Nanobody-based immunocapture assay for detection of Shiga toxin-producing Escherichia coli

MELLI, LUCIANO J.; HIRIART, Y; LAUCHE, C; PARDO, R; CHINEN, ISABEL; RIVAS, MARTA; ZYLBERMAN, VANESA; UGALDE, JUAN E; COMERCI, DIEGO J; CIOCCHINI, ANDRÉS E.

Mar del Plata

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB 2015); 2015

SAIB

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia and acute renal failure. Shiga toxins (Stx1 and/or Stx2) are the most important virulence factor of STEC strains and all the strains express one or both toxins independently of the serotype. Therefore, Shiga toxins detection assays are considered the most suitable methods for detection of STEC and HUS diagnosis. In this work, we have exploited nanobody technology for the development of a double nanobody sandwich ELISA for Stx2 detection. A panel of 13 anti-Stx2 nanobodies, obtained from a variable-domain repertoire library isolated from a llama immunized with the recombinant protein BLS-Stx2B (Brucella Lumazine Synthase-B subunit of Stx2), was evaluated in 130 capture-detection pair combinations. Based on this analysis two nanobodies were selected. Using this combination, we were able to detect the recombinant protein BLS-Stx2B (detection limit, 0.05 ng/ul) as well as the native toxin obtained from culture supernatants of STEC strains expressing Stx2. The nanobody-based immunocapture assay is a novel diagnostic tool that allows a sensitive and specific detection of Stx2 in broth cultures and may be of great value for HUS diagnosis from stool samples.