CloneID: A9
Antigen Long Description: The original antibody was generated by immunizing two alpacas with a ricin toxoid. A VHH phage-displayed library was constructed and the antibody was identified in a panning directly on plate bound RTA.
Origin Pub PMID: 6CWG
Buffer Composition: PBS only.
Uniprot Accession No.: P02879
Specificity Statement: The antibody recognizes an epitope on RTA that straddles clusters I and III. In particular, the antibody contacts the core secondary structural elements of cluster I, namely β-strand h, α-helix B and α-helix D, as well as a core element of cluster III, namely α-helix C. Ricin is a member of the type II ribosome-inactivating protein (RIP) family of plant toxins. It is a 65 kDa glycoprotein consisting of two subunits, RTA and RTB, joined by a single disulfide bond.
Application Notes (Clone): The VHH antibody was specific for RTA as confirmed by competition ELISA. The crystal structure of ricin catalytic subunit in complex with the VHH antibody was determined. The antibody had relatively weak toxin-neutralizing activity (IC50 ~750 nM), as shown by Vero cell cytotoxicity assays. The antibody binding affinity (KD) for ricin holotoxin (0.08 nM). The binding affinity, and, toxin-neutralizing activity of the antibody was mediated by CDR2 containing five consecutive Gly residues that interact with α-helix B. In order to prove this, a variant of the antibody lacking Gly AC151residue 59 (A9Δ59) was generated. Binding studies and toxinneutralizing assays confirmed that the removal of a single glycine residue from A9’s CDR2 significantly reduced binding affinity for RTA (~10-fold weaker; 1.76 nM versus 0.102 nM) and eliminated toxin-neutralizing activity (Rudolph et al., 2018; PMID: 30265352).
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