| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1137-1 | ||||
Resumo:Enterotoxigenic Escherichia coli (ETEC) causes diarrhea and diarrheal death among young children and travelers in developing countries. The major virulence factors of diarrhea-causing ETEC strains are enterotoxins, specifically a heat-labile toxin (LT) and a heat-stable toxin, making these toxins excellent targets to detection for diagnosis. The LT is an AB5 toxin, it binds to ADP and ribosylates the guanyl-nucleotide alpha regulatory binding protein of the adenylate cyclase system, thereby increasing the levels of cyclic AMP. Antibodies are an excellent molecule for the design of high-affinity, protein-based binding reagents to be use as detection tools for immunodiagnosis. Using recombinant DNA technology and methods such as Phage Display, it is also possible to obtain fragments of recombinant antibodies (rAbs), such as Fab, with in vitro immunological repertoires, without the need for direct immunization of live hosts. Here, we used a human synthetic Fab fragment library, to select antibody fragments against LT by phage display, to be use as tools for ETEC detection. Antibodies selection was performed by phage display with a naive human Fab fragment library panned against immobilized native LT. The selected phages were analyzed for affinity by single-point competitive phage ELISA assay and sequencing. The sequences showed the presence of 34 distinct clones, of which only 12 had CDRs with functional sequences. Furthermore, among these 12, two had responded with saturation at the lowest antigen concentration in the competition ELISA, the most promising being 10C and 12G. The 10C and 12G Fab sequence was amplified, cloned, and expressed in a bacterial system, followed by purification by Immobilized-metal affinity chromatography. The purification yield was 3 mg/L and 6,5 mg/L to 10E and 12G respectively. The ETEC diagnostic assays are ongoing. These results showed promising tools for specific diagnosis of ETEC. Palavras-chave: Recombinant Antibody;, Immunodiagnostic; , ETEC; , LT; , Phage Display Agência de fomento:FAPESP | |||||