To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expres...To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.展开更多
The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further charac- terized by ...The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further charac- terized by biochemical and immunological techiques. Results showed that assoiciated antigen of McAb 3H11 was mainly located on the membrane of target cells. It was sensitive to heat and digestion of proteases,but resistant to treatment with sodium periodate. Schiff’s reagent staining was negative. SDS-PAGE and IEF- analysis showed that the antigen was a 210 kD molecular weight with pl of 8. 5 protein. The molecular weight of antigen extracted from target cell with tunicamycin was not changed. We concluded that the cor- responding antigen of McAb 3H11 might be an alkaline protein.展开更多
OBJECTIVE To explore the method of preparation of 99m↑Tc labeled AntiVEGF McAb 5-FU loaded polylactic acid nanoparticles （99m↑TC-5-FU-Ab-NPs）, and investigate the biological distribution of the nanoparticles in hu...OBJECTIVE To explore the method of preparation of 99m↑Tc labeled AntiVEGF McAb 5-FU loaded polylactic acid nanoparticles （99m↑TC-5-FU-Ab-NPs）, and investigate the biological distribution of the nanoparticles in human gastric carcinoma xenografts. METHODS Anti-VEGF monoclonal antibodyes （MCAB）in 5-FU-Ab-NPs were labeled with 99m↑Tc using a modified Schwarz method. After isolation of the 99m↑TC-5-FU-Ab-NPs using a Sephadex G-250 column, the labeling percentage and radiochemical purity were determined using paper chromatography. The immunocempetence of the 99m↑TC-5-FU-Ab-NPs as tumor markers was determined using ELISA and immunohistochemistry. 99m↑TC-5-FU-Ab-NPs （experimental group）, 99m↑Tc-labelled murine multiclonal IgG loaded polylactic acid and nanoparticles （control group） were injected via the tail vein into SCID mice bearing human gastric carcinoma. A radio-immunity ECT image was developed at 2 and 6 h after the injection. Following the ECT imaging, the mice were sacrificed, their tissue and tumor radioactivity distribution determined, and percentage of the injected-dose per gram （%ID/g） and tumor/ nontumor （T/NT） ratio calculated. High performance liquid chromatography （HPLC） was used to determine the 5-FU concentration in the tumor tissue and blood in the mice of both groups. RESULTS The percentage of 99m↑TC-5-FU-Ab-NPs labeling was 90%-95%. There was no obvious decrease in the antibody activity before and after labeling. The radio-immuno-imaging （RII） showed that the tumor image had developed 2 h after injection of the 99m↑TC-5-FU-Ab-NPs, and with time it was clearer at the 6th hour following the injection. The %lD/g of the tumor tissue at both 2 h and 6 h after the injection was significantly higher compared to the control group. The tumor %lD/g and the tumor to blood activity ratio （TB） of the experimental group at 6 h following the injection increased compared to that at 2 h, and at the same time, 5-FU concentration in the tumor of the experimental group continuously increased over time, and showed a significant difference compared to the 5-FU concentration in the tumor of the control group. CONCLUSION The 99m↑TC-5-FU-Ab-NPs prepared in this study are adequate to meet the demands of the RII, and the immune targeting ability of the anti-VEGF MCAB is reliable. Six hours after injection, the 99m↑TC-5-FU-Ab-NPs showed a relatively high specific concentration shadow in the human gastric carcinoma xenografts.展开更多
文摘To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.
文摘The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further charac- terized by biochemical and immunological techiques. Results showed that assoiciated antigen of McAb 3H11 was mainly located on the membrane of target cells. It was sensitive to heat and digestion of proteases,but resistant to treatment with sodium periodate. Schiff’s reagent staining was negative. SDS-PAGE and IEF- analysis showed that the antigen was a 210 kD molecular weight with pl of 8. 5 protein. The molecular weight of antigen extracted from target cell with tunicamycin was not changed. We concluded that the cor- responding antigen of McAb 3H11 might be an alkaline protein.
基金the grants as fol-lows:The Problems-Tackling Program in Sci-ence and Technology of Guangzhou City,Chi-na(No.2003 Z 3-E0381)National Foundationof Natural Science,China(No.30670951)+1 种基金Guangdong Foundation of Natural Science,Guangdong,China(No.06021322)TheProblems-Tackling Program in Science andTechnology of Guangdong Province,China(No.2005 B31211002).
文摘OBJECTIVE To explore the method of preparation of 99m↑Tc labeled AntiVEGF McAb 5-FU loaded polylactic acid nanoparticles （99m↑TC-5-FU-Ab-NPs）, and investigate the biological distribution of the nanoparticles in human gastric carcinoma xenografts. METHODS Anti-VEGF monoclonal antibodyes （MCAB）in 5-FU-Ab-NPs were labeled with 99m↑Tc using a modified Schwarz method. After isolation of the 99m↑TC-5-FU-Ab-NPs using a Sephadex G-250 column, the labeling percentage and radiochemical purity were determined using paper chromatography. The immunocempetence of the 99m↑TC-5-FU-Ab-NPs as tumor markers was determined using ELISA and immunohistochemistry. 99m↑TC-5-FU-Ab-NPs （experimental group）, 99m↑Tc-labelled murine multiclonal IgG loaded polylactic acid and nanoparticles （control group） were injected via the tail vein into SCID mice bearing human gastric carcinoma. A radio-immunity ECT image was developed at 2 and 6 h after the injection. Following the ECT imaging, the mice were sacrificed, their tissue and tumor radioactivity distribution determined, and percentage of the injected-dose per gram （%ID/g） and tumor/ nontumor （T/NT） ratio calculated. High performance liquid chromatography （HPLC） was used to determine the 5-FU concentration in the tumor tissue and blood in the mice of both groups. RESULTS The percentage of 99m↑TC-5-FU-Ab-NPs labeling was 90%-95%. There was no obvious decrease in the antibody activity before and after labeling. The radio-immuno-imaging （RII） showed that the tumor image had developed 2 h after injection of the 99m↑TC-5-FU-Ab-NPs, and with time it was clearer at the 6th hour following the injection. The %lD/g of the tumor tissue at both 2 h and 6 h after the injection was significantly higher compared to the control group. The tumor %lD/g and the tumor to blood activity ratio （TB） of the experimental group at 6 h following the injection increased compared to that at 2 h, and at the same time, 5-FU concentration in the tumor of the experimental group continuously increased over time, and showed a significant difference compared to the 5-FU concentration in the tumor of the control group. CONCLUSION The 99m↑TC-5-FU-Ab-NPs prepared in this study are adequate to meet the demands of the RII, and the immune targeting ability of the anti-VEGF MCAB is reliable. Six hours after injection, the 99m↑TC-5-FU-Ab-NPs showed a relatively high specific concentration shadow in the human gastric carcinoma xenografts.
