Abstract
We aimed to develop and validate sensitive fluorescence techniques to assess the binding of magnetic microbeads derivatized with ristocetin from Amycolatopsis lurida to carboxyfluorescein-labeled D-Ala-D-Ala-D-Ala and in competition with Staphylococcus aureus bacteria. Glycopeptide antibiotics have been widely used to treat bacterial infections. However, new antibiotics are needed because of growing bacterial resistance and serious side effects. To screen potential candidates for new antibiotics, there is a great demand for sensitive, fast and inexpensive techniques to analyze the interactions between these molecules and bacterial cells. Fluorometry, an in-house fluorescent instrument and fluorescence microscopy were used to determine binding constants of 2.75 × 10(4), 2.21 × 10(4) and 0.81 × 10(4) M(-1), respectively, for the interaction between ristocetin and the labeled peptide. The methods detailed herein have been successfully applied to assess the binding of carboxyfluorescein- labeled D-Ala-D-Ala-D-Ala to ristocetin on microspheres. The magnetic bead-based immunoassay could be used to detect bacteria at low concentrations.
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