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Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas.

Kim DJ, Chung JH, Ryu YS, Rhim JH, Kim CW, Suh Y, Chung HK - Br. J. Cancer (2002)

Bottom Line: The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry.Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody.These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea.

ABSTRACT
SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy- and variable light-chain-encoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

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Related in: MedlinePlus

Binding of SC142 scFv to normal stomach tissue (C) and poorly differentiated human gastric adenocarcinoma tissue (D) as detected by immunohistochemical staining comparable with binding of SC142 antibody to normal stomach tissue (A) and poorly differentiated human gastric adenocarcinoma tissue (B). (A) and (C) Scale bar=60 μm; (C) and (D) Scale bar=30 μm. To detect scFv, the system of biotin-labelled SC142 scFv and streptavidin/horseradish peroxidase conjugate was used as this system eliminates nonspecific binding signal because the covalent-like interaction between biotin and streptavidin is so strong that streptavidin is highly specific for biotin. SC142 scFv was biotinylated as described in Materials and Methods.
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fig7: Binding of SC142 scFv to normal stomach tissue (C) and poorly differentiated human gastric adenocarcinoma tissue (D) as detected by immunohistochemical staining comparable with binding of SC142 antibody to normal stomach tissue (A) and poorly differentiated human gastric adenocarcinoma tissue (B). (A) and (C) Scale bar=60 μm; (C) and (D) Scale bar=30 μm. To detect scFv, the system of biotin-labelled SC142 scFv and streptavidin/horseradish peroxidase conjugate was used as this system eliminates nonspecific binding signal because the covalent-like interaction between biotin and streptavidin is so strong that streptavidin is highly specific for biotin. SC142 scFv was biotinylated as described in Materials and Methods.

Mentions: Immunohistochemical staining was used to test the ability of SC142 scFv to bind to tumour cells. It shows the ability of SC142 antibody (Figure 7BFigure 7


Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas.

Kim DJ, Chung JH, Ryu YS, Rhim JH, Kim CW, Suh Y, Chung HK - Br. J. Cancer (2002)

Binding of SC142 scFv to normal stomach tissue (C) and poorly differentiated human gastric adenocarcinoma tissue (D) as detected by immunohistochemical staining comparable with binding of SC142 antibody to normal stomach tissue (A) and poorly differentiated human gastric adenocarcinoma tissue (B). (A) and (C) Scale bar=60 μm; (C) and (D) Scale bar=30 μm. To detect scFv, the system of biotin-labelled SC142 scFv and streptavidin/horseradish peroxidase conjugate was used as this system eliminates nonspecific binding signal because the covalent-like interaction between biotin and streptavidin is so strong that streptavidin is highly specific for biotin. SC142 scFv was biotinylated as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376138&req=5

fig7: Binding of SC142 scFv to normal stomach tissue (C) and poorly differentiated human gastric adenocarcinoma tissue (D) as detected by immunohistochemical staining comparable with binding of SC142 antibody to normal stomach tissue (A) and poorly differentiated human gastric adenocarcinoma tissue (B). (A) and (C) Scale bar=60 μm; (C) and (D) Scale bar=30 μm. To detect scFv, the system of biotin-labelled SC142 scFv and streptavidin/horseradish peroxidase conjugate was used as this system eliminates nonspecific binding signal because the covalent-like interaction between biotin and streptavidin is so strong that streptavidin is highly specific for biotin. SC142 scFv was biotinylated as described in Materials and Methods.
Mentions: Immunohistochemical staining was used to test the ability of SC142 scFv to bind to tumour cells. It shows the ability of SC142 antibody (Figure 7BFigure 7

Bottom Line: The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry.Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody.These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea.

ABSTRACT
SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy- and variable light-chain-encoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

Show MeSH
Related in: MedlinePlus