title: Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA) formulation.
abstract: BACKGROUND
  Antigen 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  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. 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.
authors.affiliation: Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, Texas, USA.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Sherwood
authors.forename: Andrew
authors.initials: A
authors.lastname: Hayhurst
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D029043:Ebolavirus / Q000187:drug effects / Q000276:immunology* / Q000502:physiology / Q000528:radiation effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005720:Gamma Rays; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000737:chemistry* / Q000276:immunology*; D011420:Propiolactone / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology*; D014764:Viral Proteins / Q000737:chemistry* / Q000276:immunology*; D038301:Virus Inactivation / Q000187:drug effects / Q000528:radiation effects
chemical_list: D000956:Antigens, Viral; D009698:Nucleoproteins; D061905:Single-Domain Antibodies; D014764:Viral Proteins; D011420:Propiolactone