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Method for screening CD47/SIRP alpha blocker by HTRF (Homogeneous Time Resolved Fluorescence) one-step method

A blocking agent and one-step technology, which is applied in the field of screening CD47/SIRPalpha blocking agents by using HTRF one-step method, can solve problems such as experimental error, time-consuming, unstable, etc., and avoid false positives and false negatives, and fluorescence duration Long-term, save the effect of experimental workload

Inactive Publication Date: 2018-11-23
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] FACS requires a special flow cytometry instrument, which is expensive to modify, and a single cell passes through the column, and the throughput is very low. It is only suitable for antibody drug screening, not for small molecules. Binding to CD47 or SIRP alpha is enough, but it does not necessarily block the combination of the two, and other experimental evidence is usually required
As a traditional method, ELISA has some shortcomings that are difficult to overcome, such as: 1) There are many experimental steps, it takes a long time, and it needs to go through multiple washings, adding samples, color development, etc., and it takes at least one day; 2) It cannot reach high-pass 3) The coated protein may shield some protein sites, resulting in missing positive results, that is, false negative results; 4) Due to many steps, it is easy to cause experimental errors, leading to poor repeatability, unstable

Method used

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  • Method for screening CD47/SIRP alpha blocker by HTRF (Homogeneous Time Resolved Fluorescence) one-step method
  • Method for screening CD47/SIRP alpha blocker by HTRF (Homogeneous Time Resolved Fluorescence) one-step method
  • Method for screening CD47/SIRP alpha blocker by HTRF (Homogeneous Time Resolved Fluorescence) one-step method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1) Prepare the protein. CD47 starts from 10nM and is diluted by 3 times to obtain different concentrations from 10nM to 0nM; SIRPalpha is diluted by 3 times from 11.1nM to obtain different concentrations from 11.1nM to 0nM.

[0032] 2) Add 5ul each of the prepared protein to the 384-well detection plate, incubate at room temperature for 15 minutes, add the tag antibody coupled to Donor and Acceptor (or add 10ul after mixing the same amount), and incubate for 1 hour to read. According to the principle of HTRF method, the fluorescence ratio of 665nm / 620nm is detected, and the protein binding is judged according to the ratio. The higher the signal, the more binding, the result is figure 2 .

Embodiment 2

[0034] 1) Configure the positive blocking agent, and dilute the mouse-derived anti-human CD47blocking antibody in a gradient, starting from 100nM, and dilute 3 times;

[0035] 2) Prepare the protein, and screen the blocking agent according to the appropriate protein concentration in Example 1. CD47 is 2nM and SIRPalpha is 1nM;

[0036] 3) The prepared blocking agent and protein are sequentially added to the 384-well detection plate. After 15 minutes of incubation at room temperature, add the tag antibody coupled to Donor and Acceptor (or add 10ul after mixing the same amount), and incubate for 1 hour to read.

[0037] According to the principle of HTRF method, the fluorescence ratio of 665nm / 620nm is detected, and the protein binding is judged according to the ratio. The higher the signal, the more binding, the result is image 3 .

[0038] by figure 2 with 3 It can be seen that the present invention is suitable for screening a variety of CD47 or SIRP alpha blockers, with a small amou...

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Abstract

The invention discloses a method for screening a CD47 / SIRP alpha blocker by an HTRF (Homogeneous Time Resolved Fluorescence) one-step method, and belongs to the field of blocker screening methods. Thescreening method comprises the following steps: configuring the constructed CD47 and SIRP alpha proteins with different labels as samples to be screened; sequentially adding the CD47 and SIRP alpha proteins as the samples to be screened into a microplate; incubating at room temperature, reading by using Eliasa to detect fluorescence, and the ratio of 665 nm / 620 nm being the raw data. The method disclosed by the invention realizes the screening of CD47 / SIRP alpha various blockers at the biochemical level, and can effectively screen high expression cell line cloning in a short time, and accelerate the progress of the pharmaceutical research and development industry.

Description

Technical field [0001] The invention belongs to the field of blocking agent screening methods, and in particular relates to a method for screening CD47 / SIRPalpha blockers using HTRF one-step method. Background technique [0002] The integrin-related protein CD47 is a transmembrane glycoprotein widely expressed on cells and belongs to the immunoglobulin superfamily. When CD47 is combined with its ligand SIRP alpha (signal regulatory protein alpha), CD47 will be immune to macrophages. Cells send "don't eat me" signals to inhibit the activation of the immune system, so CD47 and SIAP alpha become potential immune checkpoints. Its blocker can block the formation of CD47 / SIRP alpha complex, so that the immune system can regain immune activity. Therefore, in the field of antibody drug development, CD47 / SIRP alpha has become one of the important targets for drug development at home and abroad. [0003] Currently, the commonly used screening methods are FACS and ELISA. FACS is a flow cyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/533
CPCG01N33/533G01N33/54306
Inventor 王维娜
Owner 浠思(上海)生物技术有限公司
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