nf-κb RelA/p65 protein site-specific phosphorylation diagnostic kit

A technology of p65 and kits, applied in fluorescence/phosphorescence, biological testing, instruments, etc., can solve problems such as less success

Active Publication Date: 2022-07-22
湖南莱拓福生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, nearly a thousand NF-κB inhibitors have been developed. Although some inhibitors have achieved promising results in experimental treatments, they are rarely successful in clinical practice.

Method used

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  • nf-κb RelA/p65 protein site-specific phosphorylation diagnostic kit
  • nf-κb RelA/p65 protein site-specific phosphorylation diagnostic kit
  • nf-κb RelA/p65 protein site-specific phosphorylation diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of a sandwich time-resolved fluorescence immunoassay (TRFIA) kit for the detection of NF-κB RelA / p65 Ser276 and Ser536 phosphorylated proteins

[0037] In this embodiment, the sandwich time-resolved fluorescence immunoassay (TRFIA) kit includes:

[0038] Plates or beads coated with specific capture monoclonal antibodies;

[0039] Biotin or other labeled specific detection antibody from another genus;

[0040] Coating buffer (1×PBS, pH7.3±0.1);

[0041] Sample diluent (1×PBS, 1%BSA, 0.05%NaN3, pH 7.3±0.1);

[0042] Blocking solution (1×PBS, 3% BSA);

[0043] Eluent PBST (1×PBS, 0.05% Tween-20);

[0044] Streptavidin-Eu / HRP / ALP;

[0045] The biotin labeling kit was purchased from Pierce Company. Streptavidin-Eu was purchased from Perkin Elmer. Prepare the time-resolved fluorescence immunoassay (TRFIA) plate for detection by yourself, dilute the capture antibody to 7-10 μg / ml with coating buffer, and add 100 μl of the above to each well of the t...

Embodiment 2

[0049] Example 2: Detection of phosphorylation levels of NF-κB RelA / p65 Ser276 and Ser536 in tumor tissues of lung cancer patients by sandwich time-resolved fluorescence immunoassay (TRFIA) kit

[0050] Preparation of detection time-resolved fluorescence immunoassay plate: Dilute the capture antibody to 7-10 μg / ml with coating buffer, add 100 μl of the above antibody dilution to each well of a time-resolved fluorescence immunoassay (TRFIA) plate, overnight at 4ºC ; Take out, put at 37ºC for 30 minutes; shake off the liquid, wash 5 times with PBST; air dry, add 100 μl blocking solution to each well, 37ºC for 2 hours; shake off the liquid, wash 2 times with PBS; air dry naturally at room temperature 2 hours; vacuum seal with foil, set at 4ºC, set aside.

[0051] Detection of tissue samples: The soluble protein solution of tumor tissue was prepared by standard methods, diluted 1:10 with PBS, and 100 μl of sample diluent or standard (different concentrations of RelA / p65 Ser276 or ...

Embodiment 3

[0055] Example 3: Detection of phosphorylation levels of NF-κB RelA / p65 Ser276 and Ser536 in tumor tissues of colon cancer patients by sandwich time-resolved fluorescence immunoassay (TRFIA) kit

[0056] Preparation of detection time-resolved fluorescence immunoassay plate: Dilute the capture antibody to 7-10 μg / ml with coating buffer, add 100 μl of the above antibody dilution to each well of a time-resolved fluorescence immunoassay (TRFIA) plate, overnight at 4ºC ; Take out, put at 37ºC for 30 minutes; shake off the liquid, wash 5 times with PBST; air dry, add 100 μl blocking solution to each well, 37ºC for 2 hours; shake off the liquid, wash 2 times with PBS; air dry naturally at room temperature 2 hours; vacuum seal with foil, set at 4ºC, set aside.

[0057] Detection of tissue samples: The soluble protein solution of tumor tissue was prepared by standard methods, diluted 1:10 with PBS, and 100 μl of sample diluent or standard (different concentrations of RelA / p65 Ser276 or...

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Abstract

The invention relates to the technical field of medicine and biology, in particular to a kit for the detection of NF-κB RelA / p65 protein site-specific phosphorylation. Specifically, the kit can specifically detect the phosphorylation status of Ser276 and Ser536 sites of NF-κB RelA / p65 protein to guide the targeted antitumor drug treatment of NF-κB inhibitors or RelA / p65 S276A / S536D Gene therapy has important clinical application value.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a kit for specifically detecting the phosphorylation of human NF-κB RelA / p65 protein sites, which is used for detecting NF-κB RelA / p65 protein Ser276 in various human tumor tissues The phosphorylation state of Ser536 site is used for targeted anti-tumor drug therapy and anti-tumor gene therapy. The kit involves immunoturbidimetric method, enzyme-linked immunosorbent assay, time-resolved immunofluorescence analysis (TRFIA) method, chemiluminescence method and electrochemiluminescence method based on sandwich ELISA. The kit also involves immunohistochemistry (IHC) method and immunocytochemistry (ICC) method. Background technique [0002] The eukaryotic transcription factor protein family NF-κB RelA / p65 (NF-κB RelA / p65 gene in GenBank accession number L19067.1) is a key regulatory protein in immune and inflammatory responses. The mammalian NF-κB / Rel family includes five subuni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/68G01N21/64
CPCG01N33/577G01N33/6893G01N21/6408G01N21/6486
Inventor 曹哲
Owner 湖南莱拓福生物科技有限公司
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