RESUMO
AIMS: Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus. METHODS AND RESULTS: To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA1 ) and fluorescent dimeric affibody (fA2 ) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA1 and fA2 had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that 'fA2 ' affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the 'fA1 ' affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results. CONCLUSIONS: The results show that 'fA1 ' affiprobe and 'fA2 ' affiprobe are two new efficient luminescent affiprobes for detecting S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits.
Assuntos
Técnicas Bacteriológicas/métodos , Citometria de Fluxo/métodos , Sondas Moleculares/metabolismo , Staphylococcus aureus/isolamento & purificação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luminescência , Sondas Moleculares/genética , Sondas Moleculares/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/citologia , Staphylococcus aureus/metabolismoRESUMO
The human immunodeficiency virus (HIV) destroys CD4+ lymphocytes and monitoring these cells is one of the best techniques for studying HIV infection. In the present study a novel bioluminescent probe, super RLuc8-sFv, is developed in order to detect human CD4+ cells by fusion of an anti-human CD4 sFv to the C-terminus of super RLuc8. The results indicate that the probe can bind to CD4+ cells via its sFv domain; also it emits visible light through its signalling domain. Super RLuc8-sFv provides a new gateway for detection of human CD4+ cells using luminometric-based assays and may reduce the difficulties involved in, and the cost of, HIV-related diagnostic tests.
Assuntos
Linfócitos T CD4-Positivos , Luciferases , Proteínas Luminescentes , Sondas Moleculares , Anticorpos de Cadeia Única , Antígenos CD4/química , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Humanos , Luciferases/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Modelos Moleculares , Sondas Moleculares/química , Ligação Proteica , Conformação Proteica , Anticorpos de Cadeia Única/químicaRESUMO
A novel and advanced Fc-binding probe FcUni-RLuc namely has been produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence HWRGWV was fused to Renilla luciferase, and the purified probe was employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells.
