Limits...
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.

Show MeSH

Related in: MedlinePlus

Relevant parts of the nucleotide and amino acid sequences of the SC142 scFv unit in pRSET-Angiogenin SfiI/NotI vector. The pRSET SfiI/NotI vector (Yi et al, 1999) was amplified with the primers as described in Materials and Methods. The vector was modified to include a HindIII site and angiogenin site after NotI site. The HIS-tag and Xpress-tag upstream from the enterokinase cleavage site can be removed by enterokinase treatment. The numbers in the left column indicate the nucleotide numbers of pRSET B (Invitrogen).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2376138&req=5

fig1: Relevant parts of the nucleotide and amino acid sequences of the SC142 scFv unit in pRSET-Angiogenin SfiI/NotI vector. The pRSET SfiI/NotI vector (Yi et al, 1999) was amplified with the primers as described in Materials and Methods. The vector was modified to include a HindIII site and angiogenin site after NotI site. The HIS-tag and Xpress-tag upstream from the enterokinase cleavage site can be removed by enterokinase treatment. The numbers in the left column indicate the nucleotide numbers of pRSET B (Invitrogen).

Mentions: Hybridoma total RNA served as the template for constructing SC142 scFv using the Recombinant Phage Antibody System (RPAS; Pharmacia, Uppsala, Sweden); incorporating many of the features described by Mccafferty et al (1990) and Winter and Milstein (1991). Isolated, agarose gel-purified VH- and VL-encoding DNA were subsequently spliced together by PCR using primers designed to introduce a linking sequence between the two gene segments and specific restriction sites at both 5′ (SfiI) and 3′ (NotI) ends of the spliced sequence. Restriction digestion with SfiI and NotI endonucleases, and agarose gel purification of the digested linked product, preceded ligation of this DNA into the SfiI- and NotI-digested pRSET-Angiogenin SfiI/NotI vector (Figure 1Figure 1


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)

Relevant parts of the nucleotide and amino acid sequences of the SC142 scFv unit in pRSET-Angiogenin SfiI/NotI vector. The pRSET SfiI/NotI vector (Yi et al, 1999) was amplified with the primers as described in Materials and Methods. The vector was modified to include a HindIII site and angiogenin site after NotI site. The HIS-tag and Xpress-tag upstream from the enterokinase cleavage site can be removed by enterokinase treatment. The numbers in the left column indicate the nucleotide numbers of pRSET B (Invitrogen).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Relevant parts of the nucleotide and amino acid sequences of the SC142 scFv unit in pRSET-Angiogenin SfiI/NotI vector. The pRSET SfiI/NotI vector (Yi et al, 1999) was amplified with the primers as described in Materials and Methods. The vector was modified to include a HindIII site and angiogenin site after NotI site. The HIS-tag and Xpress-tag upstream from the enterokinase cleavage site can be removed by enterokinase treatment. The numbers in the left column indicate the nucleotide numbers of pRSET B (Invitrogen).
Mentions: Hybridoma total RNA served as the template for constructing SC142 scFv using the Recombinant Phage Antibody System (RPAS; Pharmacia, Uppsala, Sweden); incorporating many of the features described by Mccafferty et al (1990) and Winter and Milstein (1991). Isolated, agarose gel-purified VH- and VL-encoding DNA were subsequently spliced together by PCR using primers designed to introduce a linking sequence between the two gene segments and specific restriction sites at both 5′ (SfiI) and 3′ (NotI) ends of the spliced sequence. Restriction digestion with SfiI and NotI endonucleases, and agarose gel purification of the digested linked product, preceded ligation of this DNA into the SfiI- and NotI-digested pRSET-Angiogenin SfiI/NotI vector (Figure 1Figure 1

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