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Method for detection of biological activity and titer of co-expressed recombinant human protein based on multiple time-resolved fluorescence techniques

A time-resolved fluorescence and biological activity technology, applied in biological testing, fluorescence/phosphorescence, analytical materials, etc., can solve the problem of lack of comprehensive analysis of multiple test results, and achieve the effect of cost saving and low reaction volume

Active Publication Date: 2022-02-11
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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Problems solved by technology

At present, a single fluorescence or ELISA method cannot provide a comprehensive comparison of the results of various components in the process of clone screening and process optimization; the combined use of fluorescence detection for different recombinant human proteins also lacks the ability to systematically analyze multiple detection results platform approach

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  • Method for detection of biological activity and titer of co-expressed recombinant human protein based on multiple time-resolved fluorescence techniques

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Embodiment 1

[0045]The present invention establishes a platform for evaluating and screening the activity of co-expressed recombinant human proteins, and optimizes two innovative fluorescence technologies, using recombinant human protein A (no tag) and recombinant human protein B-tag (commonly used tags, including Fc, His, Flag, etc.) fusion protein co-expression as an example, protein A and protein B are co-expressed to form an aggregate, and the molecular molar ratio of A:B in the AB aggregate is 1:n (n≥1). The platform scheme consists of the following three fluorescence methods, which are selectively used together, and a comprehensive evaluation method for the final result is attached.

[0046] Method 1: Determination of recombinant human protein B-tag by competition method

[0047] 1) Add 5ul of cell supernatant or analyte to a 384 microwell plate, which can be diluted with dilution buffer;

[0048] 2) Add 2.5ul of fluorescent signal receptor coupling tag;

[0049] 3) Add Anti-tag-cr...

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Abstract

The invention discloses a co-expressed recombinant human protein biological activity and titer detection method based on multiple time-resolved fluorescence techniques, which includes the following steps: 1. Any two or three of the following methods are adopted: method 1, Competition method to determine one or two recombinant human proteins; method two, sandwich method to determine one or two recombinant human proteins; method three, sandwich method to determine two recombinant human protein fusion protein conjugates; two, set the fluorescence wavelength, read Take the fluorescence value; 3. Data processing: get the standard curve according to the ratio of the fluorescence and the concentration of the standard, and calculate the sample concentration; 4. Sorting the samples: assign the detection values ​​of the above method to the X, Y and Z axis coordinates of the sample to obtain the sample Position in the coordinate system; define an acceptable standard interval, and all samples falling within this interval meet the requirements. The invention can systematically and high-throughput evaluate the activity / titer of each component of the recombinant human protein produced by co-expression, so as to support production cell line clone screening and purification process optimization.

Description

technical field [0001] The present invention relates to a homogeneous time-resolved fluorescence technique (fluorescence), in particular to a method for detecting the biological activity and titer of a co-expressed recombinant human protein based on multiple time-resolved fluorescence techniques, which is used for cell clone screening and purification Process evaluation. Background technique [0002] Homogeneous Time Resolved Fluorescence (Homogeneous Time Resolved Fluorescence, Fluorescence) can measure the time delay of fluorescence resonance energy transfer, which can be used for quantitative detection of homogenized samples (reference 1: Bazin H, Trinquet E, Mathis G . Time resolved amplification of cryptateemission: a versatile technology to trace biomolecular interactions. JBiotechnol. 2002; 82(3):233–50.). The technique combines two techniques of fluorescence resonance energy transfer (FRET) and time-resolved fluorescence (TRF). FRET technology utilizes the energy t...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N33/533G01N33/68
CPCG01N21/6408G01N21/6428G01N21/6452G01N33/533G01N33/68G01N2021/6415G01N2021/6439
Inventor 陆心远沈旸贾晓青黄岗周伟昌陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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