Generation of Monoclonal Antibodies to the AML1-ETO Fusion Protein: Strategies for Overcoming High Homology

Hybridoma ◽  
2011 ◽  
Vol 30 (5) ◽  
pp. 433-443 ◽  
Author(s):  
Stephen G. Simkins ◽  
Steven L. Knapp ◽  
George H. Brough ◽  
Karen L. Lenz ◽  
Lise Barley-Maloney ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
High Homology

Monoclonal antibodies against the fusion protein are protective in necrotizing mumps meningoencephalitis.

1986 ◽  
Vol 58 (1) ◽  
pp. 220-222 ◽  
Author(s):  
A Löve ◽  
R Rydbeck ◽  
G Utter ◽  
C Orvell ◽  
K Kristensson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Protein

scholarly journals Probing the Flavivirus Membrane Fusion Mechanism by Using Monoclonal Antibodies

2007 ◽  
Vol 81 (20) ◽  
pp. 11526-11531 ◽  
Author(s):  
Karin Stiasny ◽  
Samantha Brandler ◽  
Christian Kössl ◽  
Franz X. Heinz
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fusion Inhibition ◽  
Tick Borne Encephalitis ◽  
Target Membrane ◽  
Initial Interaction ◽  
Tick Borne Encephalitis Virus ◽  
Late Stages

ABSTRACT In this study, we investigated in a flavivirus model (tick-borne encephalitis virus) the mechanisms of fusion inhibition by monoclonal antibodies directed to the different domains of the fusion protein (E) and to different sites within each of the domains by using in vitro fusion assays. Our data indicate that, depending on the location of their binding sites, the monoclonal antibodies impaired early or late stages of the fusion process, by blocking the initial interaction with the target membrane or by interfering with the proper formation of the postfusion structure of E, respectively. These data provide new insights into the mechanisms of flavivirus fusion inhibition by antibodies and their possible contribution to virus neutralization.

scholarly journals Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor-Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells

2020 ◽  
Vol 26 (4) ◽  
pp. 393-398
Author(s):  
Ehsan Naghneh ◽  
Es'hagh Pourmaleki ◽  
Azam Rahimpour
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Cho Cells ◽  
Fusion Proteins ◽  
Chinese Hamster ◽  
Vascular Endothelial ◽  
Cell Clones ◽  
Fc Fusion Protein ◽  
Endothelial Growth Factor

Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene

1986 ◽  
Vol 6 (11) ◽  
pp. 3862-3872
Author(s):  
T C James ◽  
S C Elgin
Keyword(s):  
Drosophila Melanogaster ◽  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Cdna Clone ◽  
Nuclear Protein ◽  
Cdna Expression Library ◽  
General Strategy ◽  
Chromosomal Protein ◽  
Western Blots ◽  
Single Chromosome

Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.

scholarly journals Monoclonal antibodies specific for the C-terminus of the laminin B2 subunit. Evidence for glycosylation differences between murine and human laminin

1991 ◽  
Vol 277 (3) ◽  
pp. 593-596
Author(s):  
J C Brown ◽  
J H Spragg ◽  
P W Taylor
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Adult Mouse ◽  
Yolk Sac ◽  
Specific Difference ◽  
C Terminus ◽  
Mouse Tissues ◽  
Species Specific ◽  
Beta Galactosidase

We have raised a panel of monoclonal antibodies against a beta-galactosidase fusion protein (XLB2.1) containing the C-terminal 153 amino acids of the murine laminin B2 subunit. Five of the nine antibodies characterized recognize human placental laminin as well as murine Engelbreth-Holm-Swarm (EHS)-tumour laminin. Only two of the antibodies recognize both rat parietal-yolk-sac laminin and murine EHS-tumour laminin. Two antibodies recognize an epitope on the human laminin B2 subunit which is masked by N-linked oligosaccharide in murine EHS-tumour laminin. These antibodies also fail to bind to laminin from adult-mouse tissues. These results demonstrate a species-specific difference in the glycosylation of the laminin B2 subunit.

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