RESUMO
Ready to use dry-reagent PCR assays for Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas spp. and for broad-range bacteria detection were developed. The assays were based on novel switchable lanthanide probes that provide sensitive target DNA detection with exceptionally high signal-to-background ratio, thus enabling clear discrimination between positive and negative results. For example, sensitivity of three S. aureus and two S. pneumonia bacteria (colony forming units) per PCR assay was measured with fluorescence signal more than 30 times over the background signal level. The rapid and easy-to-use assays are suitable for routine clinical diagnostics without molecular biology expertise and facilities.
Assuntos
Infecções Bacterianas/diagnóstico , Técnicas Bacteriológicas/métodos , Elementos da Série dos Lantanídeos/metabolismo , Medições Luminescentes , Técnicas de Diagnóstico Molecular/métodos , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Humanos , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Sensibilidade e Especificidade , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.
