scholarly journals Adaption of a Conventional ELISA to a 96-well ELISA-Array for Measuring the Antibody Responses to Influenza virus proteins, viruses and vaccines

2019 ◽  
Author(s):  
Eric Waltari ◽  
Esteban Carabajal ◽  
Mrinmoy Sanyal ◽  
Natalia Friedland ◽  
Krista M. McCutcheon
Keyword(s):  
Viral Fusion ◽  
Flu Vaccination ◽  
Conventional Elisa ◽  
High Protein Binding ◽  
Dependent Signal ◽  
Viral Fusion Proteins ◽  
Assay Precision ◽  
Antibody Levels ◽  
Dose Dependent ◽  
Array Format

AbstractWe describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins, vaccine and virus on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance of the ELISA-Array was demonstrated by serially diluting n=8 human post-flu vaccination plasma samples starting at a 1/1000 dilution and measuring binding to the array of Influenza antigens. Plasma polyclonal antibody levels were detected using a cocktail of biotinylated anti-human kappa and lambda light chain antibodies, followed by a Streptavidin-horseradish peroxidase conjugate and the dose-dependent signal was developed with a precipitable TMB substrate. Intra- and inter-assay precision of absorbance units among the eight donor samples showed mean CVs of 4.8% and 10.8%, respectively. The plasma could be differentiated by donor and antigen with titer sensitivities ranging from 1 × 103 to 4 × 106, IC50 values from 1 × 104 to 9 × 106, and monoclonal antibody sensitivities in the ng/mL range. Equivalent sensitivities of ELISA versus ELISA-Array, compared using plasma and an H1N1 HA trimer, were achieved on the ELISA-Array printed at 0.25ng per 200um spot and 1000ng per ELISA 96-well. Vacuum-sealed array plates were shown to be stable when stored for at least 2 days at ambient temperature and up to 1 month at 4-8°C. By the use of any set of printed antigens and analyte matrices the methods of this multiplexed ELISA-Array format can be broadly applied in translational research.

Structural comparison of three viral “fusion” proteins

1991 ◽  
Vol 19 (3) ◽  
pp. 312S-312S
Author(s):  
BRUCE H NICHOLSON ◽  
MAHMOUD NAASE
Keyword(s):  
Fusion Proteins ◽  
Viral Fusion ◽  
Structural Comparison ◽  
Viral Fusion Proteins

scholarly journals New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 413 ◽  
Author(s):  
Mark A. Benhaim ◽  
Kelly K. Lee
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Viral Entry ◽  
Fusion Proteins ◽  
Structural Changes ◽  
Fusion Reaction ◽  
Type I ◽  
Viral Fusion ◽  
Technological Advances ◽  
Viral Fusion Proteins

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.

Peptide models for the membrane destabilizing actions of viral fusion proteins

Biopolymers ◽  
1992 ◽  
Vol 32 (4) ◽  
pp. 309-314 ◽  
Author(s):  
Richard M. Epand ◽  
James J. Cheetham ◽  
Raquel F. Epand ◽  
Philip L. Yeagle ◽  
Christopher D. Richardson ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Viral Fusion ◽  
Peptide Models ◽  
Viral Fusion Proteins

A chemically defined, toleragen-based approach for targeting anti-β2-glycoprotein I antibodies

Lupus ◽  
1998 ◽  
Vol 7 (2_suppl) ◽  
pp. 166-229 ◽  
Author(s):  
GM Iverson ◽  
DS Jones ◽  
D Marquis ◽  
MD Linnik ◽  
EJ Victoria
Keyword(s):  
Antiphospholipid Syndrome ◽  
Antiphospholipid Antibodies ◽  
Keyhole Limpet Hemocyanin ◽  
Cell Tolerance ◽  
Prothrombotic State ◽  
B Cell Tolerance ◽  
Glycoprotein I ◽  
Antibody Levels ◽  
Dose Dependent

Antiphospholipid syndrome is characterized by a prothrombotic state and the presence of β2-glycoprotein I (β2-GPI)-dependent antiphospholipid antibodies. The feasibility of a B cell tolerance-based approach for specific reduction of anti-β2-GPI antibodies was investigated. Anti-β2GPI antibodies isolated from a patient with antiphospholipid syndrome were used to screen peptide libraries expressed in phage, resulting in the identification of a phage that specifically bound anti-β2-GPl antibodies. The phage-displayed peptide was identified and chemically optimized to generate a synthetic 14-mer peptide with an internal thioether linkage (LJP 685) that retained the binding profile of the original phage. LJP 685 was conjugated to a defined, non-immunogenic organic platform to generate a tetravalent presentation of LJP 685 for use as a toleragen. Tetravalent LJP 685 induced a dose-dependent reduction in antibody levels in mice previously immunized and boosted with LJP 685 coupled to the carrier keyhole limpet hemocyanin. These experiments support the technical feasibility of a tolerance-based approach for reducing anti-β2GPI antibodies in vivo.

scholarly journals Characterization of EBV gB indicates properties of both class I and class II viral fusion proteins

Virology ◽  
2007 ◽  
Vol 368 (1) ◽  
pp. 102-113 ◽  
Author(s):  
Marija Backovic ◽  
George P. Leser ◽  
Robert A. Lamb ◽  
Richard Longnecker ◽  
Theodore S. Jardetzky
Keyword(s):  
Fusion Proteins ◽  
Class Ii ◽  
Class I ◽  
Viral Fusion ◽  
Viral Fusion Proteins

scholarly journals Thiol/Disulfide Exchange Is Required for Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein

2006 ◽  
Vol 81 (5) ◽  
pp. 2328-2339 ◽  
Author(s):  
Surbhi Jain ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Cell Surface ◽  
Newcastle Disease Virus ◽  
Newcastle Disease ◽  
Fusion Proteins ◽  
Protein Disulfide Isomerase ◽  
Viral Fusion ◽  
Disulfide Isomerase ◽  
F Protein ◽  
Viral Fusion Proteins ◽  
Protein Disulfide

ABSTRACT Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity—5′5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody—inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.

scholarly journals Pharmacophore-based peptide biologics neutralize SARS-CoV-2 S1 and deter S1-ACE2 interaction in vitro

2020 ◽  
Author(s):  
Masaud Shah ◽  
Sung Ung Moon ◽  
Hyun Goo Woo
Keyword(s):  
Cell Attachment ◽  
Immune Surveillance ◽  
Cell Entry ◽  
Viral Fusion ◽  
Receptor Binding Domain ◽  
Spontaneous Movement ◽  
Viral Receptor ◽  
Dose Dependent ◽  
Host Immune Surveillance

AbstractEffective therapeutics and stable vaccine are the urgent need of the day to combat COVID-19 pandemic. SARS-CoV-2 spike protein has a pivotal role in cell-entry and host immune response, thus regarded as potential drug- and vaccine-target. As the virus utilizes the S1 domain of spike to initiate cell-attachment and S2 domain for membrane fusion, several attempts have been made to design viral-receptor and viral-fusion blockers. Here, by deploying interactive structure-based design and pharmacophore-based approaches, we designed short and stable peptide-biologics i.e. CoV-spike-neutralizing peptides (CSNPs) including CSNP1, CSNP2, CSNP3, CSNP4. We could demonstrate in cell culture experiments that CSNP2 binds to S1 at submicromolar concentration and abrogates the S1-hACE2 interaction. CSNP3, a modified and downsized form of CSNP2, could neither interfere with the S1-hACE2 interaction nor bind to S1. CSNP4 exhibited dose-dependent binding to both S1 and hACE2 and abolished the S1-hACE2 interaction in vitro. CSNP4 possibly enhance the mAb-based S1 neutralization by limiting the spontaneous movement of spike receptor-binding domain (RBD), whereas CSNP2 allowed RBD-mAb binding without any steric hindrance. Taken together, we suggest that CSNP2 and CSNP4 are potent and stable candidate peptides that can neutralize the SARS-CoV-2 spike and possibly pose the virus to host immune surveillance.

scholarly journals Viral Membrane Fusion and the Transmembrane Domain

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 693 ◽  
Author(s):  
Chelsea T. Barrett ◽  
Rebecca Ellis Dutch
Keyword(s):  
Membrane Fusion ◽  
Host Cell ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Viral Fusion ◽  
Transmembrane Region ◽  
Host Cell Membrane ◽  
The Past ◽  
Viral Fusion Proteins

Initiation of host cell infection by an enveloped virus requires a viral-to-host cell membrane fusion event. This event is mediated by at least one viral transmembrane glycoprotein, termed the fusion protein, which is a key therapeutic target. Viral fusion proteins have been studied for decades, and numerous critical insights into their function have been elucidated. However, the transmembrane region remains one of the most poorly understood facets of these proteins. In the past ten years, the field has made significant advances in understanding the role of the membrane-spanning region of viral fusion proteins. We summarize developments made in the past decade that have contributed to the understanding of the transmembrane region of viral fusion proteins, highlighting not only their critical role in the membrane fusion process, but further demonstrating their involvement in several aspects of the viral lifecycle.

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