CN113687085A - Method for evaluating expression of Claudin18.2 protein of solid tumor and application - Google Patents
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Abstract
The invention discloses a method for evaluating expression of Claudin18.2 protein of solid tumor and application thereof, belonging to the field of biomedical detection. The evaluation method comprises the following steps: selecting tissue sections with different expression levels of Claudin18.2 in a specimen bank as a reference according to the type of solid tumor tissue to be detected, detecting the expression of Claudin18.2 protein in the tissue section to be detected and the reference section by utilizing immunohistochemical staining, and performing immunohistochemical quality control and accurately evaluating the expression level of Claudin18.2 protein in the tumor tissue to be detected by comparing the staining results of the reference section. The method provides a high-sensitivity, high-specificity and standardized judgment standard for detecting the solid tumor Claudin18.2 protein, can accurately evaluate the expression level of solid tumor tissue Claudin18.2 of different patients, and can be used for screening targeted Claudin18.2 medicaments for treating the solid tumor.
Description
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a method for evaluating the expression of Claudin18.2 protein of a solid tumor and application thereof.
Background
Claudins are a family of tight junction proteins in which Claudin18.2 is a stomach-specific subtype that is highly selectively expressed in the epithelium of the normal human gastric mucosa, but is normally buried in the gastric mucosa where it is difficult for the Claudin18.2 monoclonal antibody to bind to the site of Claudin 18.2. Recent research shows that Claudin18.2 has different degrees of expression in solid tumors such as gastric cancer, pancreatic cancer, bile duct cancer and the like, and the solid tumors can destroy the tight junction structure, so that Claudin18.2 epitope of tumor cells is exposed, and Claudin18.2 becomes a potential therapeutic target of the solid tumors. The anti-tumor drug taking Claudin18.2 as a target point can continuously develop early clinical research in different types of advanced solid tumors, but no effective means for evaluating Claudin18.2 expression in the solid tumors exists at present, so that a detection method capable of quickly, conveniently and accurately evaluating Claudin18.2 protein expression in the solid tumors is urgently needed to be developed.
Immunohistochemistry is a technology for detecting the location and expression intensity of a certain protein in tissues or cells by utilizing the characteristic that an antibody is specifically combined with an antigen and through the chromogenic part and the intensity of a marker on the antibody, and is a reliable method for detecting the expression of a solid tumor Claudin18.2. However, certain false positive and false negative exist in immunohistochemical detection, and the results of the same antigen in the same tissue in different batches of experiments may have differences. Therefore, the existing immunohistochemical technology and evaluation means need to be optimized, the stability and accuracy of immunohistochemical detection are improved, and the method is used for screening Claudin18.2 positive expression solid tumors and serving for targeted Claudin18.2 treatment.
Disclosure of Invention
The invention mainly aims to provide a method for quickly, accurately and stably evaluating the expression of Claudin18.2 protein of a solid tumor.
The invention also aims to provide the application of the method for evaluating the expression of the Claudin18.2 protein of the solid tumor in screening of anti-tumor drugs taking the Claudin18.2 protein as a target.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of Claudin18.2 protein in preparing a kit for solid tumor diagnosis or prognosis evaluation.
Preferably, the kit uses the claudin18.2 protein as a marker for diagnostic or prognostic assessment.
Preferably, the solid tumor comprises a solid tumor of epithelial origin.
Preferably, the kit is used for in vitro detection of the expression of Claudin18.2 protein in surgically excised tissue of a patient with a solid tumor by immunohistochemical staining.
Preferably, the kit comprises the following reagents:
reagent A, Claudin18.2 monoclonal antibody X1;
reagent B, HRP marked IgG secondary antibody X1;
reagent C PBS buffer solution x 1;
reagent D, antigen retrieval liquid X1;
reagent E, 3% hydrogen peroxide solution is multiplied by 1;
reagent F, goat serum confining liquid multiplied by 1;
reagent G is DAB concentrated solution multiplied by 1;
reagent H, DAB buffer solution multiplied by 1;
reagent I is hematoxylin staining solution multiplied by 1;
wherein, the Claudin18.2 monoclonal antibody can specifically bind to Claudin18.2 antigen on solid tumor cells.
Preferably, the immunohistochemical staining technique comprises the steps of deparaffinization, antigen retrieval, inactivation of endogenous peroxidase, blocking, primary antibody incubation, secondary antibody incubation, DAB visualization, counterstaining, dehydration, mounting and microscopy.
The invention also provides a method for evaluating the expression of the Claudin18.2 protein of the solid tumor, which comprises the following steps:
(1) carrying out immunohistochemical staining on the quality control, high expression, low expression, medium expression and negative control section of the Claudin18.2 protein and a section to be detected of a solid tumor cell membrane, observing under a mirror and collecting an image by a machine;
(2) analyzing the images by using immunohistochemical analysis software, if the staining of Claudin18.2 protein quality control slices and negative control slices is not false positive or false negative, respectively obtaining the average gray value of the Claudin18.2 protein staining of the slices to be detected and the quality control, high expression, low expression, medium expression and negative control slices as intensity indexes by using the software, taking the proportion of positive cells as a proportion index,
(3) and (3) scoring according to the following standard by combining the strength indexes in the step (2), specifically: the staining intensity of the to-be-detected section takes the average gray value of the contrast section as a judgment standard, and 0 represents that the average gray value of the to-be-detected section is less than the average gray value of the low-expression contrast section; 1+ represents that the average gray value of the low-expression control section is less than or equal to the average gray value of the section to be detected and the average gray value of the expression control section is less than or equal to the average gray value of the section to be detected; 2+ represents that the average gray value of the expression control section is less than or equal to the average gray value of the section to be detected and less than the average gray value of the high expression control section; 3+ represents that the average gray value of the section to be detected is more than or equal to the average gray value of the high-expression control section;
meanwhile, immunohistochemical analysis software such as Image J is used for obtaining the positive cell proportion of the section to be detected, the strength index and the proportion index of the section to be detected are integrated to judge the Claudin18.2 protein expression result, and the standard is as follows:
if the staining intensity of the section to be detected is more than or equal to 2+ and the positive cell ratio is more than or equal to 40%, the Claudin18.2 protein in the solid tumor cell membrane is considered to be positive;
if the staining intensity of the section to be detected is less than 2+, or the positive cell percentage is less than 40%, the Claudin18.2 protein in the solid tumor cell membrane is considered to be negative.
Preferably, the Claudin18.2 protein control section contains a section of human normal gastric mucosal epithelial tissue;
the Claudin18.2 protein high-expression control section is a solid tumor tissue section corresponding to a reference sample with the average gray value of Claudin18.2 protein staining close to 70% of the highest gray value;
the expression control section in the Claudin18.2 protein is a solid tumor tissue section corresponding to 40 percent of the highest gray value of a reference sample, wherein the average gray value of the Claudin18.2 protein staining is close to the highest gray value;
the Claudin18.2 protein low-expression control section is a solid tumor tissue section corresponding to a reference sample with the average gray value of Claudin18.2 protein staining close to 10% of the highest gray value;
the Claudin18.2 protein negative control section is a solid tumor tissue section corresponding to the reference sample with the lowest average gray value of Claudin18.2 protein staining;
wherein, the reference sample is tumor tissue slices or tissue chips of more than 100 different patient sources in the solid tumor tissue specimen library to be detected.
More preferably, the quality control, high expression, low expression, medium expression and negative control sections of the Claudin18.2 protein are paraffin-embedded sections, and the thickness is 4 μm.
More preferably, the high expression, low expression, medium expression and negative control section solid tumor tissues of the Claudin18.2 protein are all from the same type of tumor tissue of the patient to be tested.
The invention also provides application of the method for evaluating the expression of the Claudin18.2 protein of the solid tumor in screening of anti-tumor drugs taking the Claudin18.2 protein as a target.
Advantageous effects
The invention discloses a method for accurately evaluating Claudin18.2 protein expression in solid tumor tissue for the first time, which improves the operability and consistency of interpretation by providing control slices with different expression levels, reduces the influence of the subjectivity of manual interpretation on result judgment, provides a judgment standard with high sensitivity, high specificity and standardization for detecting the Claudin18.2 protein of the solid tumor, has the advantages of simple and convenient operation, convenient popularization, reliable result, high repeatability and the like, can accurately evaluate the Claudin18.2 expression level in the solid tumor tissue of different patients, and can be used as a diagnosis basis for screening targeted Claudin18.2 related drugs to treat the solid tumor to guide the use of clinically related drugs.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings required in the description of the embodiments will be briefly introduced below, it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without inventive labor, wherein:
FIG. 1 is a schematic flow chart of the method for evaluating the expression of Claudin18.2 protein of solid tumor in the invention.
FIG. 2 shows the results of immunohistochemical staining of Claudin18.2 protein expression quality control sections and Claudin18.2 protein tissue sections with different expression levels; 21: claudin18.2 expression quality control section staining result; 22: claudin18.2 high expression control section staining results; 23: expression control section staining in claudin 18.2; 24: claudin18.2 low expression control section staining results; 25: claudin18.2 negative control section staining results.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in FIG. 1, the method for evaluating the expression of Claudin18.2 protein of solid tumor in the invention comprises the following steps:
step S1: according to the type of the tumor tissue to be detected, tumor tissue slices or tissue chips of more than 100 different patient sources in a specimen bank are obtained as reference samples, Claudin18.2 expression of the tumor tissue slices or tissue chips is detected by an immunohistochemical method (such as a kit in the invention), and related images are collected;
step S2: analyzing the dyeing result of the reference sample in S1 by using immunohistochemical analysis software (such as Image J, Image pro plus, Meta Morph) to obtain a corresponding average gray value as a dyeing intensity index;
step S3: taking the tissue section corresponding to the lowest gray value in S2 as a negative control, and taking the tissue sections corresponding to the highest gray values of 10%, 40% and 70% as low, medium and high expression control sections;
step S4: detecting the expression of Claudin18.2 in the tissue section of the patient to be detected and the control section simultaneously by using the immunohistochemical method of S1, and collecting related images;
step S5: observing and evaluating the immunohistochemical result in the S4, performing quality control on the immunohistochemical result, observing whether a false positive result and a false negative result exist, and detecting again if the quality control is unqualified;
step S6: if the S5 quality control is qualified, analyzing the staining results of the to-be-detected section and the control section by using software in S2 to obtain an average gray value and a positive cell ratio, wherein the average gray value of the to-be-detected section is smaller than that of the low, medium and high expression control sections and is larger than that of the high expression control sections, and the expression intensities are respectively evaluated as 0, 1+, 2+ and 3 +; and comprehensively evaluating the Claudin18.2 expression of the section to be detected by combining the proportion index.
In a particular embodiment of the invention, the Claudin18.2 monoclonal antibody is capable of specifically binding to the Claudin18.2 antigenic molecule.
In a specific embodiment of the present invention, the sample to be tested is a solid tumor tissue, such as a solid tumor cell.
The following examples use an immunohistochemical staining method to detect claudin18.2 expression in solid tumor tissues involving the following reagents: the kit comprises a Claudin18.2 monoclonal antibody, an HRP-labeled IgG secondary antibody, a PBS buffer solution, an antigen retrieval solution, a 3% hydrogen peroxide solution, a goat serum blocking solution, a DAB concentrated solution, a DAB buffer solution and a hematoxylin staining solution; the specific process comprises dewaxing, antigen retrieval, endogenous peroxidase inactivation, sealing, primary antibody incubation, secondary antibody incubation, DAB color development, counterstaining, dehydration and mounting and microscopic examination evaluation.
Control sections for detection of claudin18.2 expression in solid tumor tissue in the examples below include:
1) the Claudin18.2 expression quality control reference section is provided with a human normal gastric mucosa epithelial tissue section;
2) claudin18.2 high expression control section is solid tumor tissue section corresponding to the reference sample which is analyzed by immunohistochemical analysis software (such as Image J, Image pro plus, Meta Morph, etc.), and the Claudin18.2 staining average gray value is closest to the highest gray value of 70% of the reference sample;
3) the expression control section in Claudin18.2 is a solid tumor tissue section corresponding to a reference sample which is analyzed by immunohistochemical analysis software, and the Claudin18.2 staining average gray value is closest to 40% of the highest gray value of the reference sample;
4) the Claudin18.2 low-expression control section is a solid tumor tissue section corresponding to a reference sample which is analyzed by immunohistochemical analysis software, and the Claudin18.2 staining average gray value is closest to the highest gray value of the reference sample by 10 percent;
5) the Claudin18.2 negative control section is a solid tumor tissue section corresponding to the Claudin18.2 staining the lowest average gray value after the analysis of immunohistochemical analysis software in a reference sample;
as a preferred example, in the control section for detecting the expression of the solid tumor Claudin18.2, the solid tumor sections except the Claudin18.2 expression quality control section are all from the same type of tumor tissue of the patient to be detected.
As a preferred example, the control sections for detecting the expression of the solid tumor Claudin18.2 are paraffin-embedded sections.
As a preferred example, the thickness of the control sections for detecting the expression of the solid tumor Claudin18.2 is 4 μm.
In the following examples, the method for quality control of immunohistochemical results and accurate determination of Claudin18.2 expression level in solid tumors by comparing staining results of control sections comprises the steps of:
1) taking out a prepared control section, carrying out immunohistochemical staining together with the section to be detected, observing under a mirror, collecting images by a machine, and analyzing and evaluating by using software;
2) if the Claudin18.2 expression quality control section and the negative control section are not stained with obvious false positive and false negative results, carrying out the next analysis and evaluation;
3) and (3) respectively acquiring the average gray value of the Claudin18.2 staining of the section to be detected and the control section by using immunohistochemical analysis software as an intensity index, and taking the proportion of positive cells as a proportion index.
Wherein, the staining intensity of the section to be detected takes the average gray value of the control section as a judgment standard, namely:
1)0: the average gray value of the to-be-detected section is less than the average gray value of the low-expression control section;
2)1+: the average gray value of the low-expression contrast section is less than or equal to the average gray value of the section to be detected, and the average gray value of the medium-expression contrast section is less than the average gray value of the low-expression contrast section;
3)2+: the average gray value of the expression control section is less than or equal to the average gray value of the section to be detected and less than the average gray value of the high expression control section;
4)3+: the average gray value of the to-be-detected section is more than or equal to the average gray value of the high-expression control section;
meanwhile, the positive cell proportion of the section to be detected is obtained by using Image J, and the Claudin18.2 expression result is judged by integrating the intensity index and the proportion index of the section to be detected. The standard is as follows:
if the staining intensity of the cell membrane of the tumor to be detected is more than or equal to 2+ and the positive cell ratio is more than or equal to 40%, the cell membrane is considered to be positive by Claudin18.2;
if the staining intensity of the cell membrane of the tumor to be detected is less than 2+ or the positive cell percentage is less than 40%, the result is considered to be Claudin18.2 negative.
Example 1
Claudin18.2 selection of solid tumor tissues with different expression levels and preparation of control sections, the steps are as follows:
control sections for detecting expression of solid tumor Claudin18.2 need to be selected from an existing solid tumor tissue sample. Obtaining solid tumor tissue sections or tissue chips from more than 100 patients in a sample bank, and detecting Claudin18.2 expression by using an immunohistochemical staining method, wherein the details are as follows:
1) paraffin section dewaxing: placing the solid tumor tissue slices on a sheet baking machine, baking the slices at 65 ℃ for 30min, and then soaking the slices in fresh xylene for 5min twice; soaking in 100%, 95%, and 75% ethanol for 5min respectively; soaking in double distilled water for 5min, and washing with PBS for 5min and 3 times to obtain dewaxed slices.
2) Antigen retrieval: injecting antigen repairing liquid into a high-temperature resistant metal container, putting the deparaffinized slices into the high-temperature resistant metal container to ensure that tissues in the slices are completely immersed, then putting the slices into a pressure cooker, keeping the temperature at 97 ℃ for 25min, then taking out the slices, cooling the slices at room temperature for 20min, and washing the slices with double distilled water for 5min and 3 times.
3) Inactivation of endogenous peroxidase: dropping 100-;
4) and (3) sealing: and (3) dropwise adding 200ul of goat serum for sealing into the section obtained in the step (3), and sealing for 15min at normal temperature.
5) Primary antibody incubation: and (3) throwing the section obtained in the step (4) away the confining liquid without cleaning, directly dripping 100-200 ul of diluted mouse anti-human Claudin18.2 monoclonal antibody, incubating for 1h or overnight at 4 ℃ in a wet box at room temperature, and then washing for 5min by PBS for 3 times.
6) And (3) secondary antibody incubation: and (3) dropwise adding 100-.
7) DAB color development: DAB solution was mixed with PBS buffer at 1: preparing DAB color developing solution according to the proportion of 50, dripping fresh DAB color developing solution on the slices incubated with the second antibody, standing for 10min at room temperature, then throwing off the DAB color developing solution, and washing for 5min with PBS for 3 times.
8) Counterdyeing: and (3) dropwise adding hematoxylin complex staining solution to the slices obtained in the step (7), washing with tap water for 5min after 1min and 30s, and returning blue.
9) Dewatering and sealing: soaking the slices in 75%, 95%, 100% ethanol and xylene for 5min, air drying, adding a drop of neutral gum, placing a cover glass on the tissue to obtain detected slices, and observing under a microscope.
10) Interpretation of results and preparation of control sections: claudin18.2 is a membrane protein, usually expressed on the surface of solid tumor cells, which can be classified into 4 grades according to the staining intensity of solid tumor cells on a section:
0, no staining, no brown coloration;
1+, low expression, appearing pale yellow;
2+, expressed as brown yellow;
3+, highly expressed, appearing tan.
Based on the above criteria, the evaluation was performed using immunohistochemical analysis software such as Image J, and the tissue sections corresponding to the lowest gray scale value were used as negative control, and the tissue sections corresponding to the 10%, 40%, and 70% of the highest gray scale value were used as low, medium, and high expression control sections.
11) Preparation of claudin18.2 expression quality control section: according to the previous research results and the preliminary experiment results, the Claudin18.2 is expressed in the human normal gastric mucosal epithelium, and the Claudin18.2 expression quality control section adopted by the invention is the human normal gastric mucosal epithelium tissue section.
Example 2
The determination of Claudin18.2 expression in the specimen to be detected is as follows:
after preparing a solid tumor tissue section to be detected, the control section prepared in example 1 was taken out, the section to be detected and the control section were simultaneously detected using the immunohistochemical detection method in example 1, and an image of the staining result was collected, as shown in fig. 2. And if the staining result of the Claudin18.2 expression quality control section has no false positive and false negative result, evaluating the staining result of the to-be-detected sheet and the control section by using immunohistochemical analysis software Image J to obtain the ratio of the average gray value to the positive cells.
Wherein, the Claudin18.2 staining intensity judgment standard of the section to be detected is as follows:
1)0, the average gray value of the section to be detected is less than the average gray value of the low-expression control section;
2)1+ the average gray value of the low-expression control section is less than or equal to the average gray value of the section to be detected and less than the average gray value of the expression control section;
3)2+ the average gray value of the expression control section is less than or equal to the average gray value of the section to be detected and less than the average gray value of the high expression control section;
4)3+ (ii) the average gray value of the section to be detected is more than or equal to the average gray value of the high-expression control section;
meanwhile, the positive cell proportion of the section to be detected is obtained by using Image J, and the Claudin18.2 expression result is judged by integrating the intensity index and the proportion index of the section to be detected. And (4) result judgment standard:
the staining intensity of the solid tumor cell membrane is more than or equal to 2+ and the positive cell ratio is more than or equal to 40%, namely the Claudin18.2 is positive;
claudin18.2 is considered negative when the staining intensity of solid tumor cell membranes is less than 2+ or the percentage of positive cells is less than 40%.
Claims (10)
- Application of Claudin18.2 protein in preparation of solid tumor diagnosis or prognosis evaluation kit.
- 2. Use according to claim 1, characterized in that the kit uses the claudin18.2 protein as a marker for diagnostic or prognostic assessment.
- 3. The use of claim 1, wherein the solid tumor comprises a solid tumor of epithelial origin.
- 4. The use according to any one of claims 1 to 3, wherein said kit is used for the in vitro detection of the expression of the Claudin18.2 protein in surgically excised tissue of a patient with a solid tumor by immunohistochemical staining.
- 5. The use according to claim 4, wherein the kit comprises the following reagents:reagent A, Claudin18.2 monoclonal antibody X1;reagent B, HRP marked IgG secondary antibody X1;reagent C PBS buffer solution x 1;reagent D, antigen retrieval liquid X1;reagent E, 3% hydrogen peroxide solution is multiplied by 1;reagent F, goat serum confining liquid multiplied by 1;reagent G is DAB concentrated solution multiplied by 1;reagent H, DAB buffer solution multiplied by 1;reagent I is hematoxylin staining solution multiplied by 1;wherein, the Claudin18.2 monoclonal antibody can specifically bind to Claudin18.2 antigen on solid tumor cells.
- 6. Use according to claim 5, wherein the immunohistochemical staining technique comprises the steps of deparaffinization, antigen retrieval, inactivation of endogenous peroxidase, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, counterstaining, dehydration, mounting and microscopy.
- 7. A method for assessing expression of the claudin18.2 protein of a solid tumor, comprising the steps of:(1) carrying out immunohistochemical staining on the quality control, high expression, low expression, medium expression and negative control section of the Claudin18.2 protein and a section to be detected of the solid tumor, and carrying out under-lens observation and machine-acquired image collection;(2) analyzing the images by using immunohistochemical analysis software, if the staining of Claudin18.2 protein quality control slices and negative control slices is not false positive or false negative, respectively obtaining the average gray value of the Claudin18.2 protein staining of the slices to be detected and the quality control, high expression, low expression, medium expression and negative control slices as intensity indexes by using the software, taking the proportion of positive cells as a proportion index,(3) and (3) scoring according to the following standard by combining the strength indexes in the step (2), specifically: the staining intensity of the to-be-detected section takes the average gray value of the contrast section as a judgment standard, and 0 represents that the average gray value of the to-be-detected section is less than the average gray value of the low-expression contrast section; 1+ represents that the average gray value of the low-expression control section is less than or equal to the average gray value of the section to be detected and the average gray value of the expression control section is less than or equal to the average gray value of the section to be detected; 2+ represents that the average gray value of the expression control section is less than or equal to the average gray value of the section to be detected and less than the average gray value of the high expression control section; 3+ represents that the average gray value of the section to be detected is more than or equal to the average gray value of the high-expression control section;meanwhile, immunohistochemical analysis software such as Image J is used for obtaining the positive cell proportion of the section to be detected, the strength index and the proportion index of the section to be detected are integrated to judge the Claudin18.2 protein expression result, and the standard is as follows:if the staining intensity of the section to be detected is more than or equal to 2+ and the positive cell ratio is more than or equal to 40%, the Claudin18.2 protein in the solid tumor cell membrane is considered to be positive;if the staining intensity of the section to be detected is less than 2+, or the positive cell percentage is less than 40%, the Claudin18.2 protein in the solid tumor cell membrane is considered to be negative.
- 8. The method of assessing expression of the Claudin18.2 protein of a solid tumor according to claim 7,the Claudin18.2 protein control section contains a human normal gastric mucosal epithelial tissue section;the Claudin18.2 protein high-expression control section is a solid tumor tissue section corresponding to a reference sample with the average gray value of Claudin18.2 protein staining close to 70% of the highest gray value;the expression control section in the Claudin18.2 protein is a solid tumor tissue section corresponding to 40 percent of the highest gray value of a reference sample, wherein the average gray value of the Claudin18.2 protein staining is close to the highest gray value;the Claudin18.2 protein low-expression control section is a solid tumor tissue section corresponding to a reference sample with the average gray value of Claudin18.2 protein staining close to 10% of the highest gray value;the Claudin18.2 protein negative control section is a solid tumor tissue section corresponding to the reference sample with the lowest average gray value of Claudin18.2 protein staining;wherein, the reference sample is tumor tissue slices or tissue chips of more than 100 different patient sources in the solid tumor tissue specimen library to be detected.
- 9. The method for evaluating the expression of Claudin18.2 protein of a solid tumor according to claim 8, wherein the quality control, high expression, low expression, medium expression and negative control sections of the Claudin18.2 protein are paraffin-embedded sections, and the thickness of each section is 4 μm; and/or the high expression, low expression, medium expression and negative control section solid tumor tissues of the Claudin18.2 protein are all from the same type of tumor tissues of a patient to be detected.
- 10. Use of the method of any one of claims 7 to 9 for assessing the expression of the Claudin18.2 protein of a solid tumor in the screening of antitumor drugs targeting the Claudin18.2 protein.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114428174A (en) * | 2022-04-07 | 2022-05-03 | 北京肿瘤医院(北京大学肿瘤医院) | Gastric cancer prognosis biomarker and application thereof |
| CN116773790A (en) * | 2023-08-18 | 2023-09-19 | 南京普恩瑞生物科技有限公司 | Preparation method and application of tumor tissue HER2 gradient detection product |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150147763A1 (en) * | 2012-05-09 | 2015-05-28 | Ganymed Pharmaceuticals Ag | Antibodies Against Claudin 18.2 Useful in Cancer Diagnosis |
| US20180140634A1 (en) * | 2015-05-11 | 2018-05-24 | Biontech Cell & Gene Therapies Gmbh | Enhancing the effect of car-engineered t cells by means of nucleic acid vaccination |
| CN111777681A (en) * | 2020-07-06 | 2020-10-16 | 上海岺樾生物医药科技有限公司 | Antibody combined with tight junction protein-18.2 and application thereof |
| CN112770723A (en) * | 2018-07-25 | 2021-05-07 | 阿克罗斯生物科学公司 | Novel monoclonal antibodies specific for CLDN18.2 and methods of use thereof |
| CN113166246A (en) * | 2018-12-28 | 2021-07-23 | 四川科伦博泰生物医药股份有限公司 | Antibody and application thereof |
-
2021
- 2021-08-27 CN CN202110996155.5A patent/CN113687085A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150147763A1 (en) * | 2012-05-09 | 2015-05-28 | Ganymed Pharmaceuticals Ag | Antibodies Against Claudin 18.2 Useful in Cancer Diagnosis |
| US20180140634A1 (en) * | 2015-05-11 | 2018-05-24 | Biontech Cell & Gene Therapies Gmbh | Enhancing the effect of car-engineered t cells by means of nucleic acid vaccination |
| CN112770723A (en) * | 2018-07-25 | 2021-05-07 | 阿克罗斯生物科学公司 | Novel monoclonal antibodies specific for CLDN18.2 and methods of use thereof |
| US20210214433A1 (en) * | 2018-07-25 | 2021-07-15 | Accurus Biosciences, Inc. | Novel cldn 18.2-specific monoclonal antibodies and methods of use thereof |
| CN113166246A (en) * | 2018-12-28 | 2021-07-23 | 四川科伦博泰生物医药股份有限公司 | Antibody and application thereof |
| CN111777681A (en) * | 2020-07-06 | 2020-10-16 | 上海岺樾生物医药科技有限公司 | Antibody combined with tight junction protein-18.2 and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 何萍;张志强;戴晓淳;傅立业;隋承光;孟凡东;姜又红;: "两种方法检测肺癌患者CAP43蛋白的结果比较", 中国现代医学杂志, no. 20 * |
| 文彬, 丘钜世, 李扬: "成骨性肿瘤中p27基因mRNA与蛋白的表达及其意义", 中国肿瘤临床, no. 06 * |
| 杨文彬;蔡锋;程传涛;曹罡;秦兆寅;: "人胰腺癌中前列腺干细胞抗原及紧密连接蛋白-4的表达", 中国医学科学院学报, no. 06 * |
| 罗刚, 周四维, 曹正国, 鲁雄兵, 孙凯, 宋晓东, 叶章群: "黏蛋白MUC1/Y在膀胱癌中的表达及其意义", 医学研究生学报, no. 04 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114428174A (en) * | 2022-04-07 | 2022-05-03 | 北京肿瘤医院(北京大学肿瘤医院) | Gastric cancer prognosis biomarker and application thereof |
| CN114428174B (en) * | 2022-04-07 | 2022-07-08 | 北京肿瘤医院(北京大学肿瘤医院) | Gastric cancer prognosis biomarker and application thereof |
| CN116773790A (en) * | 2023-08-18 | 2023-09-19 | 南京普恩瑞生物科技有限公司 | Preparation method and application of tumor tissue HER2 gradient detection product |
| CN116773790B (en) * | 2023-08-18 | 2023-11-28 | 南京普恩瑞生物科技有限公司 | Preparation method and application of tumor tissue HER2 gradient detection product |
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