Development and Evaluation of a Novel VHH-Based Immunocapture Assay for High-Sensitivity Detection of Shiga Toxin Type 2 (Stx2) in Stool Samples

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype. Therefore, Stx detection assays are considered the most suitable methods for the early detection of STEC infections. Single-domain antibodies from camelids (VHHs) exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis. In this work, we have exploited VHH technology for the development of an immunocapture assay for Stx2 detection. Thirteen anti-Stx2 VHHs previously obtained from a variable-domain repertoire library were selected and evaluated in 130 capture-detection pair combinations for Stx detection. Based on this analysis, two VHHs were selected and a double VHH-based biotin-streptavidin capture enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection was developed and optimized for Stx2 detection. This assay showed an excellent analytical and clinical sensitivity in both STEC culture supernatants and stool samples even higher than the sensitivity of a commercial ELISA. Furthermore, based on the analysis of stool samples, the VHH-based ELISA showed high correlation with stx2 detection by PCR and a commercial rapid membrane-based immunoassay. The intrinsic properties of VHHs (high target affinity and specificity, stability, and ease of expression at high yields in recombinant bacteria) and their optimal performance for Stx detection make them attractive tools for the diagnosis of HUS related to STEC (STEC-HUS).

SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications

“…Current in vitro diagnostic systems use polyclonal or monoclonal full length antibodies as detector antibodies coupled to HRP or biotin to provide a measurable and sensitive detection of the target. So far, there are several assay systems based on camelid nanobodies published ( 29 – 34 ), where nanobodies were coupled to HRP, alkaline phosphatase or beads but none of these are using camelid heavy-chain-only antibodies for detection. This study identifies suitable camelid heavy-chain-only antibody candidates specific for SARS-CoV-2 Spike protein selected by phage display from a camelid naïve library.…”

SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications

“…Current in vitro diagnostic systems use polyclonal or monoclonal full length antibodies as detector antibodies coupled to HRP or biotin to provide a measurable and sensitive detection of the target. So far, only few assay systems based on camelid nanobodies are published [20][21][22] but none of these are using camelid full length antibodies for detection. This study identifies suitable camelid antibody candidates specific for SARS-CoV-2 Spike protein selected by phage display from a camelid naïve library.…”

Food-Borne Biotoxin Neutralization in Vivo by Nanobodies: Current Status and Prospects

“…Shiga toxin-producing Escherichia coli , a subset of Escherichia coli pathogens, poses a grave threat through severe foodborne diseases, including hemolytic uremic syndrome and even fatalities. , The main virulence factors, Stx1 and Stx2, are crucial contributors to the pathogenicity of Shiga toxin-producing Escherichia coli . The latter is more often associated with severe pathology in humans and newly weaned piglets .…”