Method and kit for evaluating biological activity of LAG3 antibody

A biological activity and kit technology, applied in the field of LAG3 antibody activity detection, can solve the problems of cumbersome process, poor experimental stability, long construction period, etc., and achieve the effect of short construction time, good stability and effective detection of activity.

Active Publication Date: 2021-06-18
GUANGDONG FEIPENG PHARM CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both of these two techniques need to collect peripheral blood from healthy people for the isolation of peripheral blood lymphocytes PBMC, or further obtain induced differentiation of DC cells and isolation of high-purity T cells. It is difficult to obtain experimental materials, and the experimental process is complicated. Due to different donors, the experimental stability is poor
[0004] At present, the blocking activity of MHC class II molecules mediated by anti-hLAG3 antibodies can also be evaluated by detecting the activation signal of the downstream signaling pathway of LAG3 protein through the luciferase reporter gene system, but this method takes a lot of time to construct Two cell lines, the stable cell line expressing the LAG3 protein and the stable cell line containing the luciferase reporter gene, require a long and cumbersome construction period

Method used

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  • Method and kit for evaluating biological activity of LAG3 antibody
  • Method and kit for evaluating biological activity of LAG3 antibody
  • Method and kit for evaluating biological activity of LAG3 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construct a stable expression of people lag3 Jurkat stabilized cell line.

[0042] Jurkat cells did not express endogenous human LAG3 protein (HLAG3), and the Jurkat stabilized cell line with stable expression of HLAG3 protein (sequence such as SEQ ID NO.1) was constructed by a slow viral system, which is Jurkat-HLAG3, which is specifically as follows.

[0043] 293T cells were seeded in a 100 mm plate using a 10% DMEM complete medium in 5 × 10E6, at 37 ° C, 5% CO 2The incubator was cultured overnight. The next day, to be 293T cells grow to 80 ~ 90%, using polyethylene imine PEI, plasmid transfection, 37 ° C, 5% CO 2 Static culture in the incubator.

[0044] On the second day, the supernatant was completely sucked (the first virus supernatant), and the fresh DMEM complete medium was added and cultured in the incubator for 48 hours. After absorbing the virus, the addition of fresh fully medium continued to cultivate for 1 day, collect the superprint, and combined the supernat...

Embodiment 2

[0048] Evaluation of the Evaluation of the Birage of JURKAT-HLAG3 Stabilized Cell Lines.

[0049] In order to prevent the loss of the exogenous LAG3 gene, the part Jurkat-HLAG3 clone obtained in Example 1 was stabilized.

[0050] The secretion of cytokine IL2 is detected by Jurkat-HLAG3 and RAJI cellular mixed culture systems:

[0051] The 5 μg / ml CD3 antibody package was incubated in 96-well plates, the next day, according to 1 × 10E4 / hole Raji cell, 2 × 10E5 / hole Jurkat-HLAG3 cells, 37 ° C continuously for 3 days, enzyme-linked immunization Reaction ELISA detection expression of IL2 in supernatants, test results figure 2 Indicated.

[0052] by figure 2 It can be known, relative # 34, clonal, # 14, clone, clone jurkat-hlag3 almost no IL2 secretion, after adding Raji cells, the secretion of IL2 is significantly up-regulated, indicating that # 14 cells can be obvious Evaluation of biological activity of LAG3 antibodies. At the same time, through flow cytometry, the expression...

Embodiment 3

[0054] A method of evaluating the biological activity of LAG3 antibody.

[0055] The RPMI 1640 fully medium retained RAJI cells containing 10% FBS was 5 × 10E6 / ml of the live cell density of 10% FBS, and the mixed methacin C was added to the Raji cells to the final concentration of 2.5 μg / mL, 37 ° C cell culture. The box is standing for 30 min. After adding DPBS to 10ml, 250 x g from centrifugation 5 minutes, after discarding the supernatant, the cells were washed again with DPBS once.

[0056] Finally, the cells complete medium retained Raji cells to a live cell concentration of 2 × 10E5 / mL. JURKAT-HLAG3 # 14 (Example 2) The cell fluid, 250 × g centrifugation was centrifuged for 5 min, and the live cell concentration to 1 × 10E6 / ml were adjusted.

[0057] After mixing Raji cells and Jurkat-HLAG3 # 14 cells, followed by 100 μl / pore to the 96-well flat bottom plate. Dilution anti-HLAG3 antibodies were used in cells to the desired concentration. Press 100 μL / hole into a ...

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Abstract

The invention discloses a method and a kit for evaluating the biological activity of an LAG3 antibody, and relates to the technical field of LAG3 antibody activity detection. Specifically, the method for evaluating the biological activity of the LAG3 antibody comprises the following steps: co-incubating a mixed product of T lymphoma cells expressing LAG3 protein and cells expressing MHCII with the LAG3 antibody to be evaluated, and detecting the expression quantity of cell factors in the incubated product. According to the method, only one cell strain capable of stably expressing the LAG3 protein needs to be constructed, the system construction time is short, the stability is good, and the activity of the LAG3 antibody can be effectively detected.

Description

Technical field [0001] The present invention relates to the field of LAG3 antibody active detection, and in particular, to a method for evaluating biological activity of the LAG3 antibody and a kit thereof. Background technique [0002] LAG3 (Lymphocyte Activation Gene-3, lymphocyte activation gene 3, also known as CD223) is a member of the I-type transmembrane immunoglobulin supercoded family. It can be combined with MHC II molecules (tissue compatibility complex II molecules), and negatively regulates the immune response. The LAG3 antibody binding to the MHC II molecule can be raised by investigating LAG3 antibodies, which can up-regulate the immune response, and enhance cell resistance. [0003] The prior art is multi-ultra-antigen Staphylococcus colon toxin (SEB) to stimulate healthy PBMC secretion cytokines, or by mixing lymphocyte reactions in the mixed human LAG3 (HLAG3) antibody by mixing lymphocyte biologically active. However, both techniques need to collect healthy peo...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6872G01N33/6866G01N33/6869G01N33/56972G01N33/56977G01N2333/70503Y02A50/30
Inventor 张婵张先鹏李凡施荷
Owner GUANGDONG FEIPENG PHARM CO LTD
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