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Antibody combined with tight junction protein-18.2 and application of antibody

A technology of linking proteins and antibodies, applied in the direction of hybrid immunoglobulins, antibodies, immunoglobulins, etc., which can solve the problems of indistinguishability

Active Publication Date: 2020-10-16
CHENGDU CONMED BIOSCI CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another mouse monoclonal antibody 41-14A is used in clinical research, but this antibody is directed against the common sequence of Claudin 18C, and cannot distinguish the expression of Claudin 18.2 and Claudin 18.1 in clinical samples (US951223282)

Method used

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  • Antibody combined with tight junction protein-18.2 and application of antibody
  • Antibody combined with tight junction protein-18.2 and application of antibody
  • Antibody combined with tight junction protein-18.2 and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1 Construction of Claudin 18 stably transfected cell line

[0131] Using the synthetic gene Claudin 18.1 (UniProtKB-P56856, SynbioTech, whose amino acid sequence is shown in SEQ ID NO: 21) as a template, the upstream primer PRIMER 18.1-FO (SEQ ID NO: 23) and the downstream primer PRIMER18BA (SEQ ID NO: 24 ) to amplify the full-length fragment of human Claudin 18.1 (Met1-Val261), and the PCR product was subcloned into pCMV3 eukaryotic expression plasmid (self-constructed eukaryotic expression vector, containing CAG promoter, multiple cloning sites, prokaryotic screening markers Amp, true Nuclear screening markers such as puromycin and replication sites were sequentially synthesized at SynbioTech), and the pCMV3-Claudin 18.1 plasmid was obtained. Similarly, with the synthetic gene Claudin 18.2 (UniProtKB-P56856-2, SynbioTech, whose amino acid sequence is shown in SEQ ID NO: 22) as a template, the upstream primer PRIMER 18.2FO (SEQ ID NO: 25) and the downstream pri...

Embodiment 2

[0137] Embodiment 2 Preparation of anti-CIaudin 18.2 monoclonal antibody

[0138] 1) Immunization of mice

[0139] Will 1×10 6 HEK293-Claudin 18.2 stably transfected cells were mixed with equal volumes of TiterMax (Sigma, #T2684), and injected into the thigh root and footpad of female Balb / c mice for 6-8 weeks, and 20 μg pCMV3-Claudin 18.2 plasmid and gold powder ( Biorad, #1652264) were mixed, and the mouse abdomen was directly bombarded with a gene gun, once a week, and the immunization was continued for 3 weeks. Before fusion, the stably transfected cells with high expression of NIH3T3-Claudin 18.2 were used to inject pulse immunization into the tail vein (1×10 6 cells / 50μL / only). After 3 days, they were sacrificed by neck dislocation, and the popliteal lymph nodes, inguinal lymph nodes, iliac lymph nodes and spleen were collected, ground in DMEM and centrifuged to obtain a cell suspension. An appropriate amount of lymph node and spleen cell suspension was mixed with SP...

Embodiment 3

[0143] Example 3 Cell Paraffin Section Verification

[0144] Expand the positive clones 3H2-C5, 4F6-G11 and 7E8-F3 into 6-well plates, culture them with low IgG serum (Gibco, #16250), collect the supernatant after the cells are confluent, and use proteinA / G media for purification , the purified antibody was validated on paraffin sections of Claudin 18 stably transfected cells.

[0145]Collect HEK293-Claudin 18.2 stably transfected cells, HEK293-Claudin 18.1 stably transfected cells and HEK293 cells in the logarithmic growth phase, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash 3 times with PBS, add 10 mL of neutral formalin Lin solution, fixed overnight at 4°C; centrifuged at 1000 rpm for 5 minutes, discarded the supernatant, and gradually added HistoGel (Thermo, #HG-4000-012) from 20 μl until the cells were viscous and coagulated, and then the cell mass was dehydrated Treatment: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 90% ethanol for 40 minutes, ...

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Abstract

The present disclosure relates to an antibody or an antigen-binding portion thereof combined with a tight junction protein-18. 2 and application of the antibody or the antigen-binding portion thereof.The antibody can be combined with the tight junction protein-18. 2 with high affinity, and can be used for detecting the tight junction protein-18. 2, and diagnosing, treating or preventing diseasesrelated to cells of the tight junction protein-18. 2.

Description

technical field [0001] The present invention relates to an antibody binding to tight junction protein-18 and its use, especially an antibody binding to tight junction protein-18.2 and its use. Background technique [0002] Claudin is a cell surface protein family that establishes a paracellular barrier and controls the flow of molecules between cells. It was first reported by Shorichiro Tsukita et al. in 1998 (Furuse et al., (1998) JCell Biol 141(7) : 1539-1550). Claudin is an essential component of tight binding and plays an important role in maintaining the polarity of epithelial cells, controlling paracellular diffusion, and regulating cell growth and differentiation. [0003] Claudin-18 (Claudin 18) belongs to the Claudin family of tight junction proteins, has four transmembrane domains, the N-terminal and C-terminal are located in the cell, and has two extracellular regions. Due to the alternative splicing of exon 1, Claudin 18 has two splicing isoforms, Claudin 18.1 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C07K16/46G01N33/577G01N33/574A61K39/395A61K47/68A61K45/06A61P35/00
CPCC07K16/28G01N33/577G01N33/574A61K47/6835A61K47/6803A61K45/06A61P35/00C07K2317/565C07K2317/31C07K2317/24C07K2317/92G01N2333/705A61K2039/505
Inventor 王莹
Owner CHENGDU CONMED BIOSCI CO LTD
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