Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA) formulation.

Laura J Sherwood,Andrew Hayhurst

doi:10.1371/journal.pone.0061232

Abstract

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.

Highlights

Viruses are usually experts in energetic economy, employing multiple copies of a relatively small repertoire of key architectural templates to assemble infectious particles comprising outer coat/ envelope superstructures and internal substructures such as matrix and nucleocapsid [1,2]
The 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
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

Summary

Introduction

Viruses are usually experts in energetic economy, employing multiple copies of a relatively small repertoire of key architectural templates to assemble infectious particles comprising outer coat/ envelope superstructures and internal substructures such as matrix and nucleocapsid [1,2]. To formulate a virus specific sandwich assay only a single antibody clone specific to a surface exposed, preferably highly conserved epitope on one of these polyvalent virus components should be required. The polyvalent display of the antigenic epitope on large protein oligomers should ensure a very strong interaction with surface immobilized captor antibody increasing the sensitivity for even mediocre affinity antibodies through a Velcro like effect via massively parallel avidity. This condition is met provided the assay conditions preserve antigen polyvalency which is especially important for enveloped viruses since detergents used to reduce non-specific binding or to inactivate the agent, may destroy the envelope. 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

Results

Conclusion