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Nanobody-Based Sandwich Immunoassay for Pathogenic Escherichia coli F17 Strain Detection
- Source :
- Biosensors, Vol 13, Iss 2, p 299 (2023)
- Publication Year :
- 2023
- Publisher :
- MDPI AG, 2023.
-
Abstract
- 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.
Details
- Language :
- English
- ISSN :
- 20796374
- Volume :
- 13
- Issue :
- 2
- Database :
- Directory of Open Access Journals
- Journal :
- Biosensors
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.88e4ab87f06f40adae134a8a6fa86762
- Document Type :
- article
- Full Text :
- https://doi.org/10.3390/bios13020299