Sub-domains of ricin's B subunit as targets of toxin neutralizing and non-neutralizing monoclonal antibodies

PLoS One. 2012;7(9):e44317. doi: 10.1371/journal.pone.0044317. Epub 2012 Sep 11.


The B subunit (RTB) of ricin toxin is a galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicity assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB's high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB's sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Neutralizing / immunology*
  • Antibody Specificity / immunology
  • Apoptosis
  • Blotting, Western
  • Cell Surface Display Techniques
  • Chlorocebus aethiops
  • Epitope Mapping
  • Epitopes, B-Lymphocyte / chemistry
  • Epitopes, B-Lymphocyte / immunology
  • Female
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry*
  • Protein Subunits / immunology*
  • Receptors, Cell Surface / metabolism
  • Ricin / chemistry*
  • Ricin / immunology*
  • Vero Cells


  • Antibodies, Monoclonal
  • Antibodies, Neutralizing
  • Epitopes, B-Lymphocyte
  • Protein Subunits
  • Receptors, Cell Surface
  • Ricin