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Screening method of pH-dependent antibody targeting complement protein C5

A technology of complement protein and screening method, applied in immunoglobulin, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve problems such as heavy workload

Active Publication Date: 2020-08-04
SHANGHAI PUREMAB BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it will take several months or even longer to complete the entire transformation process, and the workload is huge

Method used

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  • Screening method of pH-dependent antibody targeting complement protein C5
  • Screening method of pH-dependent antibody targeting complement protein C5
  • Screening method of pH-dependent antibody targeting complement protein C5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Screening and Identification of Mouse Immunization and Human C5 Antibody Hybridoma Cell Line

[0095] Referring to Antibodies: A Laboratory Manual, Second Edition (Edward A. Greenfield 2012), 8-week-old Balb / c mice were immunized for a total of 42 days at intervals of 14 days. Human C5 protein was emulsified in complete or incomplete Freund's adjuvant, and it was injected unilaterally into the subcutaneous tissue of the back of the neck, the root of the tail, and the groin of the mouse, and into the peritoneal cavity. Blood was collected from the tail vein on the 35th day of immunization, and after the antibody titer was detected by ELISA, spleen cells of immunized mice were fused with myeloma cells.

[0096] Human C5 protein was used to immunize the spleen cells of Balb / c mice, and fused with myeloma cells using PEG or electrofusion method, and the fused hybridoma cells were 1x10 7 Cells / well were inoculated in HAT-containing medium and placed in a 384-well ...

Embodiment 2

[0099] Example 2 Using SKY59 as a reference antibody to screen for antibodies competing with epitopes

[0100] The ForteBio OCTET Red biomolecular interaction instrument based on the principle of biofilm interference was used to screen the mouse antibody that competed with the SKY59 epitope. SKY59 was used as the reference antibody, and the sample to be tested was the supernatant of the mouse hybridoma screened in Example 1.

[0101] Prepare buffer 1 (phosphate buffered saline (PBS) + 0.1% BSA + 0.02% Tween 20 (Tween 20) + 0.05% Priclin300, pH7.4) for diluting antigen and reference antibody. According to the method described above in the present invention, a ratio of 60%-100% or even >100% means no competition; 30-60% means partial competition; <30% means competition; greater than 100% means no competition. The results are shown in Table 2, which indicated that the supernatants of D11 and J11 clones competed with SKY59, and the supernatants of C15 clone partially competed with...

Embodiment 3

[0108] Example 3 Identification of mouse anti-sequence and construction of chimeric antibody

[0109] After expanding the screened monoclonal hybridoma cells in 12 or 24 well plates, the total RNA was extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); 5×PrimeScript RTMaster Mix (Takara) was used to The total RNA of hybridoma cells was reverse-transcribed into cDNA; using degenerate primers and Extaq PCR reagent (Takara) to amplify the antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequence; using PCR clean-up Gelextraction reagent The amplified PCR product was purified using the Macherey-Nagel kit (Macherey-Nagel); the amplified PCR product was connected to the T vector according to the instructions of the pmd19T Simple Vector Kit (Takara) and transformed into E. coli competent cells. The strain was amplified and the plasmid was extracted for DNA analysis. Monoclonal antibody variable regio...

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Abstract

The invention discloses a screening method of a pH-dependent antibody targeting complement protein C5 and an antibody obtained by the screening method. The screening method disclosed by the inventionis carried out on a biomolecule interaction analysis system; the method comprises the following steps of: setting an experiment group 1, a control group 1, an experiment group 2 and a control group 2,and enabling each group of chips to be combined with antigen complement protein C5 (A) as a reference antibody (Ab1) and a to-be-detected antibody (Ab2) of a pH-dependent C5 monoclonal antibody: theexperiment group 1 A-Ab1-Ab2; the control group 1 is A-Ab1-0; the experimental group 2 is A-0-Ab2; the control group 2 is A-0-0; and detecting the binding signal and calculating the ratio of (experimental group 1-control group 1) / (experimental group 2-control group 2) to judge whether the antibody to be detected is a pH-dependent C5 antibody or not. According to the method, the pH-dependent C5 monoclonal antibody development period can be shortened, and the labor capacity and the time cost are saved; a hybridoma cell culture supernatant can be used as a to-be-detected sample for direct identification, so that the antibody screening efficiency is further improved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular, the invention relates to a screening method for a pH-dependent antibody targeting complement C5 and an antibody obtained by the screening. Background technique [0002] Complement protein C5 is a major component of the complement system, a key part of the innate immune system. C5 is a glycoprotein of 190kDa, which consists of two polypeptide chains α and β connected by disulfide bonds, and their molecular masses are 115kDa and 75kDa, respectively. Cleavage by C5 convertase at an arginine residue 75 amino acids downstream of the N-terminus of the C5 α-chain produces 7.4 kDa C5a and 180 kDa C5b complement cleavage products. Among them, C5a is an anaphylatoxin that can stimulate vasodilation through histamine released by basophils and giant cells; C5b can form a membrane attack complex (MAC) with complement proteins C6, C7, C8, and C9 on the cell surface in turn. ), the accumulation of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/68G01N33/577
CPCC07K16/18G01N33/68G01N33/577C07K2317/56C07K2317/565C07K2317/34C07K2317/92G01N2333/4716
Inventor 王雪高攀吴建李祥烽陶春艳王骊淳任红媛
Owner SHANGHAI PUREMAB BIO TECH CO LTD
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