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Method for determining biological activity of LAG3 (Lymphocyte Activation Gene-3) protein binding molecule

A molecular biology and molecular binding technology, applied in the field of measuring LAG3 protein binding molecular biological activity, which can solve the problems of heavy workload, poor repeatability, and inability to quantify specifically

Active Publication Date: 2018-12-07
NANJING LEADS BIOLABS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the biological activity of anti-LAG3 antibody has been reflected by the traditional method of detecting PBMC or T cell cytokine release, but only relative comparison can be made, and specific quantification cannot be done.
Traditional factor detection methods are easily affected by individual differences in PBMC or T cells, with poor repeatability, heavy workload and long delay (at least 3 days)

Method used

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  • Method for determining biological activity of LAG3 (Lymphocyte Activation Gene-3) protein binding molecule
  • Method for determining biological activity of LAG3 (Lymphocyte Activation Gene-3) protein binding molecule
  • Method for determining biological activity of LAG3 (Lymphocyte Activation Gene-3) protein binding molecule

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1, the screening of Jurkat-NFAT-LAG3 cell clone

[0045] 1. Protocells

[0046] The original Jurkat cells (Clone E6-1) involved in the present invention were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and subcultured and preserved in our laboratory. The cell viability rate used by the cells is over 95%.

[0047] Jurkat cell clones were resuspended with OPTI-MEM (GIBCO, 31985062) medium, transferred to an electric shock cup provided with an electroporation instrument (Bio-Rad, Gene Pulser Xcell), the plasmid was added and mixed gently, and then electroshocked, and then all the cells were suspended The solution was transferred to a complete medium (90% 1640+10% FBS) in a clean six-well plate (Costar, 3516), and cultured statically in a 37°C incubator (Thermo, BB150I) for 24 hours.

[0048] 2. Plasmid

[0049] The plasmid pGL4.30[luc2P / NFAT-RE / Hygro] (Promega, E848A) with NFAT reporter element, and the pIRES-puro3 plasmid (Clonte...

Embodiment 2

[0061] Embodiment 2, reporter gene experimental method

[0062] 1. Cell Recovery

[0063] The cell clone involved in the present invention is the Jurkat-NFAT-LAG3 cell clone after transfection screening and identification in our laboratory, and the clone number is 35B8. It was verified as a cell clone with high expression of NFAT reporter element and LAG3 protein. The Raji cells involved in the present invention are human Burkitt's lymphoma cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and subcultured and preserved in our laboratory. After the cells were taken out of the liquid nitrogen tank, they were immediately put into a 37°C water bath to thaw until a small piece of ice remained, diluted with complete medium (90% 1640+10% FBS) and transferred to a clean 15mL centrifuge tube (Corning, 430791). Centrifuge at 400×g to discard the supernatant, resuspend the cells with an appropriate volume of complete medium and inoculate the cells on a ce...

Embodiment 4

[0085] Embodiment 4, reporter gene experiment optimization

[0086] 1. The ratio relationship between Jurkat-NFAT-LAG3 and Raji cells

[0087] The cells involved in the present invention are Jurkat-NFAT-LAG3 cell clones and Raji cells. When cell sampling, the cell number of Jurkat-NFAT-LAG3 was fixed at 1×10 5 One / well, Raji cells were sampled as 1 / 4, 1 / 6 and 1 / 8 of the number of Jurkat-NFAT-LAG3 cells, respectively. Then the reporter gene experiment was carried out under the conditions of fixed SED concentration of 100ng / mL and 150ng / mL, and the cell number matching conditions with good luminescence intensity and signal window were selected. The effect of different cell ratio conditions on the reporter gene results is as follows: Figure 4 As shown, under the two SED concentration conditions, different cell ratios can show that the intensity of the luminescent signal increases with the increase of the antibody concentration, and the cell ratios of 4:1 and 8:1 are better. ...

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Abstract

The invention discloses a method for determining the biological activity of an LAG3 (Lymphocyte Activation Gene-3) protein binding molecule. The method comprises the step of detecting the activity ofan anti-human LAG3 antibody by a co-expression NFAT (Nuclear Factor of Activated T cells) reporting element and Jurkat cells of human LAG3 protein under the condition of coexistence of SED and Raji cells. The method provided by the invention can be used for rapidly and accurately detecting the activity of the molecule bound with human LAG3 protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for measuring the biological activity of LAG3 protein binding molecules. Background technique [0002] LAG3 (Lymphocyte Activation Gene-3, Lymphocyte Activation Gene-3) is an immune checkpoint protein, which contains 4 Ig-like regions. Molecular binding. It has been confirmed that LAG3 can negatively regulate the activation and proliferation of T cells, and is usually expressed on the surface of T cells infiltrating tumor tissues, which is a sign of T cell failure. By affecting the function of LAG3, it can improve the immune system dysfunction caused by viral infection, autoimmune disease and malignant tumor, and improve the prognosis of the disease. [0003] As immune checkpoint antibody drugs play an increasingly important role in tumor treatment, a variety of LAG3-related drugs are in clinical and preclinical research. Since LAG3 negatively regulates the activity of T ce...

Claims

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Application Information

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IPC IPC(8): C12Q1/66G01N33/68
CPCC12Q1/66G01N33/6863
Inventor 康小强赖寿鹏张鹏黄潇管静
Owner NANJING LEADS BIOLABS CO LTD
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