Using the BrdU antibody technique followed by an immuno-chemical staining(BAT),the amplification o f DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU i...Using the BrdU antibody technique followed by an immuno-chemical staining(BAT),the amplification o f DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products or in situ hybridization with PCR products on slides, the amplified targetDNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differencesin the sensitivity and efficiency between BAT and dig--if-dUTP labeling in cell in situ PCR were disCussed.展开更多
The gene encoding the heavy-and light-chain Fv regions of monoclonal antibody PS-9,which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma,was clonedby PCR techniques.The light and heavy chains we...The gene encoding the heavy-and light-chain Fv regions of monoclonal antibody PS-9,which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma,was clonedby PCR techniques.The light and heavy chains were connected by a flexible linker to form asingle chain variable fragment(ScFv)gene with 720bp,which was in turn fused topCANTAB 5 phage.The single chain Fv was expressed as fusion protein displayed on thephage surface.The phagemid is used to transform compepent E.Coli TG1 cells,then infect-ed with M13K07 helper phage to rescue the phagemid and antibody ScFv gene.All rand-mized 12 clones were shown reacting with colon cancer cell line Ls174t,which expresses S-Tnantigen.The recombinant phage has been infected E.Coli HB2151 cells to produe soluble an-tibody,which can be used for immunodetection and immunotherapy for cancer.展开更多
Genes encoding single-chain antibodies have been first constructed,which consist of the heavyand light chain variable domains of antibody PS-9 joined together by a flexible peptide linker.The geneswere cloned into coa...Genes encoding single-chain antibodies have been first constructed,which consist of the heavyand light chain variable domains of antibody PS-9 joined together by a flexible peptide linker.The geneswere cloned into coat protein g3p genes of pCANTAB5 phagemids,and expressed as fusion proteins on thephage tips.Immunological assay demonstrated that the engineered antibodies specifically bound to cancer cellsLS-174-T as well as to pure bovine submaxillary gland mucin.Their specificity and affinity appeared the sameas their parent antibodies.Our results supposed that the single-chain antibodies will be a target for thediagnosis and treatment of cancer.展开更多
The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes ...The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.展开更多
文摘Using the BrdU antibody technique followed by an immuno-chemical staining(BAT),the amplification o f DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products or in situ hybridization with PCR products on slides, the amplified targetDNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differencesin the sensitivity and efficiency between BAT and dig--if-dUTP labeling in cell in situ PCR were disCussed.
基金Supported by Grant of Medical Science from the Ministry of Health of PLA.
文摘The gene encoding the heavy-and light-chain Fv regions of monoclonal antibody PS-9,which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma,was clonedby PCR techniques.The light and heavy chains were connected by a flexible linker to form asingle chain variable fragment(ScFv)gene with 720bp,which was in turn fused topCANTAB 5 phage.The single chain Fv was expressed as fusion protein displayed on thephage surface.The phagemid is used to transform compepent E.Coli TG1 cells,then infect-ed with M13K07 helper phage to rescue the phagemid and antibody ScFv gene.All rand-mized 12 clones were shown reacting with colon cancer cell line Ls174t,which expresses S-Tnantigen.The recombinant phage has been infected E.Coli HB2151 cells to produe soluble an-tibody,which can be used for immunodetection and immunotherapy for cancer.
文摘Genes encoding single-chain antibodies have been first constructed,which consist of the heavyand light chain variable domains of antibody PS-9 joined together by a flexible peptide linker.The geneswere cloned into coat protein g3p genes of pCANTAB5 phagemids,and expressed as fusion proteins on thephage tips.Immunological assay demonstrated that the engineered antibodies specifically bound to cancer cellsLS-174-T as well as to pure bovine submaxillary gland mucin.Their specificity and affinity appeared the sameas their parent antibodies.Our results supposed that the single-chain antibodies will be a target for thediagnosis and treatment of cancer.
文摘The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.
