Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

Rodríguez-Martínez, Luis Mario; Márquez-Ipiña, Alan Roberto; López-Pacheco, Felipe; Pérez-Chavarría, Roberto; González-Vázquez, Juan Carlos; Gonzalez‐González, Everardo; Santiago, Grissel Trujillo‐de; León, César Alejandro Ponce-Ponce de; Zhang, Yu Shrike; Dokmeci, Mehmet R.; Khademhosseini, Ali; Álvarez, Mario Moisés

doi:10.1371/journal.pone.0135859

Cited by 15 publications

(13 citation statements)

References 40 publications

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“…Binding analysis of solubilized and refolded scFv and VHH antibodies were performed by direct ELISA using 96 well fat bottom polystyrene plates. The commercially available recombinant version of the capsid glycoprotein (GP) of Ebola GP [29] and human serum albumin (HSA) were coated with equimolar amounts at 4 • C overnight. The unbound protein samples were washed with PBST buffer (PBS with 0.05% Tween 20), and the untenanted spaces were blocked with 3% (w/v) non-fat skim milk dissolved in PBS.…”

Section: Elisa Analysis Of Antibody Binding To Target Antigensmentioning

confidence: 99%

Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts

2020

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Antibodies have been used for basic research, clinical diagnostics, and therapeutic applications. Escherichia coli is one of the organisms of choice for the production of recombinant antibodies. Variable antibody genes have canonical and non-canonical disulfide bonds that are formed by the oxidation of a pair of cysteines. However, the high-level expression of an antibody is an inherent problem to the process of disulfide bond formation, ultimately leading to mispairing of cysteines which can cause misfolding and aggregation as inclusion bodies (IBs). This study demonstrated that fragment antibodies are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies when expressed using engineered bacterial host strains with optimal culture conditions. It was observed that moderate-solubilization and an in vitro matrix that associated refolding strategies with redox pairing more correctly folded, structured, and yielded functionally active antibody fragments than the one achieved by a direct dilution method in the absence of a redox pair. However, natural antibodies have canonical and non-canonical disulfide bonds that need a more elaborate refolding process in the presence of optimal concentrations of chaotropic denaturants and redox agents to obtain correctly folded disulfide bonds and high yield antibodies that retain biological activity.

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Section: Elisa Analysis Of Antibody Binding To Target Antigensmentioning

confidence: 99%

Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts

2020

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“…7). Rodríguez-Martínez et al [23] reported that the scFv-13C6 mAb exhibited the highest EBOV GP-binding activity, followed by scFv-13F6 and Fab-KZ52 in ELISA experiments.…”

Section: Discussionmentioning

confidence: 99%

Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display

Kunamneni

Clarke²,

Ye³

et al. 2018

Preprint

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Filoviruses, which include ebolaviruses and marburgvirus, can cause outbreaks of highly lethal hemorrhagic fever. This disease causes significant morbidity and mortality in humans and nonhuman primates, with human fatality rates reaching 90% during some outbreaks. Currently, there are a lack of licensed vaccines or antivirals for these viruses. Since early symptoms of filovirus infection mimic more common diseases, there is a strong unmet public health and biodefense need for broad-spectrum filovirus rapid diagnostics. We have generated a panel of mouse single-chain Fv-antibodies (scFvs) to filovirus glycoproteins (GPs) using cell-free ribosome display and determined their cross-reactivity profiles to all known filovirus species. Two scFvs (4-2 and 22-1)were able to detect all known Ebolavirus and Marburgvirus species. This is the first report on ribosome display scFvs that can detect a broad set of filovirus GPs, which demonstrates their potential use in the development of a new generation of rapid diagnostic immunoassays. Author summaryFiloviruses, such as Ebola virus, cause severe and often fatal hemorrhagic fever in humans. The current Ebola virus outbreak is a health crisis with global repercussions and underscores the need for rapid and inexpensive diagnostics to detect filovirus infection. For these reasons, we used a ribosomal display method to rapidly generate single-chain antibodies (scFv's) for use in diagnostics against all six pathogenic filoviruses from mice immunized with EBOV virus-like particles (VLPs). We have successfully isolated two scFvs that can detect all known Ebolavirus and Marburgvirus species using this method. This method is inexpensive, rapid, and can be used to quickly develop repertoires of high-affinity antibodies for detection of a broad set of filovirus GPs.

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“…The use of antibody fragments to neutralize viruses or prevent virus infection is not novel; proof of concept experiments have shown their potential applications in the context of different viral infections such us HIV (Lülf et al, 2014), Influenza A H1N5 (Bal et al, 2015), SARS (Sui et al, 2004), HPV (Culp et al, 2007), and West Nile virus (Gould et al, 2005). A recent contribution by Rodríguez-Martínez et al (2015) offers proof-of-principle of the application of three anti-EBOV mAb fragments, containing the variable regions the mAbs KZ52, 13C6, and 13F6, in immunological assays to specifically detect recombinant GP.…”

Section: Bottlenecks In Anti-ebola Mab Production: Scaling Up Cost mentioning

confidence: 99%

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead

Gonzalez‐González

Álvarez

Márquez-Ipiña

et al. 2015

Critical Reviews in Biotechnology

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The 2014 Ebola outbreak, the largest recorded, took us largely unprepared, with no available vaccine or specific treatment. In this context, the World Health Organization (WHO) declared that the humanitarian use of experimental therapies against Ebola Virus (EBOV) is ethical. In particular, an experimental treatment consisting of a cocktail of three monoclonal antibodies (mAbs) produced in tobacco plants and specifically directed to the Ebola virus glycoprotein (GP) was tested in humans, apparently with good results. Several mAbs with high affinity to the GP have been described. This review discusses our current knowledge on this topic. Particular emphasis is devoted to those mAbs that have been assayed in animal models or humans as possible therapies against Ebola. Engineering aspects and challenges for the production of anti-Ebola mAbs are also briefly discussed; current platforms for the design and production of full-length mAbs are cumbersome and costly.

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Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

Cited by 15 publications

References 40 publications

Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts

Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts

Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead

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