scholarly journals Abrogation of Viral Interleukin-6 (vIL-6)-Induced Signaling by Intracellular Retention and Neutralization of vIL-6 with an Anti-vIL-6 Single-Chain Antibody Selected by Phage Display

2006 ◽  
Vol 80 (17) ◽  
pp. 8510-8520 ◽  
Author(s):  
Marina Kovaleva ◽  
Ingo Bussmeyer ◽  
Björn Rabe ◽  
Joachim Grötzinger ◽  
Enge Sudarman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Phage Display ◽  
Cell Line ◽  
Primary Effusion Lymphoma ◽  
Recombinant Antibody ◽  
Cytokine Signaling ◽  
Hepatoma Cell Line ◽  
Single Chain ◽  
Synthetic Antibody ◽  
Stat3 Phosphorylation

ABSTRACT Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposi's sarcoma as well as primary effusion lymphoma and multicentric Castleman's disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.

scholarly journals Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 194-204 ◽  
Author(s):  
DS Fair ◽  
BR Bahnak
Keyword(s):  
Cell Line ◽  
Antithrombin Iii ◽  
Biologically Active ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Factor X ◽  
Single Chain ◽  
Hep G2 ◽  
Cell Extracts ◽  
Coagulation Proteins

The human hepatoma cell line, Hep G2, was analyzed for the ability to synthesize and secrete several coagulation proteins. Using specific radioimmunoassays, factor X, prothrombin, and antithrombin III were present in 8-day culture supernatants at 62, 405, and 1,220 ng/mL, respectively. Factor IX was not detected, either in supernatants or in cell extracts. Intrinsically labeled factor X was secreted as a single- chain polypeptide of 66,000 daltons, as measured by sodium dodecylsulfate-polyacrylamide gels under nonreduced and reduced conditions. Immunoblots of Hep G2 supernatants and normal human plasma also indicate the presence of single-chain factor X. These findings support the hypothesis of a postsecretion proteolytic cleavage of factor X into the two-chain form. Prothrombin and antithrombin represented their plasma protein counterparts structurally, with molecular weights of 73,000 and 61,000, respectively. Secreted factor X, prothrombin, and antithrombin III were biologically active, as determined in coagulation or chromogenic assays, and all three activities were neutralized by monospecific antibodies. Vitamin K increased the quantity of prothrombin secreted by twofold, without affecting the rate of secretion over a five-day culture period, and had an apparent transient inhibitory effect on secretion of antithrombin III. Warfarin caused a three to fourfold decrease in the rate and quantity of secreted prothrombin, but did not affect intracellular concentrations. The intracellular and extracellular concentrations and rate of secretion of antithrombin III were not modulated by warfarin. These data suggest that the Hep G2 cell line may provide a useful model for assessing the regulation of biosynthesis and secretion of human coagulation proteins.

Platelet integrin α2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

2005 ◽  
Vol 94 (12) ◽  
pp. 1318-1326 ◽  
Author(s):  
Darren L. Hughes ◽  
Prachi Stafford ◽  
Samir W. Hamaia ◽  
Anne Schoolmeester ◽  
Hans Deckmyn ◽  
...  
Keyword(s):  
Phage Display ◽  
Recombinant Antibody ◽  
Dna Vaccination ◽  
Gene Repertoire ◽  
Single Chain ◽  
Specific Antibodies ◽  
Domain Specific ◽  
Variable Gene ◽  
V Gene ◽  
Α2β1 Integrin

SummaryAntibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs).This system was validated using the I-domain of α2 integrin as a model. The α2β1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoproteinVI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunized with a DNA vaccine in which the α2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT).Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four α2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet α2β1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splicevariant specific antibodies that recognise native protein.

scholarly journals Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

2002 ◽  
Vol 68 (11) ◽  
pp. 5288-5295 ◽  
Author(s):  
Jacqui McElhiney ◽  
Mathew Drever ◽  
Linda A. Lawton ◽  
Andy J. Porter
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Phage Display ◽  
High Performance ◽  
Recombinant Antibody ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Cyanobacterial Toxin

ABSTRACT A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter−1) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.

scholarly journals Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 194-204 ◽  
Author(s):  
DS Fair ◽  
BR Bahnak
Keyword(s):  
Cell Line ◽  
Antithrombin Iii ◽  
Biologically Active ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Factor X ◽  
Single Chain ◽  
Hep G2 ◽  
Cell Extracts ◽  
Coagulation Proteins

Abstract The human hepatoma cell line, Hep G2, was analyzed for the ability to synthesize and secrete several coagulation proteins. Using specific radioimmunoassays, factor X, prothrombin, and antithrombin III were present in 8-day culture supernatants at 62, 405, and 1,220 ng/mL, respectively. Factor IX was not detected, either in supernatants or in cell extracts. Intrinsically labeled factor X was secreted as a single- chain polypeptide of 66,000 daltons, as measured by sodium dodecylsulfate-polyacrylamide gels under nonreduced and reduced conditions. Immunoblots of Hep G2 supernatants and normal human plasma also indicate the presence of single-chain factor X. These findings support the hypothesis of a postsecretion proteolytic cleavage of factor X into the two-chain form. Prothrombin and antithrombin represented their plasma protein counterparts structurally, with molecular weights of 73,000 and 61,000, respectively. Secreted factor X, prothrombin, and antithrombin III were biologically active, as determined in coagulation or chromogenic assays, and all three activities were neutralized by monospecific antibodies. Vitamin K increased the quantity of prothrombin secreted by twofold, without affecting the rate of secretion over a five-day culture period, and had an apparent transient inhibitory effect on secretion of antithrombin III. Warfarin caused a three to fourfold decrease in the rate and quantity of secreted prothrombin, but did not affect intracellular concentrations. The intracellular and extracellular concentrations and rate of secretion of antithrombin III were not modulated by warfarin. These data suggest that the Hep G2 cell line may provide a useful model for assessing the regulation of biosynthesis and secretion of human coagulation proteins.

scholarly journals SWCNH (Single walled carbon nanohorn) supervises ER (Endoplasmic reticulum) stress through triggering autophagy process of hepatocytes, especially in hepatoma cell line HepG2

2021 ◽  
Author(s):  
Jinling Dong ◽  
Ying Zhang ◽  
Zhihong Xie ◽  
Jie He ◽  
Tiantian Wu
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Western Blot ◽  
Er Stress ◽  
Cell Line ◽  
Cell Apoptosis ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Catabolic Pathway ◽  
Hepatoma Cell Line Hepg2

Abstract Backgrounds: The cellular homeostasis is major maintained by the catabolic pathway of autophagy. Our previous work indicated that SWCNH were associated with endoplasmic reticulum (ER) stress mediated by calcium flow and autophagic response. But, its mechanism was unclear. Methods: The regulation of SWCNH on the calcium flow then autophagy of liver cells were investigated through inducing ER stress with tunicamycin and SWCNH. The calcuim flow was determined using Fluo-3, then autophagy was examined with immunofluorescence or western blot for LC3, Beclin-1, ATG-5, and p62. Moreover, the apopototic protein of Bax and Bcl-2 was detected, too. Results: Tunicamycin-induced ER stress in hepatocytes was related to calcium flow, especially for hepatoma cell line HepG2. Moreover, SWCNH participated in the regulation of endoplasmic reticulum stress-related calcium flow. Besides, SWCNH induced hepatocyte autophagy and inhibited cell apoptosis, then mediated the process of hepatocyte autophagy. Conclusions: Tunicamycin-induced ER stress in hepatocytes was related to calcium flow. Moreover, SWCNH induced hepatocyte autophagy, inhibited cell apoptosis, and participated in the autophagy regulation of hepatocyte, especially for hepatoma cell line.

scholarly journals Recombinant Single-Chain Variable Fragment Antibodies Directed against Clostridium difficile Toxin B Produced by Use of an Optimized Phage Display System

2003 ◽  
Vol 10 (4) ◽  
pp. 587-595 ◽  
Author(s):  
Xiao K. Deng ◽  
Lance A. Nesbit ◽  
K. John Morrow
Keyword(s):  
Monoclonal Antibody ◽  
Clostridium Difficile ◽  
Phage Display ◽  
Recombinant Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Display System ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Toxin B ◽  
Single Chain Antibodies

ABSTRACT Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10−8 M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.

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