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Rapid modification and screening method for antibody pH-dependent binding activity

A technology that combines activity and antibodies is applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc. It can solve the problem that the sensitivity is difficult to meet the requirements, and achieve the guarantee of flux and stability, sensitivity and Stable, high throughput effect

Inactive Publication Date: 2020-01-10
SHANGHAI WUXI BIOLOGIC TECH CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the pH of the slightly acidic environment of the tumor is about 6.5, the difference from the neutral pH 7.4 is less than 1, the sensitivity of conventional screening methods is difficult to meet the requirements

Method used

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  • Rapid modification and screening method for antibody pH-dependent binding activity
  • Rapid modification and screening method for antibody pH-dependent binding activity
  • Rapid modification and screening method for antibody pH-dependent binding activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. The scFv that does not have pH-dependent binding activity is modified so that its binding at pH 6.5 is greater than that at pH 7.4

[0042] 1. First construct the wild-type antibody scFv prokaryotic expression plasmid template.

[0043] 2. Determination of antigen-antibody binding incubation buffer

[0044] In order to ensure that the unpurified antibody expressed by Escherichia coli maintains the required pH conditions in the experiment, different binding buffers were screened, buffers with different salt concentrations, different ratios were mixed with the supernatant of E. Added 0.2M PB buffer (100ml formula as follows), and mixed it with the supernatant at 1:1.

[0045] pH 0.2mol / L NaH 2 PO 4 (ml)

0.2mol / L Na 2 HPO 4 (ml)

6.0 87.7 12.3 6.5 68.5 31.5 7.4 19 81

[0046] Capture-ELISA, KD-ELISA washing buffer is 1X PBS phosphate buffered saline. It is necessary to prepare 10*PBS buffer master solution first,...

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Abstract

The invention discloses a method for transforming and screening antibody pH-dependent binding activity. The method comprises the following steps: (1) constructing a wild antibody scFv plasmid template; (2) performing site-specific mutagenesis on six CDR regions of an antibody, and replacing each amino acid in the CDR regions with histidine; (3) establishing a Capture-ELISA condition and carrying out high-throughput screening; and (4) performing KD-ELISA and FACS detection: carrying out gradient dilution on a sample, carrying out KD-ELISA detection by using buffers with different pH values, anddetermining the pH-dependent binding activity of targets cloned on the antigen surface and cell surface according to a curve drawn by detection data. The method provided by the invention can be usedfor obtaining the antibody with pH-dependent binding activity in a high-flux, high-efficiency and low-cost manner.

Description

technical field [0001] The invention relates to the engineering transformation of antibodies, in particular to a rapid transformation and screening method for pH-dependent binding activity of antibodies. Background technique [0002] Monoclonal antibodies are widely used in disease treatment. The traditional antibody-antigen binding is not condition-dependent, for example, antibodies exhibit the same antigen-binding activity within a certain pH range. The introduction of pH-dependent activity through antibody engineering has high application value. After the antibody without pH-dependent activity binds to the antigen, it prolongs the half-life of the target antigen through the "buffering" effect, and cannot remove the antigen from the plasma, but instead increases the concentration of the antigen in the plasma. However, some natural receptors can not only bind ligands, but also dissociate in a pH-dependent manner in acidic endosomes (~pH 5.5), continuously clearing ligands...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/13C12N15/10G01N33/68G01N33/543
CPCC12N15/70C12N15/102C07K16/00G01N33/6854G01N33/543C07K2317/622C07K2317/565
Inventor 徐曼张丽英刘娟根纳季·戈洛洛博夫李竞顾继杰陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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