Method for determining the biological activity of human il-33/st2 pathway inhibitors

A biologically active, inhibitory technology

Active Publication Date: 2019-09-03
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The above two traditional methods for measuring the biological activity of IL-33 / ST2 pathway inhibitors require long-term cell culture and ELISA detection. These traditional methods have cumbersome steps, large variability, high cost, long time, and durability and poor repeatability

Method used

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  • Method for determining the biological activity of human il-33/st2 pathway inhibitors
  • Method for determining the biological activity of human il-33/st2 pathway inhibitors
  • Method for determining the biological activity of human il-33/st2 pathway inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Construction of Effector Cells KU812 / NF-κB Using KU812 Cells Growing in Suspension

[0088] KU812 is a suspension of human peripheral blood basophilic leukemia cells, the transfection is very difficult, and the chemical transfection method is difficult to transfect the plasmid into the cells, so the electroporation transfection method is chosen.

[0089] Several electroporation transfection conditions were compared:

[0090] (1) Electroporation transfection condition 1: take 1×10 6 KU812 cells, 1 µg pDSRed plasmid (red fluorescent plasmid is used to identify transfection efficiency), electric shock voltage 1000v, electric shock time 50ms, electric shock frequency 1 time;

[0091] (2) Electroporation transfection condition 2: take 1×10 6 KU812 cells, 1 µg pDSRed plasmid, electric shock voltage 1200v, electric shock time 40ms, electric shock times 1 time;

[0092] (3) Electroporation transfection condition 3: take 1×10 6 KU812 cells, 1 µg pDSRed plasmid, ...

Embodiment 2

[0099] Example 2 Construction of Effector Cell HEK293 / ST2 / IL-8 Using Adherent HEK293 Cells

[0100] pcDNA3.1(+) / hST2 and pGL4 [luc2P / hIL-8 / Hygro] were co-transfected into HEK293 cells by chemical transfection method, and then in the presence of 0.5 mg / ml neomycin and 0.1 mg / ml tide The transfected cells were cultured in medium 1640+10% FBS of mycin B to obtain multiple clones. The obtained cells of different clones were respectively diluted to 40×10 using medium 1640+10% FBS 4 cells / mL, 50 µL / well in 96-well plate, 37°C 5% CO 2 Incubate overnight. On the second day, recombinant human IL-33 was added to some wells with a final concentration of 1000ng / mL (medicated group), and some wells were not added as a control (blank group), at 37°C in 5% CO 2 Incubate for 16 h. Then use Bright-Glo TM Kit, according to the instructions, add luminescent substrate 1:1 (100µL:100µL) to measure the expression of luciferase, see the results of luciferase activity assay Figure 5 .

[0...

Embodiment 3

[0102] Example 3 Comparison of effector cells KU812 / NF-κB and HEK293 / ST2 / IL-8

[0103] As a kind of adherent cells, HEK293 / ST2 / IL-8 needs to be digested and passaged with trypsin during passage. Digestion with trypsin itself is an external stimulus to cells, and IL33, as a typical emergency response protein, will increase with this external stimulus, which instead stimulates the signaling pathway in HEK293 cells, resulting in an increase in the overall response background signal, The signal-to-noise ratio is reduced. In addition, HEK293 / ST2 / IL-8 requires a one-day resting period after digestion and plating. KU812 / NF-κB is a suspension cell and does not have the above problems.

[0104] The comparison between KU812 / NF-κB and HEK293 / ST2 / IL-8 effector cells can be seen in Table 1.

[0105] Table 1 Comparison of two methods between KU812 / NF-κB and HEK293 / ST2 / IL-8

[0106]

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Abstract

The invention provides a method for rapidly measuring the biological activity of an IL-33 / ST2 pathway inhibitor. The method comprises the following steps: constructing a cell strain of a stably expressed NF-kappa B or IL-8 reporter gene, stimulating the reporter gene expression with IL-33, blocking an IL-33 signal pathway by using an IL-33 / ST2 antibody drug, and determining the biological activityof the antibody by fitting a four-parameter curve based on a measured reporter gene signal value. According to the method provided by the invention, a quicker and more accurate quantitative detectionmethod is established for the biological activity of the IL-33 / ST2 antibody drug, the experimental period is short, the operation is simple and convenient, and the cell pollution and errors caused bylong-time incubation and multi-step operation are avoided.

Description

technical field [0001] The invention relates to the field of biological activity detection of biological medicines, in particular, the invention relates to a method for rapidly, accurately and quantitatively measuring the biological activity of IL-33 / ST2 pathway inhibitors. Background technique [0002] Monoclonal antibody is a highly uniform antibody produced by a single B cell clone that only targets a specific epitope, and has become an important part of the global pharmaceutical market. The determination of the biological activity of monoclonal antibodies at the cellular level plays an important role in the discovery and development of monoclonal antibody drugs. Currently, most biological activity detection methods use cell-based biological activity assays, including cell proliferation inhibition methods. , cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, cell ELISA and reporter gene method. [0003] Interleukin 33 (IL-33) ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6869G01N2333/7155
Inventor 熊新辉张弢仲恺
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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