RESUMO
AIMS: To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR. METHODS AND RESULTS: The gene coding for specific VHH derived from the camel immunized with binding domain of botulinum neurotoxin E (BoNT/E) was amplified by error-prone PCR. Several biopanning rounds were used to screen the phage-displaying BoNT/E Hc nanobodies. The final nanobody, SHMR4, with increased affinity recognized BoNT/E toxin with no cross-reactivity with other antigens especially with related BoNT toxins. CONCLUSIONS: The constructed nanobody could be a suitable candidate for VHH-based biosensor production to detect the Clostridium botulinum type E. SIGNIFICANCE AND IMPACT OF THE STUDY: Diagnosis and treatment of botulinum neurotoxins are important. Generation of high-affinity antibodies based on the construction of secondary libraries using affinity maturation step leads to the development of reagents for precise diagnosis and therapy.
Assuntos
Anticorpos Antibacterianos/genética , Toxinas Botulínicas/imunologia , Biblioteca Gênica , Anticorpos de Domínio Único/genética , Animais , Anticorpos Antibacterianos/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Bacteriófagos/genética , Camelus/imunologia , Técnicas de Visualização da Superfície Celular , Mutagênese , Reação em Cadeia da Polimerase , Anticorpos de Domínio Único/químicaRESUMO
Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.
