Use of a surface plasmon resonance method to investigate antibiotic and plasma protein interactions

Abstract

The pharmacologic effect of an antibiotic is directly related to its unbound concentration at the site of infection. Most commercial antibiotics have been selected in part for their low propensity to interact with serum proteins. These nonspecific interactions are classically evaluated by measuring the MIC in the presence of serum. As higher-throughput technologies tend to lose information, surface plasmon resonance (SPR) is emerging as an informative medium-throughput technology for hit validation. Here we show that SPR is a useful automatic tool for quantification of the interaction of model antibiotics with serum proteins and that it delivers precise real-time kinetic data on this critical parameter.

FIG. 1.

Ranking of the interactions of reference antibiotics on HSA, AGP, and gamma globulins. The binding of rifampin, vancomycin, minocycline, fusidic acid, novobiocin, lincomycin, ofloxacin, cefotaxime, and erythromycin to HSA, AGP, and gamma globulins was tested by SPR. The nonantibiotic molecule warfarin was included as a control in all the experiments. Data were normalized to the same 10,000-RU immobilization level for HSA (white bars), AGP (gray bars), and gamma globulins (black bars). The experiments were done in triplicate, and the results are presented as the mean value ± standard error (the error was low, and the bars showing the error are not always visible).

FIG. 2.

Dose-response binding of rifampin, fusidic acid, and novobiocin on HSA. The concentration-dependent binding of rifampin (▴), fusidic acid (⋄), and novobiocin (▪) to HSA was monitored by SPR. The binding response is presented on the left axis, and the fraction of the sites occupied on the protein is shown on the right axis.