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Method of screening adenosine A2A receptor ligands by Tag-lite and analytical experimental technique

An experimental technology and ligand technology, which is applied in the field of screening adenosine A2A receptor ligands using Tag-lite binding analysis experimental technology, can solve problems such as experimental errors, complicated parameter settings, and many experimental steps, and achieve simple and safe experimental operations , Simplify the operation steps and improve the effect of experimental flux

Inactive Publication Date: 2019-05-24
浠思(上海)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] As a traditional method, isotope labeling has some insurmountable shortcomings, such as: 1) radioactive contamination, which requires a high level of safety in the operating environment; 2) high requirements for the professionalism of operators; There are many steps and it takes a long time; 4) It is difficult to achieve high throughput, and it is difficult to solve the problem of many samples to be tested; 5) The accuracy of the detection instrument is high and the parameter settings are complicated; 6) Due to many steps, it is easy to cause experimental errors, resulting in duplication of results 7) The isotopes used for labeling are prone to produce impurities during the reaction process and directly affect the accuracy of the results; 8) Radioactive experiments, after the end of the experimental process or staged experiments, may have different degrees of radioactive pollution and radioactive waste. appear, need timely decontamination treatment and radioactive waste treatment

Method used

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  • Method of screening adenosine A2A receptor ligands by Tag-lite and analytical experimental technique
  • Method of screening adenosine A2A receptor ligands by Tag-lite and analytical experimental technique
  • Method of screening adenosine A2A receptor ligands by Tag-lite and analytical experimental technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Saturation binding experiment (determination of Kd value):

[0050] The experimental result of saturation binding assay is the change of non-specific binding signal and total signal with the increase of ligand concentration under reaction equilibrium state.

[0051] Such as image 3 As shown, when performing such experiments, the fluorescently labeled ligand was diluted in a concentration gradient and incubated with a fixed total amount of labeled cells to allow the reaction to reach equilibrium. At this time, the ratio of HTRF signals obtained was the total binding signal. Use unlabeled ligand as a negative control to measure the non-specific binding signal between the labeled ligand and the receptor, non-receptor molecules, or microplates. Dilute the fluorescent labeled ligand in a solution containing a fixed total amount of labeled cells and a non-labeled ligand with a molar concentration of 100 times the labeled ligand, so that the reaction reaches equilibrium, and...

Embodiment 2

[0053] Competitive binding assay (determination of Ki value):

[0054] Such as Figure 4 As indicated, a competition-type binding assay experiment was used to determine the dissociation constant, Ki, of the samples. When performing such experiments, it is necessary to serially dilute the samples to be tested (small molecule compounds, antibodies, peptides, etc.), and incubate them with a fixed concentration of fluorescent ligands and a fixed number of cells.

[0055] Using Tag-lite binding assay technology to screen adenosine A2A receptor ligands, taking small molecule compound samples as an example, the specific steps are as follows:

[0056] (1) Prepare 1X Tag-lite working buffer (1X TLB, commercial reagent): Calculate the amount of buffer required according to the experimental requirements, dilute 5X Tag-lite working buffer to 1X with distilled water, and mix well.

[0057] (2), if Figure 5 As shown, prepare the compound (sample to be tested): use 1X TLB to serially dil...

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Abstract

The invention discloses a method of screening adenosine A2A receptor ligands by Tag-lite and an analytical experimental technique and belongs to the field of screening of adenosine A2A receptor ligands. The method herein comprises the steps of constructing a specific-tag-fused target cytomembrane surface protein expression vector; marking tag-fused cytomembrane surface proteins through an HTRF (homogeneous time-resolved fluorescence) fluorescent group donor covalently-bound tag-specific primer; incubating the marked cells or cytomembranes with binding ligands, detecting by HTRF, and calculating Kd or Ki value of a sample under test according to data results. The method herein has the advantages that the signals are strong, the results are reliable, and the new marking technique is employedso that the signals are stronger and more stable; binding condition of receptor ligands is truly reflected based on a cell homogenous detection system, and the results are reliable.

Description

technical field [0001] The invention belongs to the field of screening of adenosine A2A receptor ligands, and in particular relates to a method for screening adenosine A2A receptor ligands by using Tag-lite binding analysis experiment technology. Background technique [0002] Adenosine receptors (Adenosine receptors, ARs) belong to the integral membrane protein family G protein-coupled receptors (Gprotein-coupled receptors, GPCRs), which can be divided into four subtypes: A1, A2A, A2B and A3. All four classes of adenosine receptors are activated by extracellular adenosine and play important roles in a wide range of physiological processes, such as sleep regulation, angiogenesis, and immune regulation. Therefore, adenosine receptors are very important potential targets in the study of various pathophysiology, such as sleep disorders, cancer, inflammation, etc. Adenosine A2A receptors are highly expressed in the striatum of the brain, immune cells in the spleen, thymus, lymph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 张冰洁
Owner 浠思(上海)生物技术有限公司
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