The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Hyo Jeong Hong - One of the best experts on this subject based on the ideXlab platform.
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characterization of Chimeric Antibody producing cho cells in the course of dihydrofolate reductase mediated gene amplification and their stability in the absence of selective pressure
Biotechnology and Bioengineering, 1998Co-Authors: Hyo Jeong HongAbstract:Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of Chimeric Antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 μM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and Antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific Antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 μM, and thereafter, it gradually increased to 20 μg/106 cells/day at 4 μM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 μM MTX in regard to Antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 μM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 μM MTX could maintain high volumetric Antibody productivity over three months because of improved cell growth rate during long-term culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:73–84, 1998.
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Characterization of a murine-human Chimeric Antibody with specificity for the pre-S2 surface antigen of hepatitis B virus expressed in baculovirus-infected insect cells
Virus Research, 1995Co-Authors: Seok Kwon Kang, Hyo Jeong HongAbstract:Abstract We have produced and characterized a murine-human Chimeric Antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) in baculovirus-infected insect cells. Recombinant baculovirus carrying the cDNA coding for the heavy or light chain of the Chimeric Antibody was constructed and co-infected into insect cells. The Chimeric Antibody (BV-S2) expressed in the cells was purified by an affinity chromatography on Protein A-Sepharose 4B column and characterized by N-terminal amino acid sequencing, affinity determination for pre-S2 peptide, endoglycosidase digestion and Clq binding assay, which were then compared with those of the Chimeric Antibody H69K that has the same amino acid sequence as BV-S2, but produced from transfected murine myeloma cells. The N-linked glycosylation of the BV-S2 Antibody was also analyzed by culturing the baculovirus-infected cells in the presence of tunicamycin. The results showed that the BV-S2 was secreted following correct removal of the leader peptides, contained N-linked carbohydrate at the heavy chain, and had the same binding affinity and Clq binding ability as H69K, suggesting that the BV-S2 Chimeric Antibody is functional and thus may be useful in the prevention of HBV infection.
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Stability of Chimeric Antibody Producing Transfectomas during a Long-Term Culture
Animal Cell Technology: Developments Towards the 21st Century, 1995Co-Authors: Hyo Jeong HongAbstract:To test the feasibility of large-scale production of Chimeric Antibody for the post-exposure prophylaxis of hepatitis B virus (HBV) infection, the stability of H69K-1 transfectoma concerning Antibody production was examined during a long-term, repeated-fed culture without selection pressure using antibiotics. Antibody production gradually decreased for the first 8 weeks and thereafter, was stabilized for the next 4 weeks. The specific Antibody productivity (qMAb) at the end of long-term culture was 4.63 µg/106 cells/day, which is approximately one half of the initial qMAb. This loss of MAb was due to the appearance of a nonproducing population of transfectoma which was monitored during the long-term culture by both limiting dilution method and flow cytometric method. Since stable Antibody production was achieved in a long-term culture after a significant loss of Antibody productivity, selection pressure is necessary for a long-term, continuous culture. Accordingly, a batch culture appears to be more appropriate for a large-scale Chimeric Antibody production by H69K-1 transfectoma.
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Cloning, expression and characterization of a murine-human Chimeric Antibody with specificity for pre-S2 surface antigen of hepatitis B virus
Molecular Immunology, 1993Co-Authors: Sung Sup Park, Namgung Uk, Hyo Jeong HongAbstract:Abstract The cloning, expression and characterization of a murine-human Chimeric Antibody with specificity for the pre-S2 surface antigen (Ag) of hepatitis B virus (HBV) is described. The heavy and light chain variable region (VH and VL) genes encoding the murine monoclonal Antibody (mAb) were isolated and combined with human γ 1 and κ constant region genes, respectively. The expression vectors containing the Chimeric heavy and light chain genes were sequentially electroporated into murine Sp2/0 hybridoma cells and transfectomas secreting Chimeric Antibody were isolated. The Chimeric Antibody was purified and characterized by ELISA, Western analysis and competition immunoassay, demonstrating that the transfectoma functionally express and secrete murine-human Chimeric Antibody which retained the specificity and affinity of the parental murine mAb.
Jianming Gu - One of the best experts on this subject based on the ideXlab platform.
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Construction and expression of mouse-human Chimeric Antibody SZ-51 specific for activated platelet P-selectin.
Thrombosis and Haemostasis, 1997Co-Authors: Jianming Gu, Peixia Li, Xiaomin Zhang, Changgeng RuanAbstract:: A murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human Chimeric reagents. The E. coli expression vector pHEN1-SZ51Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG gamma 1CH1 and C kappa genes. The constructs were introduced into E. coli HB2151 for expression of soluble Chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse Antibody to the appropriate constant regions of human Ig gamma 1 and kappa. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then contransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/l of culture, was selected for the large scale production of Chimeric Antibody. Immunoblotting analysis demonstrated that both of the Chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole Chimeric Antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 Chimeric Antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.
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Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Human Activated Platelets.
Acta Biochimica et Biophysica Sinica, 1996Co-Authors: Jianming Gu, Peixia Li, Chang-gen RuanAbstract:: SZ-51, a murine monoclonal Antibody (McAb) specific for alpha-granule membrane protein (GMP-140) on the surface of the activated human platelets, has shown promise for thrombus imaging and thrombolysis. In order to reduce the immunogenicity of the murine McAb SZ-51 in man and to obtain a high level of Antibody production, we constructed two chimeras (alpha-Lys17-51BVK/Hu, alpha-Lys30-51VH/Hu) by joining the variable regions gene of mouse Antibody to the constant regions gene of human immunoglobulin (Ig)(gamma1,k). Both Chimeric genes were cloned into two selectable expression vectors separately, which were co-transfected into a non-Ig secreting murine myeloma cell line SP2/0 with the Lipofectin reagent. One transfectoma, which showed stable antigen (GMP-140) binding ability and a high level expression of 5 mg/L, was obtained. Immunoblotting analysis demonstrated that the Chimeric Antibody in the supernatant, like the native mouse SZ-51, had the characteristic of binding to GMP-140. In addition, the Chimeric Antibody can bind competitively to activated platelets with (125)I-labeled mouse SZ-51. Therefore, the SZ-51 Chimeric Antibody may be a potential agent for the diagnosis and therapy of thrombotic diseases in future.
Nina V. Tikunova - One of the best experts on this subject based on the ideXlab platform.
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Chimeric Antibody 14D5 Protects Mice against the Far-Eastern, Siberian, and European Tick-borne Encephalitis Virus
Acta Biomedica Scientifica, 2019Co-Authors: A. L. Matveev, Ivan K. Baykov, I. V. Kozlova, Oleg V. Stronin, E. K. Doroshchenko, O. V. Lisak, O. V. Suntsova, Yulia Savinova, Lyudmila A. Emelyanova, Nina V. TikunovaAbstract:Tick-borne encephalitis virus (TBEV), belonging to the Flaviviridae family, is the most significant pathogen transmitted by Ixodes ticks and causing one of the most severe human neuroinfections. In Russia, serum immunoglobulin produced from the donor blood is currently used for post-exposure prophylactic and therapy of tick-borne encephalitis virus. However, it is known that preparations obtained from donated blood have certain disadvantages, and therefore development of novel preparations for post exposure prophylaxis and therapy of tick-borne encephalitis is required. To develop an alternative preparation, which does not include donor blood, a Chimeric Antibody ch14D5 against glycoprotein E of TBEV was constructed.This study was aimed to investigate protective efficacy of the Chimeric Antibody ch14D5 against the Far-Eastern, Siberian, and European subtypes of TBEV in in vivo experiments.A peripheral mouse model of tick-borne encephalitis was used in this study: the Chimeric Antibody ch14D5 was administrated intravenously in mice one day after their intraperitoneal infection with TBEV strains Sofjin, Vasilchenko, and Absettarov. Anti-TBEV serum immunoglobulin was used as a control preparation, which was administered in the same way. Protective efficacy of the Chimeric antibodies 14D5 was assessed using the log-rank test. In the study, the presence or absence of Antibody-dependent enhancement of infection (ADE) was examined when mice, infected with different subtypes of the TBEV, got the Antibody ch14d5.Obtained results demonstrated high efficacy of the ch14D5 Antibody in post-exposure prophylaxis of the disease in mice infected with any of the used TBEV strains, as well as the absence of ADE.It was shown that protective efficacy of Antibody ch14D5 is higher than that of the anti-TBEV serum immunoglobulin, and Antibody ch14D5 could be used for development of a therapeutic preparation for post-exposure prophylaxis.
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ANALYSIS OF DOMAIN SPECIFICITY OF THE PROTECTIVE Chimeric Antibody ch14D5a AGAINST GLYCOPROTEIN E OF TICK-BORNE ENCEPHALITIS VIRUS
Vavilov Journal of Genetics and Breeding, 2018Co-Authors: Ivan K. Baykov, A. L. Matveev, Lyudmila A. Emelyanova, L. M. Sokolova, E. M. Karelina, Igor V. Babkin, Ya. А. Khlusevich, V. F. Podgornyy, Nina V. TikunovaAbstract:A drug for the prevention and therapy of tick-borne encephalitis virus is being developed on the basis of the protective Chimeric Antibody ch14D5a. At the same time, the epitope recognized by this Antibody on the surface of glycoprotein E has not been localized yet. The aim of this work was to identify the domain of glycoprotein E, to which the protective Antibody ch14D5a binds. As a result, four recombinant variants of glycoprotein E were generated using the bacterial expression system: (1) the rE protein containing the domains D1, D2, and D3 of glycoprotein E; (2) the rED1+2 protein containing domains D1 and D2; (3) the rED3_301 protein, which is domain D3 of glycoprotein E, and (4) the rED3_294 protein comprising domain D3 and a hinge region connecting domains D1 and D3. The rED3_294 and rED3_301 proteins were obtained in soluble monomeric form. The rE and rED1+2 proteins were extracted from the inclusion bodies of Escherichia coli. Using Western blot analysis and surface plasmon resonance analysis, it was demonstrated that the protective Chimeric Antibody ch14D5a and its Fab fragment bound specifically to domain D3 of glycoprotein E. Since the antibodies recognizing epitopes on the surface of domain D3 do not tend to cause Antibody-dependent enhancement of the infection as compared to antibodies directed to domains D1 and D2, the data obtained confirm the promise of using the Antibody ch14D5a in the development of a therapeutic preparation against the tick-borne encephalitis virus.
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A protective Chimeric Antibody to tick-borne encephalitis virus
Vaccine, 2014Co-Authors: Ivan K. Baykov, Matveev Le, Alexander B. Ryzhikov, A. L. Matveev, Oleg V. Stronin, Marat F. Kasakin, Vladimir A. Richter, Nina V. TikunovaAbstract:The efficiency of several mouse monoclonal antibodies (mAbs) specific to the tick-borne encephalitis virus (TBEV) glycoprotein E in post-exposure prophylaxis was assessed, and mAb14D5 was shown to be the most active of all those studied. It was proven that the hybridoma cell line 14D5 produced one immunoglobulin H chain and two L chains. They were used to construct Chimeric antibodies ch14D5a and ch14D5b, the affinity constants of which were 2.6 × 1010 M−1 and 1.0 × 107 M−1, respectively, according to the SPR-based ProteOn biosensor assay. The neutralization index (IC50) of ch14D5a was 0.04 μg/ml in the focus reduction neutralization test. In in vivo experiments, ch14D5a at a dose of 10 μg/mouse resulted in a 100% survival of the mice infected with 240 LD50 of TBEV. This Chimeric Antibody is promising for further development of prevention and therapeutic drugs against TBEV.
Changgeng Ruan - One of the best experts on this subject based on the ideXlab platform.
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Construction and expression of mouse-human Chimeric Antibody SZ-51 specific for activated platelet P-selectin.
Thrombosis and Haemostasis, 1997Co-Authors: Jianming Gu, Peixia Li, Xiaomin Zhang, Changgeng RuanAbstract:: A murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human Chimeric reagents. The E. coli expression vector pHEN1-SZ51Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG gamma 1CH1 and C kappa genes. The constructs were introduced into E. coli HB2151 for expression of soluble Chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse Antibody to the appropriate constant regions of human Ig gamma 1 and kappa. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then contransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/l of culture, was selected for the large scale production of Chimeric Antibody. Immunoblotting analysis demonstrated that both of the Chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole Chimeric Antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 Chimeric Antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.
Peixia Li - One of the best experts on this subject based on the ideXlab platform.
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Construction and expression of mouse-human Chimeric Antibody SZ-51 specific for activated platelet P-selectin.
Thrombosis and Haemostasis, 1997Co-Authors: Jianming Gu, Peixia Li, Xiaomin Zhang, Changgeng RuanAbstract:: A murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human Chimeric reagents. The E. coli expression vector pHEN1-SZ51Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG gamma 1CH1 and C kappa genes. The constructs were introduced into E. coli HB2151 for expression of soluble Chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse Antibody to the appropriate constant regions of human Ig gamma 1 and kappa. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then contransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/l of culture, was selected for the large scale production of Chimeric Antibody. Immunoblotting analysis demonstrated that both of the Chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole Chimeric Antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 Chimeric Antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.
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Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Human Activated Platelets.
Acta Biochimica et Biophysica Sinica, 1996Co-Authors: Jianming Gu, Peixia Li, Chang-gen RuanAbstract:: SZ-51, a murine monoclonal Antibody (McAb) specific for alpha-granule membrane protein (GMP-140) on the surface of the activated human platelets, has shown promise for thrombus imaging and thrombolysis. In order to reduce the immunogenicity of the murine McAb SZ-51 in man and to obtain a high level of Antibody production, we constructed two chimeras (alpha-Lys17-51BVK/Hu, alpha-Lys30-51VH/Hu) by joining the variable regions gene of mouse Antibody to the constant regions gene of human immunoglobulin (Ig)(gamma1,k). Both Chimeric genes were cloned into two selectable expression vectors separately, which were co-transfected into a non-Ig secreting murine myeloma cell line SP2/0 with the Lipofectin reagent. One transfectoma, which showed stable antigen (GMP-140) binding ability and a high level expression of 5 mg/L, was obtained. Immunoblotting analysis demonstrated that the Chimeric Antibody in the supernatant, like the native mouse SZ-51, had the characteristic of binding to GMP-140. In addition, the Chimeric Antibody can bind competitively to activated platelets with (125)I-labeled mouse SZ-51. Therefore, the SZ-51 Chimeric Antibody may be a potential agent for the diagnosis and therapy of thrombotic diseases in future.