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Updated: Feb 13, 2026

Immunization of Alpacas Lama pacos with Protein Antigens and Production of Antigen-specific Single Domain Antibodies
Published on: January 26, 2019
Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment
Robert Alvin Bernedo-Navarro1, Ema Romão2, Tomomasa Yano3
1Laboratory of Bacterial Genetics, Institute of Biology, University of Campinas (UNICAMP), São Paulo 13083-862, Brazil. alvinbn@gmail.com.
Background:
Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans.
Results:
An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113-rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb113₂ exhibits a higher toxin neutralization capacity compared to monovalent Nb113.
Conclusions:
The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb113₂ with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.
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