Nanobody-Based Sandwich Immunoassay for Pathogenic <em>Escherichia coli</em> F17 Strain Detection
Asma Dhehibi,
Abdelmounaaim Allaoui,
Amal Raouafi,
Mohammed Terrak,
Balkiss Bouhaouala-Zahar,
Mohamed Hammadi,
Noureddine Raouafi,
Imed Salhi
Affiliations
Asma Dhehibi
Livestock and Wildlife Laboratory (LR16IRA04), Arid Lands Institute (I.R.A), University of Gabès, Médenine 4119, Tunisia
Abdelmounaaim Allaoui
Laboratory of Microbiology, African Genome Centre, Mohammed VI Polytechnic University (UM6P), Lot 660—Hay Moulay Rachid, Ben Guerir 43150, Morocco
Amal Raouafi
Sensors and Biosensors Group, Analytical Chemistry and Electrochemistry Lab (LR99ES15), University of Tunis El Manar, Tunis El Manar 2092, Tunisia
Mohammed Terrak
InBioS-Centre for Protein Engineering, University of Liege, B-4000 Liege, Belgium
Balkiss Bouhaouala-Zahar
Laboratory of Venoms and Theranostic Applications (LR20IPT01), Place Pasteur, BP74, Pasteur Institute of Tunis, University of Tunis El Manar, Tunis 1002, Tunisia
Mohamed Hammadi
Livestock and Wildlife Laboratory (LR16IRA04), Arid Lands Institute (I.R.A), University of Gabès, Médenine 4119, Tunisia
Noureddine Raouafi
Sensors and Biosensors Group, Analytical Chemistry and Electrochemistry Lab (LR99ES15), University of Tunis El Manar, Tunis El Manar 2092, Tunisia
Imed Salhi
Livestock and Wildlife Laboratory (LR16IRA04), Arid Lands Institute (I.R.A), University of Gabès, Médenine 4119, Tunisia
Rapid and specific detection of pathogenic bacteria in fecal samples is of critical importance for the diagnosis of neonatal diarrhea in veterinary clinics. Nanobodies are a promising tool for the treatment and diagnosis of infectious diseases due to their unique recognition properties. In this study, we report the design of a nanobody-based magnetofluorescent immunoassay for the sensitive detection of pathogenic Escherichia coli F17-positive strains (E. coli F17). For this, a camel was immunized with purified F17A protein from F17 fimbriae and a nanobody library was constructed by phage display. Two specific anti-F17A nanobodies (Nbs) were selected to design the bioassay. The first one (Nb1) was conjugated to magnetic beads (MBs) to form a complex capable of efficiently capturing the target bacteria. A second horseradish peroxidase (HRP)-conjugated nanobody (Nb4) was used for detection by oxidizing o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Our results show that the immunoassay recognizes E. coli F17 with high specificity and sensitivity, with a detection limit of 1.8 CFU/mL in only 90 min. Furthermore, we showed that the immunoassay can be applied to fecal samples without pretreatment and remains stable for at least one month when stored at 4 °C.
