary PD-L1 antibody.

The staining effect of the sections was observed under a microscope and semi-quantitatively analyzed to classify the staining marker immune features into three classes, weak (1+), medium (2+), and strong (3 +). The corresponding semi-quantitative analysis results are shown in Table 3.

Grouping	B-CPAP cells	MCF-7 cells
			Incubating at room temperature for 0.5h	1+	<1+
Incubating at room temperature for 1h	2+～3+	<1+
			Incubation at room temperature for 2h	2+～3+	1+

TABLE 3

The staining results are shown in FIG. 13, wherein A1-A3 represent the staining results of B-CPAP cells at 0.5h, 1h, and 2h, respectively, and B1-B3 represent the staining results of MCF-7 cells at 0.5h, 1h, and 2h, respectively, according to the graph: the negative control MCF-7 cells are free of any brown coloration after the primary antibody is incubated for 0.5 hour at room temperature, which indicates that the experimental system has no problems and can effectively avoid nonspecific background coloration, namely false positive, the PDL 1monoclonal antibody immunostaining result on the B-CPAP cells is weak, and the negative control MCF-7 cells are free of any brown coloration after the primary antibody is incubated for 1 hour at room temperature, which indicates that the experimental system has no problems and can effectively avoid nonspecific background coloration, namely false positive, the PDL 1monoclonal antibody immunostaining has obvious brown coloration, and the negative control MCF-7 cells are weakly brown coloration after the primary antibody is incubated for 2 hours at room temperature, which indicates that the nonspecific background coloration, namely false positive. Indicating that the incubation time of the primary antibody had a significant effect on the immunostaining of PDL 1.

Example 5: effect of incubation time of the second antibody on immunohistochemical detection Effect

This example uses cultured thyroid cancer cell line B-CPAP and human breast cancer cell line MCF-7 formaldehyde-fixed paraffin-embedded tissue sections, divided into 3 groups, and incubated at room temperature for different periods of primary antibody incubation, wherein B-CPAP cells were used to detect PDL1 protein expression, and MCF-7 cells were used as a negative control because they do not express PD-L1. PD-L1 was assayed according to the immunohistochemical procedure of example 1, in which 3 groups of the enzyme-labeled secondary antibodies were incubated at room temperature for 15, 30 and 60min, respectively.

The staining effect of the sections was observed under a microscope and semi-quantitatively analyzed to classify the staining marker immune features into three classes, weak (1+), medium (2+), and strong (3 +). The corresponding semi-quantitative analysis results are shown in Table 4.

Grouping	B-CPAP cells	MCF-7 cells
			Incubating at room temperature for 15min	1+	<1+
Incubating at room temperature for 30min	2+～3+	<1+
			Incubating at room temperature for 60min	2+～3+	1+

TABLE 4

The staining results are shown in FIG. 14, wherein A1-A3 represent the staining results of B-CPAP cells at 15min, 30min, and 60min, respectively, and B1-B3 represent the staining results of MCF-7 cells at 15min, 30min, and 60min, respectively, according to the following results: the secondary antibody is incubated at room temperature for 15 minutes without any brown coloration in the MCF-7 cells as a negative control, which indicates that the experimental system has no problems and can effectively avoid non-specific background coloration, namely false positive, the result of the immunostaining with the PDL 1monoclonal antibody is weak, the secondary antibody is incubated at room temperature for 30 minutes without any brown coloration in the negative control, which indicates that the experimental system has no problems and can effectively avoid non-specific background coloration, namely false positive, the immunostaining with the PDL 1monoclonal antibody has obvious brown coloration, and the secondary antibody is incubated at room temperature for 60 minutes and has weak brown coloration, which indicates that the non-specific background coloration, namely false positive. Indicating that the secondary antibody incubation time had a significant effect on the immunostaining of PDL 1.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A high-stability PD-L1 antibody reagent, which is characterized by comprising a mouse anti-human PD-L1monoclonal antibody and an antibody stabilizer, wherein the antibody stabilizer is selected from the group consisting of: peroxidase Stabilizing Buffer, Sigma-P9209; the mouse anti-human PD-L1monoclonal antibody is selected from the group consisting of: PD-L1Monoclonal Antibody, Thermo Fisher: 14-5983-80; the concentration of the mouse anti-human PD-L1monoclonal antibody in the antibody stabilizer is 0.4-0.8 mu g/ml.

2. The highly stable PD-L1 antibody reagent of claim 1, wherein the concentration of the murine anti-human PD-L1monoclonal antibody and the antibody stabilizer is 0.4 μ g/ml.

3. Use of the high stability antibody reagent of any one of claims 1-2 in the preparation of a PD-L1 immunohistochemical kit.

4. A PD-L1 immunohistochemical kit comprising the high stability antibody reagent of any one of claims 1-2.

5. The immunohistochemistry kit of claim 4, further comprising HRP-goat anti-mouse IgG conjugated to the mouse anti-human PD-L1monoclonal antibody.

6. The immunohistochemistry kit of claim 4 or 5, further comprising: endogenous peroxidase blocking agent and blocking liquid.

7. Use of the immunohistochemical kit of claim 4 or 5 in the preparation of a detection reagent for predicting the therapeutic effect of the PD-L1 inhibitor or a product for predicting the therapeutic effect of non-small cell lung cancer.