CN110320365A - NF- κ B RelA/p65 protein loci pecific phosphorylation diagnostic kit - Google Patents
NF- κ B RelA/p65 protein loci pecific phosphorylation diagnostic kit Download PDFInfo
- Publication number
- CN110320365A CN110320365A CN201910607155.4A CN201910607155A CN110320365A CN 110320365 A CN110320365 A CN 110320365A CN 201910607155 A CN201910607155 A CN 201910607155A CN 110320365 A CN110320365 A CN 110320365A
- Authority
- CN
- China
- Prior art keywords
- rela
- ser536
- kit
- cancer
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- 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
-
- 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
- G01N33/53—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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to technical field of pharmaceutical biotechnology, specifically, being related to a kind of kit of NF- κ B RelA/p65 protein loci pecific phosphorylation detection.Specifically, the kit can specifically detect the phosphorylation state of NF- κ B RelA/p65 Protein S er276 and the site Ser536, to instruct NF- kB inhibitor targeting antineoplastic medicine object to treat or RelA/p65 S276A/S536D gene therapy, there is important clinical value.
Description
Technical field
The present invention relates to technical field of pharmaceutical biotechnology, specifically, being related to a kind of specific detection people NF- κ B RelA/
P65 protein loci phosphorylation agent box, for detect in various mankind tumor tissues NF- κ B RelA/p65 Protein S er276 with
Ser536 site phosphorylation state is treated for the treatment of targeting antineoplastic medicine object with Antioncogene.The kit is related to base
In the immunoturbidimetry of sandwich ELISA, enzyme-linked immunization, Time-resolved Fluoroimmunoassay (TRFIA) method, chemoluminescence method and
Electrochemiluminescince.The kit further relates to immunohistochemistry (IHC) method and immunocytochemistry (ICC) method.
Background technique
Eukaryotic transcription factor protein family NF- κ B RelA/p65(NF- κ B RelA/p65 gene is in the GenBank number of logging in
For the key regulatory albumen that L19067.1) is in immune response and inflammatory reaction.The NF- κ B/Rel family of mammal wraps
5 subunits are included, they are Rel (cRel), p65 (RelA, NF- κ B3), RelB, p50/p105 (NF- κ B1) respectively
With p52/p100 (NF- κ B2).Each subunit by the Rel homeodomain of its N-terminal (Rel homology domain,
RHD homologous or heterodimer) is formed.
So far from NF- κ B discovery, people conduct extensive research its structure and function.Studies have found that NF- κ B
Activation liver cancer, breast cancer, cancer of pancreas, the kinds of tumors such as the cancer of the brain, gastric cancer, colorectal cancer, bladder cancer, prostate cancer promote cancer thin
The growth and transfer of born of the same parents.Thus, the inhibition to NF- κ B is so that pancreatic cancer cell becomes the Apoptosis induced by chemicals
It is sensitive.However, some researches show that NF- κ B is the key factor of p53 inducing apoptosis of tumour cell, and suppression is played in some tumours
Cancer effect.Up to now, NF- kB inhibitor nearly thousand have been developed, although some inhibitor obtained in experimental therapy it is gratifying
As a result, still seldom succeeding in clinic.It has recently been demonstrated that the RelA/ as mono- important subunit of NF- κ B
P65, posttranslational modification can subtly regulate and control the transcriptional activation of NF- κ B, and in tumour, inflammatory reaction and inflammatory reaction phase
It plays an important role during the occurrence and development of related disorders.
Modification after NF- κ B transcription includes phosphorylation, acetylation and methylation.RelA/p65 be in NF- κ B family most
Important subunit/member, RelA/p65 phosphorylation are to activate adjustment mechanism after a kind of conclusive translation.RelA/ so far
P65 shares 12 phosphorylation sites and is determined, including 9 serines and 3 threonines.Wherein, Ser205, Thr254,
Ser276, Ser281 and Ser311 are located at or adjoin the end N- of homeodomain (RHD), and Thr435, Ser468, Thr505,
Ser529, Ser535, Ser536 and Ser547 phosphorylation site are then located at the end C- of trans-activation domain.
Ser536 is mono- important phosphorylation site of RelA/p65, can by a variety of phosphorylating kinase phosphorylations, such as
I kappa b kinase, TANK combination kinases (TBK1), ribosomal subunit kinases 1(RSK1) etc. can all activate Ser536.There is result of study table
Bright, in normal colonic mucosa, stage from crypts bottom to top, RelA/p65 Ser536 phosphorylation is stepped up, will
Expression in apoptosis and the mature epithelial cell for the bowel lumen that falls off peaks.But in contrast, in colon cancer cell
The significant reduction of RelA/p65 Ser536 phosphorylation.In the breast cancer (MCF7) and colon cancer (HCT116) cell of culture, NF- κ
B RelA/p65 Ser536 phosphorylation (i.e. NF- κ B/p65 S536D) causes theatrical Apoptosis, cell autophagy, and thin
Born of the same parents' aging.NF- κ B RelA/p65(, that is, NF- κ B/p65 S536D of Ser536 phosphorylation) is injected into mice-transplanted tumor, can be had
Effect ground inhibits tumour growth.On the contrary, NF- κ B RelA/p65 Ser276 phosphorylation then promote tumour cell growth, proliferation with
Existence.It is believed that the phosphorylation of Ser536 assigns NF- κ B RelA/p65 tumor inhibition effect；And the phosphorylation of Ser276 is then
NF- κ B RelA/p65 tumor enhancement may be assigned；Rush cancer or suppression cancer of the NF- κ B RelA/p65 in tumour generation are made
With phosphorylation activation depending on its activity specific site.Clinically the targeting in these sites is detected or modified, it can be to spy
Determine tumour and carry out targeted drug or gene therapy, there is important clinical value.
Summary of the invention
In view of the above-mentioned problems, we develop a kind of specific detection agents box, i.e. NF- κ B for mankind tumor tissue
RelA/p65 protein loci pecific phosphorylation kit, for detecting NF- κ B RelA/p65 egg in various mankind tumor tissues
White Ser276 and Ser536 site phosphorylation state.
Specifically, the present invention relates to following contents:
1. a kind of immunoturbidimetry based on sandwich ELISA, enzyme-linked immunization, Time-resolved Fluoroimmunoassay (TRFIA)
Method, chemoluminescence method and Electrochemiluminescince, for detect in mankind tumor tissue NF- κ B RelA/p65 Protein S er276 with
Ser536 site phosphorylation state.
2. a kind of immunohistochemistry (IHC) method and immunocytochemistry (ICC) method based on immunology principle, is used for
Detect NF- κ B RelA/p65 Protein S er276 and Ser536 site phosphorylation state in mankind tumor tissue.
3. it is a kind of based on item 1-2, for detecting NF- κ B RelA/p65 Protein S er276 in the various tumor tissues of the mankind
With the kit of Ser536 site phosphorylation state, it includes specific detection antibodies and reagent.
4. kit described in any one of 1-3, wherein the cancer is selected from the group: human breast cancer, lung cancer, brain
The various human malignancies such as cancer, gastric cancer, colorectal cancer, cancer of the esophagus, bladder cancer, prostate cancer.
5. kit described in any one of 1-4, testing result can be used for based on NF- κ B RelA/p65 targeting
Anti-tumor drug treatment is treated with Antioncogene.
6. kit described in any one of 1-5, wherein the kit also includes the site NF- κ B RelA/p65 spy
Anisotropic phosphorylated protein standard items.
7. kit described in any one of 1-6 comprising:
It is coated with the plate or microballon of specificity capture monoclonal antibody；
The specific detection antibody of another Species origin of biotin or other labels.
8. kit described in 7, further include:
It is coated with buffer (1 × PBS, pH7.3 ± 0.1)；
Sample diluting liquid (1 × PBS, 1%BSA, 0.05%NaN3, pH 7.3 ± 0.1)；
Confining liquid (1 × PBS, 3% BSA)；
Eluent PBST (1 × PBS, 0.05% Tween-20)；
Streptavidin-Eu/HRP/ALP；
IHC and ICC confining liquid
IHC and ICC substrate and color developing agent.
9. according to the kit of item 7 or 8, wherein capture antibody is exempted from come overall length NF- κ B RelA/p65 proteantigen of using by oneself
Epidemic disease mouse, and cell fusion is carried out, the strain of monoclonal hybrid cell is separated, and prepare, specific recognition NF- κ B RelA/p65
The monoclonal antibody of albumen；Antibody is detected come Ser276 or Ser536 site phosphorylation NF- κ B RelA/p65 small peptide antigen of using by oneself
Immune rabbit, and cell fusion is carried out, separation monoclonal hybrid cell strain, and the monoclonal antibody purification prepared.
10. the kit according to any one of item 1-9, wherein the detection NF- κ B RelA/p65Ser276 or
Ser536 site phosphorylation includes:
A) NF- κ B in biological sample (tumor tissues) of the specific detection agents measurement from subject is utilized
The level of RelA/p65 Ser276 or Ser536 site phosphorylation；
B) by the level measured in a) compared with the respective horizontal in normal specimens (Carcinoma side normal tissue).
11. being used for the selectively targeted drug or gene antineoplaston of cancer according to the kit of item 10.
Detailed description of the invention
Fig. 1: the sensitivity of time resolution immunofluorescence assay (TRFIA) kit.With the NF- κ B RelA/p65 of purifying
Ser276 phosphorylated protein is as measuring samples, to determine detection sensitivity and the range of linearity.
Fig. 2: the phosphorylation level of NF- κ B RelA/p65 Ser276 and Ser536 in an example Tumor Tissues of Patients with Lung Cancer.
It is detected with above-mentioned TRFIA.
Fig. 3: the phosphorylation water of NF- κ B RelA/p65 Ser276 and Ser536 in an example colorectal cancer patients tumor tissues
It is flat.It is detected with above-mentioned TRFIA.
Phosphorylation level of Fig. 4: the NF- κ B RelA/p65 Ser276 in Human Lung Cancer tissue, this is to use Human Lung Cancer
Histotomy carries out Immunohistochemical detection acquired results.
Phosphorylation level of Fig. 5: the NF- κ B RelA/p65 Ser276 in human liver cancer tissue, this is to use human liver cancer
Histotomy carries out Immunohistochemical detection acquired results.
Specific embodiment
Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but is not to limit of the invention
System, only illustrates.It is conventional method unless otherwise specified in following embodiments.
Embodiment 1: the sandwich time point of preparation detection NF- κ B RelA/p65 Ser276 and Ser536 phosphorylated protein
Distinguish fluorescence immunoassay (TRFIA) kit
In the present embodiment, sandwich time-resolved fluorescent immunoassay (TRFIA) kit includes:
It is coated with the plate or microballon of specificity capture monoclonal antibody；
The specific detection antibody of another Species origin of biotin or other labels；
It is coated with buffer (1 × PBS, pH7.3 ± 0.1)；
Sample diluting liquid (1 × PBS, 1%BSA, 0.05%NaN3, pH 7.3 ± 0.1)；
Confining liquid (1 × PBS, 3% BSA)；
Eluent PBST (1 × PBS, 0.05% Tween-20)；
Streptavidin-Eu/HRP/ALP；
Wherein biotin labeling reagent box is purchased from Pierce company.Streptavidin-Eu is purchased from Perkin Elmer company.
Time-resolved fluorescent immunoassay (TRFIA) plate of detection is prepared by oneself, is diluted to coating buffer by antibody is captured
The 100 above-mentioned antibody diluents of μ l, 4oC is added in 7-10 μ g/ml in every hole of time-resolved fluorescent immunoassay (TRFIA) plate
Overnight；It takes out, is placed in 37oC 30 minutes；Liquid is got rid of, is washed 5 times with PBST；Sky is dry, 100 μ l confining liquids of every hole addition, and 37
OC 2 hours；Liquid is got rid of, is washed 2 times with PBS；At room temperature, natural air drying 2 hours；With masking foil vacuum sealing, it is put in 4oC,
It is spare.Agents useful for same autogamy: coating buffer (1 × PBS, pH7.3 ± 0.1)；Sample dilution/antibody diluent (1 × PBS,
1%BSA、0.05%NaN3, pH 7.3 ± 0.1)；Confining liquid (1 × PBS, 3% BSA)；Eluent PBST (1 × PBS, 0.05%
Tween-20)；Standard items (various concentration RelA/p65 Ser276 phosphorylated protein antigen: 0,0.3125,0.625,1.25,
2.5、5、10、20 ng/ml)。
Wherein mouse is immunized come overall length NF- κ B RelA/p65 proteantigen of using by oneself in capture antibody, and carries out cell fusion,
It separates the strain of monoclonal hybrid cell and prepares, the monoclonal antibody of specific recognition NF- κ B RelA/p65 albumen；Detection is anti-
Rabbit is immunized come Ser276 or Ser536 site phosphorylation NF- κ B RelA/p65 small peptide antigen of using by oneself in body, and carries out cell fusion,
Separation monoclonal hybrid cell strain, and the monoclonal antibody purification prepared.
(bibliography: the system of Chen Hongyuan, Rui Wen, Hua Wenfeng, Du Jun, Cai Shaohui mouse-anti-human T CR/DC3 monoclonal antibody
Standby and its Preliminary Applications " ACAD J GCP " 2008,6,594-597；Hu Jian, Wang Qingmei, Huang Li, Zeng Yuanqing, Hu Zheng,
Luo Dixian rabbit-anti aldehyde -one restore 1 member B10(AKR1B10 of enzyme family) polyclonal antibody preparation and identification " cell and molecule
Journal of Immunology " 2016,32(11)；Jaspert R,Geske T,Teichmann A,et al. Laboratory scale
production of monoclonal antibodies in a tumbling chamber. J Immunol Methods,
1995,178:77).
The NF- κ B RelA/p65 Ser276 of 0.031ng/ml can be detected in time-resolved fluorescent immunoassay (TRFIA)
Phosphorylated protein (referring to Fig. 1).
Embodiment 2: in sandwich time-resolved fluorescent immunoassay (TRFIA) kit detection Tumor Tissues of Patients with Lung Cancer
The phosphorylation level of NF- κ B RelA/p65 Ser276 and Ser536
The preparation of detection time resolved fluorometric immunization test board: being diluted to 7-10 μ g/ml for antibody is captured with coating buffer,
The 100 above-mentioned antibody diluents of μ l are added in every hole of time-resolved fluorescent immunoassay (TRFIA) plate, 4oC is overnight；It takes out,
It is placed in 37oC 30 minutes；Liquid is got rid of, is washed 5 times with PBST；Sky is dry, 100 μ l confining liquids of every hole addition, and 37oC 2 hours；
Liquid is got rid of, is washed 2 times with PBS；At room temperature, natural air drying 2 hours；With masking foil vacuum sealing, it is put in 4oC, it is spare.
The detection of tissue samples: tumor tissues prepare soluble protein solution with standard method, are diluted with PBS with 1:10,
(various concentration RelA/p65 Ser276 or Ser536 phosphorylated protein is anti-for every hole 100 μ l sample diluting liquids of addition or standard items
It is former: 0,0.3125,0.625,1.25,2.5,5,10,20 ng/ml), 37oC, 60 minutes；Liquid is got rid of, is washed with washing buffer
It washs 5 times, sky is dry；Detection antibody is diluted with antibody diluent with 1:500, every hole addition 100 μ l detection antibody diluent, 37oC,
60 minutes；Liquid is got rid of, is washed 5 times with washing buffer, sky is dry；Streptavidin-Eu antibody diluent is with 1:80000
Dilution, 100 μ l Streptavidin-Eu dilutions of every hole addition, 37oC, 60 minutes；Liquid is got rid of, is washed with washing buffer
It washs 5 times, sky is dry；Reaction solution is added in 100 μ l；OD value is measured with microplate reader with 450 nm wavelength.Standard curve is drawn, according to mark
Directrix curve calculates the concentration of RelA/p65 Ser276 and Ser536 phosphorylated protein antigen in tissue samples, and × 10, obtains
To tissue RelA/p65 Ser276 and Ser536 phosphorylated protein antigen final concentration.
Analysis of test results
Every an example patient measures RelA/p65 Ser276 and Ser536 phosphorylation egg in tumour and normal tissue with mentioned reagent box
White concentration, and relative value (tumor tissues/normal tissue multiple) is calculated, to indicate the phosphorylation in each site in tumor tissues
It is horizontal.
Refer to Fig. 2: in the present embodiment in an example Tumor Tissues of Patients with Lung Cancer NF- κ B RelA/p65 Ser276 with
The phosphorylation level of Ser536, it is about 0.5 that wherein Ser276 measured value, which is about 3.8, Ser536 measured value,.
Embodiment 3: colorectal cancer patients tumor tissues are detected with sandwich time-resolved fluorescent immunoassay (TRFIA) kit
The phosphorylation level of middle NF- κ B RelA/p65 Ser276 and Ser536
The preparation of detection time resolved fluorometric immunization test board: being diluted to 7-10 μ g/ml for antibody is captured with coating buffer,
The 100 above-mentioned antibody diluents of μ l are added in every hole of time-resolved fluorescent immunoassay (TRFIA) plate, 4oC is overnight；It takes out,
It is placed in 37oC 30 minutes；Liquid is got rid of, is washed 5 times with PBST；Sky is dry, 100 μ l confining liquids of every hole addition, and 37oC 2 hours；
Liquid is got rid of, is washed 2 times with PBS；At room temperature, natural air drying 2 hours；With masking foil vacuum sealing, it is put in 4oC, it is spare.
The detection of tissue samples: tumor tissues prepare soluble protein solution with standard method, are diluted with PBS with 1:10,
(various concentration RelA/p65 Ser276 or Ser536 phosphorylated protein is anti-for every hole 100 μ l sample diluting liquids of addition or standard items
It is former: 0,0.3125,0.625,1.25,2.5,5,10,20 ng/ml), 37oC, 60 minutes；Liquid is got rid of, is washed with washing buffer
It washs 5 times, sky is dry；Detection antibody is diluted with antibody diluent with 1:500, every hole addition 100 μ l detection antibody diluent, 37oC,
60 minutes；Liquid is got rid of, is washed 5 times with washing buffer, sky is dry；Streptavidin-Eu antibody diluent is with 1:80000
Dilution, 100 μ l Streptavidin-Eu dilutions of every hole addition, 37oC, 60 minutes；Liquid is got rid of, is washed with washing buffer
It washs 5 times, sky is dry；Reaction solution is added in 100 μ l；OD value is measured with microplate reader with 450 nm wavelength.Standard curve is drawn, according to mark
Directrix curve calculates the concentration of RelA/p65 Ser276 and Ser536 phosphorylated protein antigen in tissue samples, and × 10, obtains
To tissue RelA/p65 Ser276 and Ser536 phosphorylated protein antigen final concentration.
Analysis of test results
Every an example patient measures RelA/p65 Ser276 and Ser536 phosphorylation egg in tumour and normal tissue with mentioned reagent box
White concentration, and relative value (tumor tissues/normal tissue multiple) is calculated, to indicate the phosphorylation in each site in tumor tissues
It is horizontal.
Refer to Fig. 3: in the present embodiment in an example colorectal cancer patients tumor tissues NF- κ B RelA/p65 Ser276 with
The phosphorylation level of Ser536, it is about 2.1 that wherein Ser276 measured value, which is about 0.8, Ser536 measured value,.
Embodiment 4: with NF- κ B RelA/p65 Ser276 phosphorylation water in Immunohistochemical detection Human Lung Cancer tissue
It is flat
Human Lung Cancer histotomy is placed in APES 1:50 acetone soln to impregnate 3 minutes and dewax, then is placed in dimethylbenzene and impregnates
10 minutes, dehydrated alcohol, 70% ethyl alcohol, 50% ethyl alcohol were replaced respectively and is impregnated 10 minutes, is finally impregnated 5 minutes with purified water；With 3%
H2O2It is added drop-wise on histotomy, respectively 10 minutes removal Endogenous peroxides, PBS is rinsed 3 times；With consistent with secondary antibody source
Serum in moisture preservation box 37 DEG C of incubation closings in 30 minutes slice, remove confining liquid；Primary antibody after dilution is added dropwise places slice
It is incubated overnight for 4 DEG C in moisture preservation box, PBS is rinsed 3 times；Secondary antibody 37 DEG C of incubations in moisture preservation box of biotin labeling after diluting are added dropwise
30 minutes, PBS was rinsed 3 times；The streptavidin that HRP is marked after diluting is added dropwise, 37 DEG C are incubated for 20 minutes, and PBS is rinsed 3 times；Add
Enter chromogenic reagent 5 minutes, then color development stopping reaction, it is clean with clear water repeated flushing；1-2 minutes are redyed with haematoxylin again, carefully
Karyon terminates after becoming blue, clean with clear water repeated flushing；With 50% ethyl alcohol, 70% ethyl alcohol, dehydrated alcohol and xylene solution point
It Jin Pao not be dehydrated within 1 minute, room temperature dries slice, then is taken pictures with microscope.
Analysis of test results
Refer to Fig. 4: for the present embodiment with Immunohistochemical detection an example Tumor Tissues of Patients with Lung Cancer, arrow show NF- κ B
The phosphorylation site of RelA/p65 Ser276.
Embodiment 5: with NF- κ B RelA/p65 Ser276 phosphorylation water in Immunohistochemical detection human liver cancer tissue
It is flat
Human Lung Cancer histotomy is placed in APES 1:50 acetone soln to impregnate 3 minutes and dewax, then is placed in dimethylbenzene and impregnates
10 minutes, dehydrated alcohol, 70% ethyl alcohol, 50% ethyl alcohol were replaced respectively and is impregnated 10 minutes, is finally impregnated 5 minutes with purified water；With 3%
H2O2It is added drop-wise on histotomy, respectively 10 minutes removal Endogenous peroxides, PBS is rinsed 3 times；With consistent with secondary antibody source
Serum in moisture preservation box 37 DEG C of incubation closings in 30 minutes slice, remove confining liquid；Primary antibody after dilution is added dropwise places slice
It is incubated overnight for 4 DEG C in moisture preservation box, PBS is rinsed 3 times；Secondary antibody 37 DEG C of incubations in moisture preservation box of biotin labeling after diluting are added dropwise
30 minutes, PBS was rinsed 3 times；The streptavidin that HRP is marked after diluting is added dropwise, 37 DEG C are incubated for 20 minutes, and PBS is rinsed 3 times；Add
Enter chromogenic reagent 5 minutes, then color development stopping reaction, it is clean with clear water repeated flushing；1-2 minutes are redyed with haematoxylin again, carefully
Karyon terminates after becoming blue, clean with clear water repeated flushing；With 50% ethyl alcohol, 70% ethyl alcohol, dehydrated alcohol and xylene solution point
It Jin Pao not be dehydrated within 1 minute, room temperature dries slice, then is taken pictures with microscope (referring to Fig. 5).
Analysis of test results
Refer to Fig. 5: the present embodiment show NF- κ B with Immunohistochemical detection an example liver cancer patient tumor tissues, arrow
The phosphorylation site of RelA/p65 Ser276.
Present invention finds a kind of new NF- κ B RelA/p65 Ser276 and Ser536 phosphorylated protein tumor tissues mark
Remember that object develops a kind of NF- κ B RelA/p65 protein loci specificity phosphorus for its differential expression in tumor tissues
Acidizing reagent box, for quantitatively or semi-quantitatively detect in various mankind tumor tissues the NF- κ B RelA/p65 Ser276 with
The level of Ser536 phosphorylated protein, to instruct clinical antineoplastic targeted drug or gene therapy.The method of the invention
High specificity, high sensitivity are simple and fast.Entire requirement of the experimental implementation for laboratory apparatus and reagent be not high, general
Laboratory can carry out, and accuracy is high, as a result by microplate reader quantitative analysis, eliminate subjectivity.
Claims (10)
1. a kind of for detecting NF- κ B RelA/p65 Protein S er276 and Ser536 site phosphorylation in mankind tumor tissue
Diagnostic kit, it includes the anti-of Ser276 and the Ser536 site phosphorylation of specific detection people's NF- κ B RelA/p65 albumen
Body, the antibody are the anti-NF- κ B RelA/p65 monoclonal antibody of mouse；Rabbit-anti NF- κ B RelA/p65 Ser276 monoclonal is anti-
Body；The anti-NF- κ B RelA/p65 Ser536 monoclonal antibody of mouse.
2. kit according to claim 1, wherein the kit also includes: NF- κ B RelA/p65 locus specificity
Phosphorylated protein standard items are coated with the plate or microballon of specificity capture monoclonal antibody；Biotin or other label it is another
The specific detection antibody of one Species origin.
3. kit according to claim 2, the kit also includes:
It is coated with buffer: 1 × PBS, pH7.3 ± 0.1；
Sample diluting liquid: 1 × PBS, 1%BSA, 0.05%NaN3, pH 7.3 ± 0.1；
Confining liquid: 1 × PBS, 3% BSA；
Eluent PBST:1 × PBS, 0.05% Tween-20；
Streptavidin-Eu/HRP/ALP；
IHC and ICC confining liquid；
IHC and ICC substrate and color developing agent.
4. kit according to any one of claim 1-3, wherein the human tumor be breast cancer, lung cancer, the cancer of the brain,
Gastric cancer, colorectal cancer, cancer of the esophagus, bladder cancer or prostate cancer.
5. kit according to claim 4, it is characterised in that: wherein the capture antibody is come the overall length NF- κ B that uses by oneself
Mouse is immunized in RelA/p65 proteantigen, and carries out cell fusion, separates the strain of monoclonal hybrid cell, and prepare, specificity
Identify the monoclonal antibody of NF- κ B RelA/p65 albumen；Antibody is detected come Ser276 the or Ser536 site phosphorylation NF- κ that uses by oneself
Rabbit is immunized in B RelA/p65 small peptide antigen, and carries out cell fusion, separation monoclonal hybrid cell strain, and the purifying list prepared
Clonal antibody.
6. kit according to claim 5, the kit is immunoturbidimetry based on sandwich ELISA, enzyme-linked
Immunization, time-resolved fluoroimmunoassay, chemiluminescence detecting method or electrochemical luminescence detection kit.
7. kit according to claim 5, wherein the kit further includes immunohistochemistry (IHC) and is immunized
Cytochemistry (ICC) kit.
8. one kind is for detecting the site NF- κ B RelA/p65 Protein S er276 or Ser536 phosphoric acid in mankind tumor tissue
The method of change state, it is characterised in that: the described in any item kits of claim 1-7 are utilized, using based on sandwich ELISA
Immunoturbidimetry, enzyme-linked immunization, Time-resolved Fluoroimmunoassay (TRFIA) method, chemoluminescence method and Electrochemiluminescince
Carry out NF- κ B RelA/p65 Protein S er276 and Ser536 site phosphorylation state in detection mankind tumor tissue.
9. method according to claim 9, it is characterised in that: wherein the detection NF- κ B RelA/p65Ser276 or
Ser536 site phosphorylation includes:
A) NF- κ B RelA/p65 in biological sample of the specific detection agents measurement from subject is utilized
The level of Ser276 or Ser536 site phosphorylation；
B) by the level measured in a) compared with the respective horizontal in normal specimens；
Wherein the biological sample is tumor tissues, and normal specimens are Carcinoma side normal tissue.
10. the described in any item kits of claim 1-7 are in the selectively targeted drug or gene antineoplaston of cancer
Application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910607155.4A CN110320365B (en) | 2019-07-06 | 2019-07-06 | NF-kB RelA/p65 protein site-specific phosphorylation diagnostic kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910607155.4A CN110320365B (en) | 2019-07-06 | 2019-07-06 | NF-kB RelA/p65 protein site-specific phosphorylation diagnostic kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110320365A true CN110320365A (en) | 2019-10-11 |
| CN110320365B CN110320365B (en) | 2022-07-22 |
Family
ID=68122972
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910607155.4A Active CN110320365B (en) | 2019-07-06 | 2019-07-06 | NF-kB RelA/p65 protein site-specific phosphorylation diagnostic kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110320365B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102049039A (en) * | 2009-10-30 | 2011-05-11 | 中国医学科学院基础医学研究所 | Application of p65 in preparation of medicament for up-regulating SIRT1 expression |
| CN102688491A (en) * | 2011-03-18 | 2012-09-26 | 梅思尔斯平移研究有限公司 | MET inhibitors for enhancing radiotherapy efficacy |
| CN103555731A (en) * | 2013-11-08 | 2014-02-05 | 湖南莱拓福生物科技有限公司 | Ser536 phosphorylated gene of NF-kB RelA/p65 and use thereof |
| CN104650234A (en) * | 2014-10-27 | 2015-05-27 | 湖南莱拓福生物科技有限公司 | Anti-AKR1B10 protein monoclonal antibody and applications thereof |
-
2019
- 2019-07-06 CN CN201910607155.4A patent/CN110320365B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102049039A (en) * | 2009-10-30 | 2011-05-11 | 中国医学科学院基础医学研究所 | Application of p65 in preparation of medicament for up-regulating SIRT1 expression |
| CN102688491A (en) * | 2011-03-18 | 2012-09-26 | 梅思尔斯平移研究有限公司 | MET inhibitors for enhancing radiotherapy efficacy |
| CN103555731A (en) * | 2013-11-08 | 2014-02-05 | 湖南莱拓福生物科技有限公司 | Ser536 phosphorylated gene of NF-kB RelA/p65 and use thereof |
| CN104650234A (en) * | 2014-10-27 | 2015-05-27 | 湖南莱拓福生物科技有限公司 | Anti-AKR1B10 protein monoclonal antibody and applications thereof |
Non-Patent Citations (3)
| Title |
|---|
| YIWEN BU 等: "Targeting NF-κB RelA/p65 phosphorylation overcomes RITA resistance", 《CANCER LETTERS》 * |
| 李丽: "磷酸化RelA/p65 Ser536在胃癌中的表达及作用", 《中国优秀博硕士学位论文全文数据库(硕士)——医药卫生科技辑》 * |
| 罗远材 等: "HPV18 E6蛋白与蛋白激酶R对核因子NF-κB p65表达及磷酸化调控作用比较", 《中华实验和临床病毒学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110320365B (en) | 2022-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109307776A (en) | A kind of gastrin 17 detection kit of improvement | |
| AU2007256391B2 (en) | Use of protein SATB2 as a marker for colorectal cancer | |
| CN104650234B (en) | Anti- AKR1B10 protein monoclonal antibodies and its application | |
| Li et al. | Expression of aryl hydrocarbon receptor in relation to p53 status and clinicopathological parameters in breast cancer | |
| JP2008539271A (en) | csPCNA isotype antibodies and uses thereof | |
| Guo et al. | Expression of gastric cancer-associated MG7 antigen in gastric cancer, precancerous lesions and H. pylori-associated gastric diseases | |
| WO2020048340A1 (en) | Serum tk1 detection kit based on fully automated chemiluminescence analyzer | |
| Kobayashi et al. | Reappraisal of the immunophenotype of pancreatic intraductal papillary mucinous neoplasms (IPMNs)—gastric pyloric and small intestinal immunophenotype expression in gastric and intestinal type IPMNs— | |
| CN110283909A (en) | The application of ZBTB20 albumen or its specific antibody in cardia cancer detection kit | |
| RU2567005C2 (en) | Novel tumour biomarker | |
| CN104345154B (en) | A kind of double-antibody sandwich test kit detecting many tumors relevant " the box-like mark of polypeptide-protein groups " | |
| WO2020200323A1 (en) | Very early cell marker of gastric cancer and early cell marker of gastric precancerous lesion and application thereof in diagnostic kit | |
| WO2015174585A1 (en) | Kit comprising antibody specifically binding to complement factor b protein and antibody specifically binding to carbohydrate antigen 19-9 protein for diagnosing pancreatic cancer | |
| BRPI0608564A2 (en) | method for determining the presence of cancer cells, method for determining the likelihood of cancer metastasis from a tissue sample and method for use of phosphorylated p68 rna helicase to screen cancer cells for cancer metastasis | |
| CN110320365A (en) | NF- κ B RelA/p65 protein loci pecific phosphorylation diagnostic kit | |
| WO2013038696A1 (en) | Molecular marker for early indentification of pleural mesothelioma patients, and expression analysis method for same | |
| KR20120116518A (en) | Xage-1a marker for early diagnosis of lung cancer and uses thereof | |
| JP7432578B2 (en) | Cancer markers and their uses | |
| CN103797369A (en) | Method for measuring anti-WT1 antibody | |
| CN111487411B (en) | Novel application of CEACAM1 polypeptide | |
| CN115112889A (en) | Application of antigen combination in preparation of lung cancer detection reagent or kit | |
| CN111879950A (en) | Application of DDRGK1 as molecular marker for predicting prognosis of gastric cancer patient by using platinum chemotherapeutic drugs | |
| US8252542B2 (en) | Serpin B 13 as a marker for squamous cell carcinoma of the lung | |
| CN111650372A (en) | Application of apolipoprotein C1 as biomarker for diagnosis and prognosis evaluation of gastric cancer | |
| CN105699649B (en) | Serum marker for preoperative detection of liver cancer microvascular invasion |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |