CN110320365B - NF-kB RelA/p65 protein site-specific phosphorylation diagnostic kit - Google Patents
NF-kB RelA/p65 protein site-specific phosphorylation diagnostic kit Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Abstract
The invention relates to the technical field of medical biology, in particular to a kit for detecting site-specific phosphorylation of NF-kB RelA/p65 protein. Specifically, the kit can specifically detect the phosphorylation states of Ser276 and Ser536 sites of NF-kB RelA/p65 protein, is used for guiding the treatment of NF-kB inhibitor targeted antitumor drugs or RelA/p65S 276A/S536D gene therapy, and has important clinical application value.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to a kit for specifically detecting phosphorylation sites of human NF-kB RelA/p65 protein, which is used for detecting phosphorylation states of Ser276 and Ser536 sites of NF-kB RelA/p65 protein in various human tumor tissues and is used for targeted antitumor drug treatment and antitumor gene treatment. The kit relates to an immunoturbidimetry method, an enzyme-linked immunosorbent assay (ELISA), a time-resolved immunofluorescence analysis (TRFIA) method, a chemiluminescence method and an electrochemical luminescence method based on sandwich ELISA. The kit also relates to Immunohistochemical (IHC) and Immunocytochemical (ICC) methods.
Background
The protein family NF-kB RelA/p65 of eukaryotic transcription factors (the accession number of NF-kB RelA/p65 gene in GenBank is L19067.1) is a key regulatory protein in immune response and inflammatory response. The NF-. kappa.B/Rel family of mammals comprises 5 subunits, which are respectively Rel (cRel), p65 (RelA, NF-. kappa.B 3), RelB, p 50/p 105 (NF-. kappa.B 1) and p 52/p 100 (NF-. kappa.B 2). Each subunit constitutes a homo-or heterodimer via its N-terminal Rel Homology Domain (RHD).
From the discovery of NF-. kappa.B, extensive studies have been made on its structure and function to date. Research shows that NF-kappa B can activate various tumors such as liver cancer, breast cancer, pancreatic cancer, brain cancer, gastric cancer, colorectal cancer, bladder cancer, prostate cancer and the like, and promote the growth and metastasis of cancer cells. Thus, inhibition of NF-. kappa.B sensitizes pancreatic cancer cells to apoptosis induced by chemical drugs. However, it has been shown that NF-. kappa.B is a key factor in the induction of apoptosis by p53 and plays a role in cancer suppression in some tumors. To date, nearly thousands of NF- κ B inhibitors have been developed, with few clinical successes, although some have yielded promising results in experimental treatment. Recent research shows that the post-translational modification of RelA/p65, which is an important subunit of NF-kappa B, can finely regulate the transcriptional activation of NF-kappa B and play an important role in the occurrence and development processes of tumors, inflammatory responses and inflammatory response-related diseases.
Post-transcriptional modifications of NF-. kappa.B include phosphorylation, acetylation, and methylation. RelA/p65 is the most important subunit/member of the NF- κ B family, and RelA/p65 phosphorylation is a crucial post-translational activation regulatory mechanism. A total of 12 phosphorylation sites have been identified so far for RelA/p65, including 9 serines and 3 threonines. Where Ser205, Thr254, Ser276, Ser281 and Ser311 are located at or adjacent to the N-terminus of the homeodomain (RHD), and Thr435, Ser468, Thr505, Ser529, Ser535, Ser536 and Ser547 phosphorylation sites are located at the C-terminus of the transactivation domain.
Ser536 is an important phosphorylation site of RelA/p65, and can be phosphorylated by a variety of phosphorylation kinases, such as IkB kinase, TANK binding kinase (TBK 1), ribosomal subunit kinase 1 (RSK 1), etc., which activate Ser 536. There are studies showing that RelA/p65 Ser536 phosphorylation increases gradually in the normal colonic mucosa, from the bottom to the top of the crypt, with peak expression in mature epithelial cells that are about to die and shed into the intestinal lumen. In contrast, however, RelA/p65 Ser536 phosphorylation was significantly reduced in colon cancer cells. Phosphorylation of NF-. kappa.B RelA/p65 Ser536 (i.e., NF-. kappa.B/p 65S 536D) in cultured breast (MCF 7) and colon (HCT 116) cancer cells caused dramatic apoptosis, autophagy, and cellular senescence. The NF-kB RelA/p65 (namely NF-kB/p 65S 536D) phosphorylated by Ser536 is injected into a mouse transplanted tumor, so that the tumor growth can be effectively inhibited. In contrast, phosphorylation of NF-kB RelA/p65Ser276 promotes growth, proliferation and survival of tumor cells. It is believed that phosphorylation of Ser536 confers NF-. kappa.B RelA/p65 tumor suppression; the phosphorylation of Ser276 probably endows NF-kB RelA/p65 tumor promotion function; the carcinogenic or carcinogenic effects of NF-. kappa.B RelA/p65 in tumorigenesis depend on phosphorylation activation of its specific active site. The clinical targeted detection or modification of the sites can carry out targeted medicine or gene therapy on specific tumors, and has important clinical value.
Disclosure of Invention
Aiming at the problems, a specific detection kit, namely an NF-kB RelA/p65 protein site-specific phosphorylation kit is developed aiming at human tumor tissues and is used for detecting the phosphorylation states of Ser276 and Ser536 sites of NF-kB RelA/p65 protein in various human tumor tissues.
Specifically, the present invention relates to the following:
1. an immunoturbidimetric method, an enzyme-linked immunosorbent assay, a time-resolved immunofluorescence analysis (TRFIA) method, a chemiluminescence method and an electrochemiluminescence method based on sandwich ELISA are used for detecting phosphorylation states of Ser276 and Ser536 sites of NF-kB RelA/p65 protein in human tumor tissues.
2. An Immunohistochemical (IHC) method and an Immunocytochemistry (ICC) method based on an immunological principle are used for detecting phosphorylation states of Ser276 and Ser536 sites of NF-kB RelA/p65 protein in human tumor tissues.
3. A kit for detecting phosphorylation states of Ser276 and Ser536 sites of NF-kB RelA/p65 protein in various human tumor tissues based on items 1-2, which comprises a specific detection antibody and a reagent.
4. The kit of any one of claims 1-3, wherein the cancer is selected from the group consisting of: human malignant tumors such as breast cancer, lung cancer, brain cancer, gastric cancer, colorectal cancer, esophageal cancer, bladder cancer, prostate cancer and the like.
5. The kit of any one of items 1 to 4, wherein the detection result can be used for NF-kB RelA/p65 targeting antitumor drug treatment and antitumor gene treatment.
6. The kit of any one of items 1-5, wherein the kit further comprises a NF- κ B RelA/p65 site-specific phosphorylated protein standard.
7. The kit of any one of items 1-6, comprising:
a plate or bead coated with a specific capture monoclonal antibody;
biotin or other labeled specific detection antibody from another species source.
8. The kit of item 7, further comprising:
coating buffer (1 XPBS, pH7.3 + -0.1);
sample dilutions (1 × PBS, 1% BSA, 0.05% NaN3, pH7.3 ± 0.1);
blocking solution (1 × PBS, 3% BSA);
eluent PBST (1 XPBS, 0.05% Tween-20);
Streptavidin-Eu/HRP/ALP；
IHC and ICC confining liquid
IHC and ICC substrates and color developing agents.
9. The kit according to item 7 or 8, wherein the capture antibody is derived from a monoclonal antibody specifically recognizing NF-. kappa.B RelA/p65 protein prepared by immunizing a mouse with a full-length NF-. kappa.B RelA/p65 protein antigen, and performing cell fusion to isolate a monoclonal hybrid cell line; the detection antibody is from a purified monoclonal antibody prepared by immunizing a rabbit with a small peptide antigen of NF-kB RelA/p65 phosphorylated at a Ser276 or Ser536 site, carrying out cell fusion, and separating a monoclonal hybrid cell strain.
10. The kit of any one of claims 1-9, wherein said detecting phosphorylation of an NF-kb RelA/p65Ser276 or Ser536 site comprises:
a) measuring the level of phosphorylation of the NF- κ B RelA/p65Ser276 or Ser536 site in a biological sample (tumor tissue) from the subject using the specific detection reagent;
b) comparing the level measured in a) with the corresponding level in a normal sample (paracancerous normal tissue).
11. The kit according to item 10, which is used for specific targeted drug or gene antitumor therapy of cancer.
Drawings
FIG. 1: sensitivity of time-resolved immunofluorescence assay (TRFIA) kit. The purified NF-kB RelA/p65Ser276 phosphorylated protein is used as a sample to be detected to determine the detection sensitivity and the linear range.
FIG. 2 is a schematic diagram: one example of the phosphorylation levels of NF-. kappa.B RelA/p65Ser276 and Ser536 in tumor tissues of lung cancer patients. Detection was performed with TRFIA as described above.
FIG. 3: phosphorylation levels of Ser276 and Ser536 in tumor tissue of one colon cancer patient, NF-. kappa.B RelA/p 65. Detection was performed with TRFIA as described above.
FIG. 4: phosphorylation of NF-. kappa.B RelA/p65Ser276 in human lung carcinoma tissue, as determined by immunohistochemical examination of human lung carcinoma tissue sections.
FIG. 5: the phosphorylation level of NF-kB RelA/p65Ser276 in human liver cancer tissues is the result of immunohistochemical detection on human liver cancer tissue sections.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary. In the following examples, unless otherwise specified, all the methods are conventional.
Example 1 preparation of Sandwich time-resolved fluorescence immunoassay (TRFIA) kit for detecting NF-kB RelA/p65Ser276 and Ser536 phosphorylated proteins
In this example, a sandwich time-resolved fluorescence immunoassay (TRFIA) kit includes:
a plate or bead coated with a specific capture monoclonal antibody;
biotin or another species-derived specific detection antibody labeled;
coating buffer (1 XPBS, pH7.3 + -0.1);
sample dilutions (1 × PBS, 1% BSA, 0.05% NaN3, pH7.3 ± 0.1);
blocking solution (1 × PBS, 3% BSA);
eluent PBST (1 XPBS, 0.05% Tween-20);
Streptavidin-Eu/HRP/ALP；
the biotin labeling kit is purchased from Pierce company. Streptavidin-Eu was purchased from Perkin Elmer. A time-resolved fluorescence immunoassay (TRFIA) plate for detection is prepared by the self, a coating buffer solution is used for diluting a capture antibody to be 7-10 mu g/ml, 100 mu l of the antibody diluent is added into each hole of the time-resolved fluorescence immunoassay (TRFIA) plate, and the temperature is 4 ℃ for standing overnight; taking out, and placing at 37 ℃ for 30 minutes; spin off the liquid and wash 5 times with PBST; drying in the air, adding 100 mu l of confining liquid into each hole, and keeping the temperature at 37 ℃ for 2 hours; the liquid was spun off and washed 2 times with PBS; naturally drying for 2 hours at room temperature; and sealing by using tin foil paper in vacuum, and placing at 4 ℃ for later use. The used reagents are self-prepared: coating buffer (1 XPBS, pH7.3 + -0.1); sample dilutions/antibody dilutions (1 XPBS, 1% BSA, 0.05% NaN)3pH 7.3. + -. 0.1); blocking solution (1 × PBS, 3% BSA); eluent PBST (1 XPBS, 0.05% Tween-20); standard (different concentrations RelA/p65Ser276 phosphorylated protein antigen: 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 ng/ml).
Wherein the capture antibody is a monoclonal antibody which is prepared by immunizing a mouse with a full-length NF-kB RelA/p65 protein antigen, carrying out cell fusion and separating a monoclonal hybrid cell strain and specifically recognizes the NF-kB RelA/p65 protein; the detection antibody is from a purified monoclonal antibody prepared by immunizing a rabbit with a small peptide antigen of NF-kB RelA/p65 phosphorylated at a Ser276 or Ser536 site, carrying out cell fusion, and separating a monoclonal hybrid cell strain.
(reference: Chenhong Yuan, Rui Wen, Huawenfeng, Du Jun, Chua Shaohui, mouse anti-human TCR/DC3 monoclonal antibody preparation and its preliminary application, Guangdong institute of medicine, proceedings 2008, 6, 594, 597; Seiki, King-Ning-N, Huangli, Zanyuan Qing, Huzheng, Ludi-xian. Rabbit anti-aldehyde-ketone reductase family 1 member B10 (AKR 1B 10) polyclonal antibody preparation and identification. J. cell & immunology mol. 2016, 32 (11); JaspR, Gesk T, Teichmann A, et al.
0.031ng/ml of NF-. kappa.B RelA/p65Ser276 phosphorylated protein was detected by time-resolved fluorescence immunoassay (TRFIA) (see FIG. 1).
Example 2: detection of phosphorylation levels of NF-kB RelA/p65Ser276 and Ser536 in tumor tissues of lung cancer patients by sandwich time-resolved fluorescence immunoassay (TRFIA) kit
Preparation of detection time-resolved fluoroimmunoassay plate: diluting the capture antibody to 7-10 μ g/ml with a coating buffer solution, adding 100 μ l of the above antibody diluent into each well of a time-resolved fluoroimmunoassay (TRFIA) plate, and standing overnight at 4 ℃; taking out, and placing at 37 ℃ for 30 minutes; spin off the liquid and wash 5 times with PBST; drying in the air, adding 100 mu l of confining liquid into each hole, and keeping the temperature at 37 ℃ for 2 hours; the liquid was spun off and washed 2 times with PBS; naturally air-drying for 2 hours at room temperature; vacuum sealing with tinfoil paper, and placing at 4 deg.C for use.
Detection of tissue samples: tumor tissue was prepared by standard methods as soluble protein solution, diluted 1:10 with PBS, 100. mu.l of sample dilution or standard (different concentrations of RelA/p65Ser276 or Ser536 phosphorylated protein antigen: 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 ng/ml) was added to each well, 37 ℃ for 60 min; throwing off the liquid, washing for 5 times by using a washing buffer solution, and drying in the air; diluting a detection antibody with an antibody diluent at a ratio of 1:500, adding 100 mu l of the detection antibody diluent into each hole, and performing 60 minutes at 37 ℃; throwing off the liquid, washing for 5 times by using a washing buffer solution, and drying in the air; diluting Streptavidin-Eu with an antibody diluent at a ratio of 1:80000, adding 100 mu l of Streptavidin-Eu diluent into each hole, and carrying out 60 minutes at 37 ℃; throwing off the liquid, washing for 5 times by using a washing buffer solution, and drying in the air; adding 100 mul of reaction solution; OD was measured at a wavelength of 450 nm using a microplate reader. And drawing a standard curve, calculating the concentrations of the RelA/p65Ser276 and Ser536 phosphorylated protein antigens in the tissue sample according to the standard curve, and multiplying by 10 to obtain the final concentrations of the RelA/p65Ser276 and Ser536 phosphorylated protein antigens in the tissue.
Analysis of detection results
The above kit was used in each patient to determine the concentrations of the phosphorylated proteins RelA/p65Ser276 and Ser536 in tumor and normal tissues, and to calculate relative values (fold tumor/normal tissue) indicating the phosphorylation levels at each site in tumor tissue.
Please refer to fig. 2: in one example of this embodiment, the phosphorylation levels of Ser-536 and NF- κ B RelA/p65Ser276 in tumor tissues of lung cancer patients were determined to be approximately 3.8 for Ser276 and approximately 0.5 for Ser 536.
Example 3: detection of phosphorylation levels of NF-kB RelA/p65Ser276 and Ser536 in tumor tissues of colon cancer patients by using sandwich time-resolved fluorescence immunoassay (TRFIA) kit
Preparation of detection time-resolved fluoroimmunoassay plate: diluting the capture antibody to 7-10 mu g/ml by using a coating buffer solution, adding 100 mu l of the antibody diluent into each hole of a time-resolved fluoroimmunoassay (TRFIA) plate, and standing overnight at 4 ℃; taking out, and placing at 37 ℃ for 30 minutes; the liquid was spun off and washed 5 times with PBST; drying in the air, adding 100 mu l of confining liquid into each hole, and keeping the temperature at 37 ℃ for 2 hours; the liquid was spun off and washed 2 times with PBS; naturally air-drying for 2 hours at room temperature; and sealing by using tin foil paper in vacuum, and placing at 4 ℃ for later use.
Detection of tissue samples: tumor tissue was prepared by standard methods as soluble protein solution, diluted 1:10 with PBS, 100. mu.l of sample dilution or standard (different concentrations of RelA/p65Ser276 or Ser536 phosphorylated protein antigen: 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 ng/ml) was added to each well, 37 ℃ for 60 min; throwing off the liquid, washing for 5 times by using a washing buffer solution, and drying in the air; diluting the detection antibody with an antibody diluent at a ratio of 1:500, adding 100 mul of the detection antibody diluent into each hole, and performing 60 minutes at 37 ℃; throwing off the liquid, washing for 5 times by using a washing buffer solution, and drying in the air; diluting Streptavidin-Eu with an antibody diluent at a ratio of 1:80000, adding 100 mu l of Streptavidin-Eu diluent into each hole, and carrying out 60 minutes at 37 ℃; throwing off the liquid, washing for 5 times by using a washing buffer solution, and drying in the air; adding 100 mul of reaction solution; OD was measured at a wavelength of 450 nm using a microplate reader. And drawing a standard curve, calculating the concentrations of the RelA/p65Ser276 and Ser536 phosphorylated protein antigens in the tissue sample according to the standard curve, and multiplying by 10 to obtain the final concentrations of the RelA/p65Ser276 and Ser536 phosphorylated protein antigens in the tissue.
Analysis of detection results
The above kit was used in each patient to determine the concentrations of the phosphorylated proteins RelA/p65Ser276 and Ser536 in tumor and normal tissues, and to calculate relative values (fold tumor/normal tissue) indicating the phosphorylation levels at each site in tumor tissue.
Please refer to fig. 3: one example of this example demonstrates the phosphorylation levels of NF-. kappa.B RelA/p65Ser276 and Ser536 in tumor tissue from a colon cancer patient, where Ser276 measures approximately 0.8 and Ser536 measures approximately 2.1.
Example 4: immunohistochemistry detection of NF-kB RelA/p65Ser276 phosphorylation level in human lung cancer tissues
Soaking human lung cancer tissue slices in APES 1:50 acetone solution for 3 min for dewaxing, soaking in xylene for 10 min, respectively soaking in anhydrous ethanol, 70% ethanol and 50% ethanol for 10 min, and finally soaking in purified water for 5 min; with 3% H2O2Dripping the solution on the tissue slices, removing endogenous peroxides in 10 minutes respectively, and washing with PBS for 3 times; incubating the sections in a moisture preservation box at 37 ℃ for 30 minutes by using serum with the same source as the secondary antibody, and removing the sealing liquid; dropwise adding the diluted primary antibody, placing the slices in a moisture preservation box for overnight incubation at 4 ℃, and washing with PBS for 3 times; dropwise adding the diluted biotin-labeled secondary antibody, incubating for 30 minutes in a humidity preservation box at 37 ℃, and washing for 3 times by PBS; dropwise adding diluted streptavidin marked by HRP, incubating for 20 minutes at 37 ℃, and washing for 3 times by PBS; adding a color developing agent for developing for 5 minutes, stopping the color developing reaction, and repeatedly washing with clear water; counterstaining with hematoxylin for 1-2 min, stopping after the cell nucleus turns blue, and repeatedly washing with clear water; soaking in 50% ethanol, 70% ethanol, anhydrous ethanol and xylene solution for 1 min respectively for dehydration, air drying at room temperature, and taking pictures with microscope.
Analysis of detection results
Please refer to fig. 4: this example demonstrates immunohistochemical detection of tumor tissue in one lung cancer patient, with arrows indicating the phosphorylation sites of Ser276 in NF-. kappa.B RelA/p 65.
Example 5: immunohistochemistry detection of NF-kB RelA/p65Ser276 phosphorylation level in human liver cancer tissues
Soaking human lung cancer tissue slices in APES 1:50 acetone solution for 3 min for dewaxing, soaking in xylene for 10 min, and respectively replacing with anhydrous ethanolSoaking in 70% ethanol and 50% ethanol for 10 min, and soaking in purified water for 5 min; with 3% H2O2Dripping the mixture on a tissue slice, removing endogenous peroxides in 10 minutes respectively, and washing with PBS for 3 times; incubating the cut section for 30 minutes in a moisture preservation box at 37 ℃ by using serum with the same source as the secondary antibody, and removing sealing liquid; dropwise adding the diluted primary antibody, placing the slices in a moisture preservation box for overnight incubation at 4 ℃, and washing with PBS for 3 times; dropwise adding the diluted biotin-labeled secondary antibody, incubating for 30 minutes at 37 ℃ in a humidity preservation box, and washing for 3 times by PBS; dropwise adding diluted streptavidin marked by HRP, incubating for 20 minutes at 37 ℃, and washing for 3 times by PBS; adding a color developing agent for developing for 5 minutes, stopping the color developing reaction, and repeatedly washing with clear water; counterstaining with hematoxylin for 1-2 min, stopping after the cell nucleus turns blue, and repeatedly washing with clear water; the slices were dehydrated by soaking in 50% ethanol, 70% ethanol, absolute ethanol and xylene solution for 1 minute, dried at room temperature, and photographed with a microscope (see fig. 5).
Analysis of detection results
Please refer to fig. 5: in this example, a tumor tissue of a liver cancer patient was examined by immunohistochemistry, and the phosphorylation site of NF-. kappa.B RelA/p65Ser276 is indicated by an arrow.
The invention discloses a novel tumor tissue marker of NF-kB RelA/p65Ser276 and Ser536 phosphorylated proteins, and develops a NF-kB RelA/p65 protein site-specific phosphorylation kit aiming at differential expression of the tumor tissue marker in tumor tissue, which is used for quantitatively or semi-quantitatively detecting the levels of the NF-kB RelA/p65Ser276 and Ser536 phosphorylated proteins in various human tumor tissues and guiding clinical antitumor targeted drugs or gene therapy. The method provided by the invention has the advantages of strong specificity, high sensitivity, simplicity and rapidness. The whole experiment operation has low requirements on experimental instruments and reagents, can be carried out in common laboratories, has high accuracy, and eliminates subjectivity by quantitatively analyzing results by an enzyme-labeling instrument.
Claims (5)
1. A diagnostic kit for detecting phosphorylation of Ser276 and Ser536 sites of NF-kb RelA/p65 protein in human tumor tissue, comprising an antibody for specifically detecting phosphorylation of Ser276 and Ser536 sites of human NF-kb RelA/p65 protein, wherein the antibody is a murine anti-NF-kb RelA/p65 monoclonal antibody; rabbit anti-NF-kB RelA/p65Ser276 monoclonal antibody; rabbit anti-NF-kB RelA/p65 Ser536 monoclonal antibody;
the kit further comprises: a plate or bead coated with a specific capture monoclonal antibody; the specific capture monoclonal antibody is prepared by immunizing a mouse with a full-length NF-kB RelA/p65 protein antigen, carrying out cell fusion and separating a monoclonal hybrid cell strain, and specifically recognizes the NF-kB RelA/p65 protein;
the kit further comprises: biotin or another species-derived specific detection antibody labeled; the detection antibody is a purified monoclonal antibody prepared by immunizing a rabbit with a small peptide antigen of NF-kB RelA/p65 phosphorylated by a Ser276 or Ser536 site, performing cell fusion, and separating a monoclonal hybrid cell strain;
the kit further comprises:
coating buffer solution: 1 XPBS, pH7.3 + -0.1;
sample diluent: 1 XPBS, 1% BSA, 0.05% NaN3, pH 7.3. + -. 0.1;
sealing liquid: 1 × PBS, 3% BSA;
eluent PBST: 1 XPBS, 0.05% Tween-20;
Streptavidin-Eu。
2. the kit of claim 1, wherein the kit further comprises: NF-kB RelA/p65 site-specific phosphorylated protein standard.
3. The kit of claim 1 or 2, wherein the human tumor is breast cancer, lung cancer, brain cancer, gastric cancer, colorectal cancer, esophageal cancer, bladder cancer, or prostate cancer.
4. The kit according to claim 1, which is a detection kit based on time-resolved immunofluorescence assay.
5. Use of the kit of any one of claims 1 to 4 for the preparation of a formulation for the specific targeted drug or gene anti-tumor therapy of cancer.
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