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Method for screening VHH/scFv by using mixed antibody capture method

A capture method and antibody technology, applied in the field of biomedicine, can solve the problems of difficult to meet the requirements of mass screening, easy omission, poor affinity, etc., to save time and cost, expand the detection range, and improve the effect of accuracy

Pending Publication Date: 2022-02-25
上海药明生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latter two methods, no matter which one is chosen, require additional steps, such as purification or IgG construction, which not only generates additional time and cost, but also makes it difficult for the throughput of these methods to meet the requirements of a large number of screenings, and these screenings The method is also prone to missing samples with poor expression in microorganisms but good affinity

Method used

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  • Method for screening VHH/scFv by using mixed antibody capture method
  • Method for screening VHH/scFv by using mixed antibody capture method
  • Method for screening VHH/scFv by using mixed antibody capture method

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Optimizing the capture conditions of mixed antibody capture VHH / scFv

[0038] First, prepare mixed antibodies with different mixing ratios for capturing VHH, and mix individual anti-Myc tag antibodies and individual anti-His tag antibodies according to different ratios, so that the ratios of anti-Myc tag antibody content to anti-His tag antibody content are respectively : 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, re-prepared with only anti The solution of His tag antibody and the solution containing only anti-Myc tag antibody, and ensure that the saturation concentration of all single or mixed antibodies is 30 μg / ml, waiting to be tested on the machine; secondly, after activating the Cytiva CM5 chip, use amino-coupling reagents to The capture antibody is coated on the chip; finally, the apparent dissociation rate constant of the coated capture antibody and VHH is detected, and the VHH flows through the chip. After a period of time, it dissociates ...

Embodiment 2

[0042] Example 2 Unlabeled antigen as analyte for VHH / scFv screening using mixed antibody capture method

[0043] Under the optimal capture conditions obtained in the study in Example 1, the anti-Myc tag antibody and the anti-His tag antibody were mixed at a ratio of 5:1 to prepare a chip. Next, the supernatant VHH / scFv is captured. After the capture is completed, use the unlabeled antigen as the analyte to k d Detection screening.

[0044] like figure 2 As shown, it is a schematic diagram of the overall process of the mixed antibody capture method provided by the present invention. First, mix the anti-Myc tag antibody and the anti-His tag antibody at a ratio of 5:1, and ensure that the antibody saturation concentration is 30 μg / ml. The kit is used to coat the mixed antibody onto the CM5 chip in a covalently coupled manner; secondly, the VHH / scFv supernatant flows through the Fc2 channel to be captured by the mixed antibody; finally, the unlabeled antigen is used as the Th...

Embodiment 3

[0054] Example 3 Half-Fc tag antigen as analyte for VHH / scFv screening using mixed antibody capture method

[0055] Under the optimal capture conditions obtained in the study in Example 1, the anti-Myc tag antibody and the anti-His tag antibody were mixed at a ratio of 5:1 to prepare a chip. Next, the supernatant VHH / scFv was captured. After the capture was completed, the half-Fc tagged antigen was used as the analyte to perform k d Detection screening. Wherein, the half Fc tag is human Fc.

[0056] First, mix anti-Myc tag antibody and anti-His tag antibody at a ratio of 5:1, and ensure that the antibody saturation concentration is 30 μg / ml, and use an amino coupling kit to coat the mixed antibody in a covalently coupled manner to the CM5 chip; secondly, let the VHH / scFv flow through the Fc2 channel, so that it is captured by the mixed antibody to the Fc2 channel; finally, use the half-Fc-labeled antigen as the analyte for detection, so that multiple concentrations of unlabe...

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Abstract

The invention discloses a method for screening VHH / scFv by using a mixed antibody capture method, which comprises the following steps: firstly, optimizing the capture condition of a mixed antibody, secondly, covalently coupling a plurality of antibodies mixed in a certain proportion to a chip under the optimal capture condition, simultaneously capturing VHH / scFv with corresponding tags through the mixed antibody, and finally, carrying out SPR detection by taking the antigen as an analyte. According to the screening method, the VHH / scFv can be stably captured, purification or IgG construction of the VHH / scFv is not needed in operation, time and cost are saved, omission of samples with poor expression but good affinity can be prevented, and dynamic binding detection and screening of a large number of VHH / scFv samples in the antibody research and development stage are facilitated.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for screening VHH / scFv by using a mixed antibody capture method. Background technique [0002] Surface plasmon resonance technology, abbreviated as SPR in English, detects the change of the SPR angle through the optical system to indirectly detect the mass change of the substance adsorbed on the metal surface, and then through further fitting calculations, the kinetic parameters of the combination of the two molecules can finally be obtained , so as to detect the interaction between the ligand and the analyte on the biosensing chip. At present, SPR has been used to study the interaction between a variety of biomolecules, including receptors, antibodies, antigens, enzymes, growth factors , glycoproteins, nucleic acids, small molecule drugs, membranes, cells, and viruses. Due to the advantages of no need for labeling, real-time detection, small sample volume, high sens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/552
CPCG01N33/6854G01N21/554
Inventor 荣乃燕陈佳婧周涛刘洁颖王卓智
Owner 上海药明生物医药有限公司
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