Method for detecting cell-binding activity of therapeutic antibody drugs using electrochemiluminescence
A therapeutic antibody, binding activity technology, applied in the direction of chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc. problems, to achieve the effect of shortening time and improving accuracy
Active Publication Date: 2018-04-27
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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Problems solved by technology
As a conventional binding activity method, ELISA has some insurmountable problems in detecting the binding activity of macromolecular antibody drugs to their receptors: 1) Some epitopes (such as receptors) are difficult to obtain commercial products, or receptor extraction It is difficult to prepare; 2) When doing cell-based binding assay (cell-based binding assay), cells cannot be coated on the plate, especially suspension cells; 3) Repeated cleaning is required during the experiment, multi-step, cumbersome and time-consuming
At present, another cell binding test is detected by flow cytometry. Although flow cytometry does not need to be washed many times, there is a problem with the technical platform of the instrument itself: when some receptors are highly expressed on the cell surface , it will exceed the detection limit of the instrument, and it will not be detected on the platform during the activity test, so it cannot be quantified; at the same time, it needs to be calibrated before each use of the flow cytometer, which is time-consuming
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[0029] In the following examples, the reagents used are commercial products unless otherwise specified. Among them, MSD company refers to MSD (Meso Scale Discovery, Gaithersburg, MD) company.
[0030] In addition, the pH in the 1×PBS buffer used below was 7.2.
[0031] The method for detecting the cell-binding activity of an anti-MHCII antibody (a therapeutic antibody drug, manufactured by Wuxi Pharmatech Biotechnology Co., Ltd.) by electrochemiluminescence method of the present invention comprises the steps of:
[0032] 1) Raji cells (purchased from ATCC) in 2×10 4 At a density of 30 μL / well, spread in the Single-SPOT 96-well plate of MSD Company, and incubate at room temperature for 1 h;
[0033] 2) Add 1×PBS buffer solution containing 30% FBS (fetal bovine serum) at a volume concentration of 50 μL / well, incubate at room temperature for 30 minutes, and block;
[0034]3) Wash 3 times with 1×PBS buffer, add a series of diluted therapeutic antibody drugs—anti-MHCII antibody ...
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The invention discloses a method for detecting the cell conjugation activity of therapeutic antibody medicines by utilizing electrochemiluminescence. The method comprises the steps of (1) spreading cells in a 96-hole plate, and incubating; (2) adding confining liquid, incubating and sealing; (3) cleaning the processed cells by buffer solution, adding therapeutic antibody medicines, and incubating; (4) cleaning the processed cells by the buffer solution, adding ruthenium marked anti-human IgG, and incubating; (5) cleaning the processed cells by the buffer solution, adding MSD read buffer solution without a surface active agent; and (6) reading the 96-hole plate by MSD Sector Imager6000 to obtain the cell conjugation activity result. According to the method, the experiment accuracy and the experiment sensitivity are greatly improved, the time is greatly shortened, and the defect of the instruments for detecting a high-expression receptor existing on the surface of the cell by a flow cytometer is overcome.
Description
technical field [0001] The invention relates to a method for detecting the cell-binding activity of therapeutic antibody drugs, in particular to a method for detecting the cell-binding activity of therapeutic antibody drugs by electrochemiluminescence. Background technique [0002] At present, the domestic biopharmaceutical industry mainly uses enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of macromolecular antibody drugs and their receptors. As a conventional binding activity method, ELISA has some insurmountable problems in detecting the binding activity of macromolecular antibody drugs to their receptors: 1) Some epitopes (such as receptors) are difficult to obtain commercial products, or receptor extraction It is difficult to prepare; 2) When doing cell-based binding assays, cells cannot be coated on the plate, especially suspension cells; 3) Repeated cleaning is required during the experiment, with multiple steps, which is cumbersome and time-...
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IPC IPC(8): G01N21/76
Inventor 陈敏李丽丽吉静娴阙红周伟昌陈智胜贾晓青
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD