Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

Ikegami, Tetsuro; Niikura, Masahiro; Saijo, Masayuki; Miranda, Mary Elizabeth; Calaor, Alan B.; Hernandez, M.; Acosta, Luz P.; Manalo, Daria L.; Kurane, Ichiro; Yoshikawa, Yasuhiro; Morikawa, Shigeru

doi:10.1128/cdli.10.4.552-557.2003

Cited by 35 publications

(21 citation statements)

References 33 publications

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“…It has been demonstrated that the C-terminal half of the filovirus NP has strong antigenicity (Saijo et al, 2001). Multiple studies have identified conformational and linear epitopes for antibodies in this NP region for several viruses within the genus Ebolavirus (Ikegami et al, 2003; Niikura et al, 2001, 2003). …”

Section: Introductionmentioning

confidence: 99%

Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein

et al. 2013

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Filoviruses (viruses in the genus Ebolavirus and Marburgvirus in the family Filoviridae) cause severe haemorrhagic fever in humans and nonhuman primates. Rapid, highly sensitive, and reliable filovirus-specific assays are required for diagnostics and outbreak control. Characterisation of antigenic sites in viral proteins can aid in the development of viral antigen detection assays such immunochromatography-based rapid diagnosis. We generated a panel of mouse monoclonal antibodies (mAbs) to the nucleoprotein (NP) of Ebola virus belonging to the species Zaire ebolavirus. The mAbs were divided into seven groups based on the profiles of their specificity and cross-reactivity to other species in the Ebolavirus genus. Using synthetic peptides corresponding to the Ebola virus NP sequence, the mAb binding sites were mapped to seven antigenic regions in the C-terminal half of the NP, including two highly conserved regions among all five Ebolavirus species currently known. Furthermore, we successfully produced species-specific rabbit antisera to synthetic peptides predicted to represent unique filovirus B-cell epitopes. Our data provide useful information for the development of Ebola virus antigen detection assays.

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Section: Introductionmentioning

confidence: 99%

Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein

et al. 2013

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“…All anti-Zaire sdAb except C exhibit cross-reactivity with Ivory Coast, and all except sdAb A react with Bundibugyo. Overall therefore, Ebolavirus NP comprises a mix of specific and cross-reactive epitopes as seen for a human Fab derived from a survivor of the Zaire Kikwit 1995 outbreak from repertoire cloning and phage display selection on gamma-irradiated virus [63], and mouse monoclonal antibodies generated via hybridomas from mice immunized with recombinant NP [64], [65], [66].…”

Section: Resultsmentioning

confidence: 99%

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

Sherwood

Hayhurst

2013

PLoS ONE

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BackgroundAntigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism.Methods and FindingsIn parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections.ConclusionsThe C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus.

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“…This made the preparation of a mixture of monoclonal antibodies with different specificities necessary. Two novel monoclonal antibodies, Res2-6C8 and Res2-1D8, specific to the nucleoprotein of EBOV-R partly solved the problem [11] .…”

Section: Elisa Assaymentioning

confidence: 99%

Laboratory detection and diagnosis of filoviruses

2011

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Ebola virus (EBOV) and Marburg virus (MARV), belonging to the Filoviridae family, emerged four decades ago and caused severe viral hemorrhagic fever in human and other primates. As high as 50-90% mortality, filoviruses can cause significant threats to public health. However, so far no specific and efficient vaccine has been available, nor have other treatment methods proved to be effective. It is of great importance to detect these pathogens specific, rapidly and sensitively in order to control future filovirus outbreaks. Here, recent progresses in the development of detection and diagnosis methods for EBOV and MARV are summarized.

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Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

Cited by 35 publications

References 33 publications

Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein

Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

Laboratory detection and diagnosis of filoviruses

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