Nanobody and F(ab')2-Based Immunocapture assays for diagnosis of STEC-associated Hemolytic Uremic Syndrome

LUCIANO J MELLI; LAUCHE C; HIRIART Y; BASCHKIER A; PARDO R; MILIWEBSKY E; MARTA RIVAS; FERNANDO GOLDBAUM; ZYLBERMAN V;; JUAN E. UGALDE; DIEGO J. COMERCI; ANDRÉS E. CIOCCHINI

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2017

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic uremic syndrome (HUS). The ability to produce Shiga toxins (Stx1 and/or Stx2) are the key virulence trait of STEC strains, and the O157:H7 and non-O157:H7 STEC strains can produce both or only one of the two types of toxins in their different variants (Stx1: Stx1a, Stx1c and Stx1d; Stx2: Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f and Stx2g). Therefore, Shiga toxins detection assays are considered the most suitable methods for diagnosis of STEC-associated diseases. In this work, we developed two capture ELISAs for Stx1 and Stx2 detection from broth culture and stool samples. In a first approach, we exploited nanobody technology for the development of a double nanobody capture ELISA for Stx2a detection, the most prevalent variant of Stx2. Using this immunocapture nanobody-based ELISA, we were able to detect the native toxin from culture supernatants of STEC strains expressing Stx2a and from stools samples obtained from HUS patients. In order to detect the entire repertory of Shiga toxin variants, we developed a capture ELISA based on a F(ab´)2 fragments that specifically recognize Stx1 and Stx2. The F(ab´)2 fragments were obtained from the peptic digestion of IgGs obtained from horses immunized with the recombinant proteins BLS-Stx1B and BLS-Stx2B (Brucella Lumazine Synthase-B subunit of Stx1 and 2). Using this approach we were able to detect with high sensitivity Stx1a and Stx1c as well as the Stx2 variants Stx2a, Stx2b, Stx2c and Stx2f greatly expanding the Stx detection spectrum. Both immunocapture assays are novel diagnostics that allow a sensitive and specific detection of Shiga toxins and may be of great value for diagnosis of HUS associated with STEC. Furthermore, the nanobodies and F(ab')2 fragments evaluated in this work may constitute excellent diagnostic tools for the development of simple, rapid and accurate lateral flow immunoassays.