Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof

A sialoglycoprotein and monoclonal antibody technology, applied in the biological field, can solve problems such as unsatisfactory clinical applications and limited scope of application of antibodies

Inactive Publication Date: 2015-03-18
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
View PDF2 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies on the application of these antibodies are rarely reported
However, the antibodies sold by Santa have a limited scope of application and cannot meet the needs of clinical applications.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof
  • Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof
  • Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, the preparation of monoclonal antibody

[0079]The ASGPR cDNA (the ASGPR fragment encoding the amino acid sequence such as SEQ ID NO: 1) was obtained by RT-PCR amplification, and the pGEX-4T-1 plasmid (purchased from Invitrogen, USA) was used as a vector, and inserted into the BamHI and EcoRI positions of the vector In the point, the ASGPR expression recombinant vector with GST tag was constructed, transformed into E.coli BL21 competent cells, screened and cultured, induced by isopropylthiogalactoside (IPTG), purified by GST affinity column, 12% SDS-PAGE detection fusion protein expression.

[0080] SQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL(SEQ ID NO:1)

[0081] Mouse immunization, cell fusion, screening of positive hybridoma cell clones, ascites production, and antibo...

Embodiment 2

[0083] Embodiment 2, the detection of monoclonal antibody

[0084] ① Identification of monoclonal antibody subclasses

[0085] According to the instructions of the monoclonal antibody subclass identification kit.

[0086] As a result, it was determined that the monoclonal antibody of the present invention was of the IgG1 subtype.

[0087] ②ELISA

[0088] According to conventional ELISA method.

[0089] ③Western blot

[0090] Follow routine immunoblotting methods.

[0091] ④ Flow cytometry (FACS)

[0092] Prepare human liver cell line WRL68 and human liver cancer cell lines Hep3B, Huh7, HepG2, PLC / PRF / 5 (the above cell lines were purchased from Shanghai Cell Bank, Chinese Academy of Sciences). The expression of ASGPR in some cell lines has been reported [Park JH, Cho EW, Shin SY, Lee YJ, Kim KL. Detection of the asialoglycoprotein receptor on cell lines of extrahepatic origin. Biochem Biophys Res Commun, 1998, 244: 304- 311].

[0093] Routinely cultivate the above cell ...

Embodiment 3

[0107] Example 3. Isolation and Identification of Circulating Hepatoma Cells Using Monoclonal Antibodies

[0108] Main materials: 5mL peripheral blood of liver cancer patient volunteers, anticoagulated with EDTA. Ficoll-Paque Plus, available from GE Healthcare. The mouse anti-human ASGPR monoclonal antibody (IgG1) of the present invention. Magnetic separator, anti-Mouse IgG1 MicroBeads, MS magnetic separation column, 30 μm filter, CK3-6H5 (antibody against epithelial tissue-derived tumor cells) were all purchased from Miltenyi, Germany. 400R desktop centrifuge (with cytocentrifuge smear attachment), purchased from Heraeus, Germany. Polylysine slides were purchased from Fujian Maixin Company. SP two-step immunohistochemistry kit was purchased from Fuzhou Maixin Biotechnology Company. PBS washing solution (containing 2mM EDTA and 0.5%BSA) was self-prepared.

[0109] step:

[0110] (A) Ficoll separation of peripheral blood mononuclear cells

[0111] 1) Mix 5mL EDTA antico...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as a preparation method and application thereof. The monoclonal antibody can be used for well recognizing an ASGPR antigen, cannot perform cross reaction with other proteins, and is very high in specificity and sensitivity. The antibody disclosed by the invention can be applied to identification of primary hepatocellular carcinoma and metastatic liver cancer, separation and detection of circulating liver cancer cells, liver imaging and targeted delivery of liver medicines or genes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a monoclonal antibody that specifically recognizes and binds to the extracellular segment of a human asialoglycoprotein receptor (ASGPR), a preparation method and an application thereof. Background technique [0002] Asialoglycoprotein receptor (ASGPR), also known as hepatic agglutinin or glycogen agglutinin, is specifically expressed on the surface of hepatic parenchymal cells in the sinusoidal space and basolateral side of the mammalian liver, and its main physiological function is to clear peripheral Glycoproteins, lipoproteins and apoptotic cells containing D-galactose residues or N-acetylgalactosamine residues in blood circulation. The unique physiological functions of ASGPR and its high hepatocyte specificity make it of great importance in the identification of hepatocellular carcinoma and metastatic liver cancer, the isolation and detection of circulating liver cancer cells, liv...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20A61K47/48G01N33/68G01N33/577C12N5/09C12R1/91
Inventor 殷正丰李军张妤钱海华孙斌陈磊
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com