Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting

Minsker, Kevin; Rustandi, Richard R.; Ha, Sha; Loughney, John W.

doi:10.1016/j.vaccine.2020.08.001

Cited by 11 publications

(2 citation statements)

References 31 publications

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“…A relatively new technology called Simple Western (SW), developed by ProteinSimple, eliminates the recovery issue by performing all traditional western blot steps in a capillary . The SW technology has been previously described and has been applied for various vaccine developments. − Major advantages of SW technology over previous methodologies are it is fast, automated, and quantitative. In short, samples are loaded directly into a capillary via hydrodynamic injection and all subsequent steps (protein separation, blocking, antibody probing, substrate detection) are performed in the capillary effectively eliminating the recovery issue from a traditional western blot.…”

Section: Introductionmentioning

confidence: 99%

Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots

Gillespie¹,

Wang²,

Hofmann³

et al. 2023

ACS Omega

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral agent that is responsible for the coronavirus disease-2019 (COVID-19) pandemic. One of the live virus vaccine candidates Merck and Co., Inc. was developing to help combat the pandemic was V590. V590 was a live-attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV) in which the envelope VSV glycoprotein (G protein) gene was replaced with the gene for the SARS-CoV-2 spike protein (S protein), the protein responsible for viral binding and fusion to the cell membrane. To assist with product and process development, a quantitative Simple Western (SW) assay was successfully developed and phase-appropriately qualified to quantitate the concentration of S protein expressed in V590 samples. A strong correlation was established between potency and S-protein concentration, which suggested that the S-protein SW assay could be used as a proxy for virus productivity optimization with faster data turnaround time (3 h vs 3 days). In addition, unlike potency, the SW assay was able to provide a qualitative profile assessment of the forms of S protein (S protein, S1 subunit, and S multimer) to ensure appropriate levels of S protein were maintained throughout process and product development. Finally, V590 stressed stability studies suggested that time and temperature contributed to the instability of S protein demonstrated by cleavage into its subunits, S1 and S2, and aggregation into S multimer. Both of which could potentially have a deleterious effect on the vaccine immunogenicity.

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Section: Introductionmentioning

confidence: 99%

Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots

Gillespie¹,

Wang²,

Hofmann³

et al. 2023

ACS Omega

Self Cite

View full text Add to dashboard Cite

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“…For this purpose, an EBOV combinatorial library constructed from B cells recovered from an immunized donor was run through our microdevice to select high‐affinity phages against EBOV‐GP, which comprises two subunits GP1 and GP2. GP1 has three domains, a receptor binding domain, a glycan cap, and a nonstructural mucin‐like domain [22] which was removed for this study. We found that this microdevice concept (easily fabricated and disposable) utilizing alternative lateral ( X ) and vertical ( Y ) electric fields can be used to efficiently separate antigen‐bound from unbound members of such libraries [12].…”

Section: Introductionmentioning

confidence: 99%

Selection of phage‐displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices

et al. 2023

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A method development aimed for high‐throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets, and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target‐specific binding affinities. Here, we present a phage‐selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high‐affinity phage‐displayed antibodies against virus glycoproteins, including human immunodeficiency virus‐1 glycoprotein 120 or the Ebola virus glycoprotein (EBOV‐GP). Phages were differentially and laterally swept depending on their antigen affinity; the high‐affinity phages were recovered at channels proximal to the application site, whereas low‐affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage‐selection is rapid, sensitive, and effective. Therefore, this is an efficient and cost‐effective method that allowed highly controlled assay conditions for isolating and sorting high‐affinity ligands displayed in phages.

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A Novel Electrophoresis Devices Combined with Immunosensor for the Rapid Detection of Carcinoembryonic Antigen

Wu,

Lu,

et al. 2024

12th Asian-Pacific Conference on Medical and Biological Engineering

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Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting

Cited by 11 publications

References 31 publications

Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots

Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots

Selection of phage‐displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices

A Novel Electrophoresis Devices Combined with Immunosensor for the Rapid Detection of Carcinoembryonic Antigen

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