A method for determining the biological activity of human IL-36/IL36R/IL1RACP pathway inhibitors

A biological activity and inhibitor technology, applied in biochemical equipment and methods, microbial determination/inspection, microorganisms, etc., can solve the problems of high cost, poor durability and repeatability, and large variability

Active Publication Date: 2020-09-25
NANJING NOVOACINE BIO-TECH CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The above six traditional IL-36 / IL36R / IL1RAcP pathway inhibitor biological activity assay methods all require long-term cell culture and cytokine MSD ELISA detection, with cumbersome steps, large variability, high cost, long time, and method durability and poor reproducibility, not suitable for high-throughput screening experiments

Method used

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  • A method for determining the biological activity of human IL-36/IL36R/IL1RACP pathway inhibitors
  • A method for determining the biological activity of human IL-36/IL36R/IL1RACP pathway inhibitors
  • A method for determining the biological activity of human IL-36/IL36R/IL1RACP pathway inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Exploration of electric shock transfection conditions for Jurkat cells

[0085] Jurkat cells are suspended human peripheral blood leukemia T cells, and the transfection is very difficult. The chemical transfection method is difficult to transfect the plasmid into the cells, so the electroporation transfection method is chosen.

[0086] (1) Electric shock transfection condition 1: take 2×10 6 Jurkat cells (Cell Bank, Chinese Academy of Sciences), 10 μg pDSRed plasmid (Clontech, 632406) (red fluorescent plasmid is used to identify transfection efficiency), electric shock voltage 1100v, electric shock time 50ms, electric shock frequency 1 time;

[0087] (2) Electric shock transfection condition 2: take 2×10 6 1 Jurkat cell, 10 μg pDSRed plasmid, electric shock voltage 1300v, electric shock time 20ms, electric shock times 2 times;

[0088] (3) Electric shock transfection condition 3: take 2×10 6 1 Jurkat cell, 10 μg pDSRed plasmid, electric shock voltage 1400v,...

Embodiment 2

[0090] Example 2 Preparation of effector cells expressing Jurkat / IL36R / IL1RAcP / NF-κB

[0091] Using the electroporation transfection condition 3 screened in Example 1, 3.33 μg pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega (Beijing) Biotechnology Co., Ltd.) plasmid, 3.33 μg pcDNA3.1 ( +) / IL36R plasmid and 3.33 μg pcDNA3.1(+) / IL1RAcP were transfected into Jurkat cells (Cell Bank, Chinese Academy of Jurkat / IL36R / IL1RAcP / NF-κB effector cells were obtained by culturing in mL Zeocine medium.

[0092] Dilute recombinant human IL-36β (ACROBiosystems, Cat. No. IL8-H5115) to 200ng / mL, 66.66ng / mL, 22.22ng / mL, 7.40ng / mL, 2.46ng / mL, 0.82ng in 1640+10% FBS medium / mL, 0.26ng / mL and other concentrations were added to a 96-well plate (Corning, cat. 3610) at 50 μL / well, and then 100,000 per well and 50 μL / well were used for different Jurkat / IL36R / IL1RAcP / NF-κB effects Cell clones were added to the above 96-well plate, 37°C, 5% CO 2 After incubation for 6 h, 100 μL / well of Bio-Lite chromogenic reagen...

Embodiment 3

[0097] Example 3 Optimization and establishment of IL36R monoclonal antibody biological activity assay method

[0098] The Jurkat / IL36R / IL1RAcP / NF-κB-25# screened out in Example 2 was selected as the effector cell strain used in optimizing and establishing the IL36R monoclonal antibody biological activity assay method.

[0099] (1) Optimize cell density:

[0100] IL36β preparation: use 1640+10% FBS medium to dilute 0.2mg / mL IL36β to 100ng / mL;

[0101] Drug preparation: use 1640+10% FBS medium to dilute IL-36R monoclonal antibody BI-655130 to 50000ng / mL, 12500ng / mL, 3125ng / mL, 781.25ng / mL, 195.31ng / mL, 48.82ng / mL, 12. 20ng / mL, 3.02ng / mL and other concentrations, the above-mentioned IL36β and different concentrations of BI-655130 were mixed 1:1 according to 100μL and 100μL, and added to a 96-well plate according to 50μL / well;

[0102] Preparation of effector cell suspension: Dilute Jurkat / IL36R / IL1RAcP / NF-κB-25# effector cells to 4×10 5 pcs / mL, 1×10 6 pcs / mL, 2×10 6 c...

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Abstract

The present invention provides a method for determining the biological activity of human IL-36 / IL36R / IL1RAcP pathway inhibitors, the method comprising: using cells stably expressing IL36R, IL1RacP and NF-κB reporter genes as effector cells, Incubate after mixing with IL-36 and human IL-36 / IL36R / IL1RAcP pathway inhibitor, and determine the biological activity of the human IL-36 / IL36R / IL1RAcP pathway inhibitor according to the measured signal value of the reporter gene . The assay method provided by the present invention does not require any human primary tissue-derived cells or other components, the color development result is stable, the experimental period is short, the operation is simple, and the cell contamination and errors caused by long-term incubation and multi-step operation are avoided; Moreover, the measured biological activity is related to the clinical curative effect and meets the relevant technical requirements of NMPA.

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-36 / IL36R / IL1RAcP 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] The Interleuk...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02C12N5/10G01N21/64
CPCC07K14/7155C12N5/0636C12N9/0069C12N2510/00C12Y113/12007G01N21/6486G01N33/502G01N33/505
Inventor 熊新辉王晋张弢王骊淳
Owner NANJING NOVOACINE BIO-TECH CO LTD
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