Method of using HTRF to screen LAG3/MHCII inhibitor at high throughput

An inhibitor and high-throughput technology, applied in the direction of biological testing, material inspection products, etc., can solve the problems that are difficult to solve due to the large number of samples in drug screening, the inability to achieve high throughput, and poor repeatability of results, so as to save experimental workload and fluorescence Long lasting effect with low false negative rate

Inactive Publication Date: 2018-09-11
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

As a traditional method, ELISA has some shortcomings that are difficult to overcome, such as: 1) There are many experimental steps and time-consuming
It takes at least one day to wash the plate, add samples, develop color, etc. many times; 2) It cannot achieve high throughput, and it is difficult to solve the problem of many samples in drug screening; 3) The coated protein may shield some protein sites 4) Due to many steps, it is easy to cause experimental errors, resulting in poor reproducibility and instability of results

Method used

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  • Method of using HTRF to screen LAG3/MHCII inhibitor at high throughput
  • Method of using HTRF to screen LAG3/MHCII inhibitor at high throughput
  • Method of using HTRF to screen LAG3/MHCII inhibitor at high throughput

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Embodiment 1

[0031] 1) For protein preparation, LAG3 was serially diluted 3-fold from 30nM to obtain 30nM, 10nM, 3.3nM and 0nM respectively; MHCII was serially diluted 3-fold from 100nM to 0.02nM.

[0032] 2) Add 5ul each of the prepared proteins into a 384-well detection plate, incubate at room temperature for 15 minutes, add the tag antibody coupled to Donor and Acceptor (or add 10ul after equal volume mixing), incubate for 1 hour and read.

[0033] According to the principle of HTRF method, detect the fluorescence ratio of 665nm / 620nm, according to this ratio, judge the binding of protein, the higher the signal, the more binding, see the results figure 2 .

Embodiment 2

[0035] 1) Configure inhibitors, and serially dilute the humanized positive monoclonal antibody anti-LAG3blocking antibody of BMS, starting from 100nM, dilute 4 times to 0.00002nM;

[0036] 2) Prepare the protein, and perform follow-up inhibitor screening according to the appropriate protein concentration of Example 1, LAG3 is 10nM, and MHCII is 20nM;

[0037] 3) The prepared inhibitor and protein were added to the 384-well detection plate in sequence, and after incubating at room temperature for 15 minutes, the tag antibody conjugated with Donor and Acceptor was added (or 10ul was added after equal volume mixing). Incubate for 1 hr and read.

[0038] According to the principle of HTRF method, detect the fluorescence ratio of 665nm / 620nm, according to this ratio, judge the binding of protein, the higher the signal, the more binding, see the results image 3 .

Embodiment 3

[0040] The mouse monoclonal antibody anti-LAG3blocking antibody of Abcam is carried out inhibition curve determination, according to the method of embodiment 2, detects, and the result sees Figure 4 .

[0041] Depend on figure 2, 3, 4, it can be seen that the present invention is applicable to the screening of various LAG3 inhibitors, and the amount of protein is less, and the sensitivity of the method is high. The experiment proves that the screening of LAG3 / MHCII inhibitors by the method of the present invention is simple and feasible.

[0042] In summary, the current main screening methods for LAG3 / MHCII are traditional ELISA and FACS, and most of them are cell experiments, which require complex cell line construction. However, the present invention uses HTRF for screening at the biochemical level for the first time, which solves the problems of cumbersome operation, long time, and low throughput of traditional methods, and realizes low cost, high throughput, high sensi...

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Abstract

The invention discloses a method of using HTRF to screen a LAG3 / MHCII inhibitor at high throughput and belongs to the field of screening methods of inhibitors. The method is characterized by comprising the steps of: 1) preparing LAG3 and MHCII proteins, which have been constructed and have different tags, as to-be-screened samples; 2) successively adding the to-be-screened samples, LAG3 and MHCIIproteins, and anti-tag antibodies corresponding to the two proteins, into a micro-well plate, and respectively conjugating a donor and an acceptor; 3) performing room-temperature inoculation, readinga number on a microplate reader, wherein the ratio value of 665 nm / 620 nm is an original data. The method is applied to the field of screening cell strains, can effectively save labor and material cost and shorten time, and also accelerates the development of biopharmaceutical industry.

Description

technical field [0001] The invention belongs to the field of inhibitor screening methods, in particular to a method for high-throughput screening of LAG3 / MHCII inhibitors using HTRF. Background technique [0002] At present, in the field of drug development of immune checkpoint inhibitors, LAG3 has become one of the hottest targets in drug development at home and abroad. Many foreign and local pharmaceutical companies in China are developing LAG3 / MHCII inhibitors. However, at this stage, there is no homogeneous method for directly screening LAG3 / MHCII inhibitors at the biochemical level in the biopharmaceutical industry at home and abroad. The structural domain of the method is not clear, and the second is that due to the difficulty of developing a homogeneous method, it is necessary to experiment with a variety of tagged proteins and donors and acceptors of fluorescent energy. When screening inhibitors of protein interactions, FACS and ELISA methods are often used. FACS ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 王维娜
Owner 浠思(上海)生物技术有限公司
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