Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for develo...Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.展开更多
AIM： To investigate the clinical significance and presence of mutations in the surface （S） and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs. METHODS： Twenty-three patients...AIM： To investigate the clinical significance and presence of mutations in the surface （S） and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs. METHODS： Twenty-three patients with chronic hepatitis B were studied. Of the 23 patients, i i were both positive for hepatitis B virus （HBV） surface antigen （HBsAg） and antibody to HBV surface antigen （anti-HBs）, 12 were negative for anti-HBs while positive for HBsAg. DNA was extracted from 200 μL serum of the patients. Nucleotide of the surface and overlapping polymerase gene from HBV-infected patients was amplified by PCR, and the PCR products were sequenced. RESULTS： Forty-one mutations were found within the surface gene protein of HBV in 15 patients （10 with coexisting HBsAg and anti-HBs）. Six （14.6%） out of 41 mutations were located at ＂α＂ determinant region in 5 patients （4 positive for HBsAg and anti-HBs）. Eleven mutations （26.8%） occurred in the downstream or upstream of ＂α＂ determinant region. Lamivudine （LMV）- selected mutations were found in three patients who developed anti-HBs, which occurred in amino acid positions （196, 198, 199） of the surface protein and in YMDD motif （M204I/V） of the polymerase protein simultaneously. Presence of these mutations did not relate to changes in ALT and HBV DNA levels.CONCLUSION： Besides mutations in the ＂α＂ determinant region, mutations at downstream or upstream of the ＂α＂ determinant region may contribute to the development of anti-HBs. These mutations do not block the replicating competency of HBV in the presence of high titer of anti-HBs.展开更多
Wistar rats were inoculated with purified YWK-I antibody. The anti-idiotypie antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs. Specifi...Wistar rats were inoculated with purified YWK-I antibody. The anti-idiotypie antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs. Specificity of anti-Id antibody was established by ELISA. The 84 kD protein inhibited the binding of anti-Id to YWK-I mAb, but failed to repress antibody against normal mouse Ig binding to YWK-I mAb. In competitive inhibition assay, 84 kD protein had shown the ability to compete with anti-Id binding to YWK-I mAb in a dose-dependent manner. Crude sperm extract showed a lower competitive ability. No effeet was found with the irrelevant 36 kD sperm protein. The antisera from the Ba1b/e mice immunized with AId contained Ab3 that reacted with 84 kD sperm protein. The binding of anti-Id to YWK-I mAb was inhibited by Ab3 in a dose-dependent fashion and Ab3 was shown to be able to induce human sparm agglutination. These results indicate that anti-Id which may mimio an epitope of the 84 kD protein could be exploited as an antigen to raise antibodies against sperm protein.展开更多
Objective. This study is to investigate the functional mimicry by using antiidiotypic antibodies of enzymes. Methods.Monoclonal antiidiotypic antibodies against antiHEL(hen eggwhite lysozyme, HEL) antibodies were obta...Objective. This study is to investigate the functional mimicry by using antiidiotypic antibodies of enzymes. Methods.Monoclonal antiidiotypic antibodies against antiHEL(hen eggwhite lysozyme, HEL) antibodies were obtained by fusion of Sp2/0 myeloma cells with spleen cells of syngeneic mice immunized with monoclonal antiHEL antibodies against HELs different antigenic epitopes. Then bacteriolysis of the antiidiotypic antibodies were observed. Results.Eight hybridomas strains secreting antiidiotypic antibodies were selected and characterized. It was shown that two of eight antiidiotypic antibodies secreted by two hybridomas(1A 10 C 9 and 2A 11 C 1B 3) could mimic HEL catalytic activity to lyse Micrococcus lysodeikticus and that the catalytic effect of mixed antiidiotypic antibodies of 1A 10 C 9 and 2A 11 C 1B 3 was stronger than that of one of them, but less than HEL. Conclusion. The results demonstrated that the antiidiotypic antibodies that could mimic enzyme activity existed in the idiotype network during antienzymatic immune response.展开更多
The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is nuclear. We describe here the structural models for the complex of a cata...The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is nuclear. We describe here the structural models for the complex of a catalytic sntibody Fv fragment with a phosphonate transition -state analogue, constructed by using gene cloning, sequencing and molecular modeling, mainly based on a known X-ray structure of a catalytic atibody. Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism: In L91 and Tyr H97 play important roles to stabilize the β-naphthyl group of hapten through r-stack; His H35 donates a pair of free electrons at the atom NEZ to an active water and let it to be a partial hydroxide, which attacks the carbon atom of the carbonyl group of the substrate. Both His H35 and Arg L96 can form hydrogen bonds and stabilize the Anoinc tetrahedral intermediate formed during turnover. This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.展开更多
基金This work was supported by the National Natural Sciences Founda- tion of China(NSFC, No. 39800057, No. 30200338) the National "863" High-tech Project Foundation (No. 102-10-01 -06) +1 种基金National Distinguished Youth Program of NSFC(No. 39525020) This wor
文摘Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.
基金Supported by the National Natural Science Foundation of China,No.30271182
文摘AIM： To investigate the clinical significance and presence of mutations in the surface （S） and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs. METHODS： Twenty-three patients with chronic hepatitis B were studied. Of the 23 patients, i i were both positive for hepatitis B virus （HBV） surface antigen （HBsAg） and antibody to HBV surface antigen （anti-HBs）, 12 were negative for anti-HBs while positive for HBsAg. DNA was extracted from 200 μL serum of the patients. Nucleotide of the surface and overlapping polymerase gene from HBV-infected patients was amplified by PCR, and the PCR products were sequenced. RESULTS： Forty-one mutations were found within the surface gene protein of HBV in 15 patients （10 with coexisting HBsAg and anti-HBs）. Six （14.6%） out of 41 mutations were located at ＂α＂ determinant region in 5 patients （4 positive for HBsAg and anti-HBs）. Eleven mutations （26.8%） occurred in the downstream or upstream of ＂α＂ determinant region. Lamivudine （LMV）- selected mutations were found in three patients who developed anti-HBs, which occurred in amino acid positions （196, 198, 199） of the surface protein and in YMDD motif （M204I/V） of the polymerase protein simultaneously. Presence of these mutations did not relate to changes in ALT and HBV DNA levels.CONCLUSION： Besides mutations in the ＂α＂ determinant region, mutations at downstream or upstream of the ＂α＂ determinant region may contribute to the development of anti-HBs. These mutations do not block the replicating competency of HBV in the presence of high titer of anti-HBs.
文摘Wistar rats were inoculated with purified YWK-I antibody. The anti-idiotypie antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs. Specificity of anti-Id antibody was established by ELISA. The 84 kD protein inhibited the binding of anti-Id to YWK-I mAb, but failed to repress antibody against normal mouse Ig binding to YWK-I mAb. In competitive inhibition assay, 84 kD protein had shown the ability to compete with anti-Id binding to YWK-I mAb in a dose-dependent manner. Crude sperm extract showed a lower competitive ability. No effeet was found with the irrelevant 36 kD sperm protein. The antisera from the Ba1b/e mice immunized with AId contained Ab3 that reacted with 84 kD sperm protein. The binding of anti-Id to YWK-I mAb was inhibited by Ab3 in a dose-dependent fashion and Ab3 was shown to be able to induce human sparm agglutination. These results indicate that anti-Id which may mimio an epitope of the 84 kD protein could be exploited as an antigen to raise antibodies against sperm protein.
文摘Objective. This study is to investigate the functional mimicry by using antiidiotypic antibodies of enzymes. Methods.Monoclonal antiidiotypic antibodies against antiHEL(hen eggwhite lysozyme, HEL) antibodies were obtained by fusion of Sp2/0 myeloma cells with spleen cells of syngeneic mice immunized with monoclonal antiHEL antibodies against HELs different antigenic epitopes. Then bacteriolysis of the antiidiotypic antibodies were observed. Results.Eight hybridomas strains secreting antiidiotypic antibodies were selected and characterized. It was shown that two of eight antiidiotypic antibodies secreted by two hybridomas(1A 10 C 9 and 2A 11 C 1B 3) could mimic HEL catalytic activity to lyse Micrococcus lysodeikticus and that the catalytic effect of mixed antiidiotypic antibodies of 1A 10 C 9 and 2A 11 C 1B 3 was stronger than that of one of them, but less than HEL. Conclusion. The results demonstrated that the antiidiotypic antibodies that could mimic enzyme activity existed in the idiotype network during antienzymatic immune response.
文摘The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is nuclear. We describe here the structural models for the complex of a catalytic sntibody Fv fragment with a phosphonate transition -state analogue, constructed by using gene cloning, sequencing and molecular modeling, mainly based on a known X-ray structure of a catalytic atibody. Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism: In L91 and Tyr H97 play important roles to stabilize the β-naphthyl group of hapten through r-stack; His H35 donates a pair of free electrons at the atom NEZ to an active water and let it to be a partial hydroxide, which attacks the carbon atom of the carbonyl group of the substrate. Both His H35 and Arg L96 can form hydrogen bonds and stabilize the Anoinc tetrahedral intermediate formed during turnover. This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.
