Receptor-ligand interactions studied with homogeneous fluorescence-based assays suitable for miniaturized screening.

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and beta(2)-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1 microl without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Different susceptibilities of genetic variants of hepatitis C virus (HCV) to interferon (IFN).

Genetic variants of HCV may have different degrees of resistance to IFN and may therefore influence the outcome of IFN therapy. However, selection of HCV variants by IFN has not been investigated in detail. In this paper, heteroduplex analysis was used to monitor major changes of HCV populations in 4 chronically infected patients under IFN therapy. We found that a major variant of the HCV 5' non-coding region (5' NCR) emerged in a responder. In other patients although no new variant of the 5' NCR was identified, significant changes occurred within the core and E1 region of the HCV genome. Disappearance and emergence of HCV variants may reflect their different susceptibilities to IFN. Our results indicate that responses of HCV populations to IFN are complex and need to be characterized by analysis of multiple HCV genome regions.

Analysis of hepatitis C virus quasispecies populations by temperature gradient gel electrophoresis.

Hepatitis C virus (HCV) forms complex quasispecies populations which consist of a large number of closely related genetic variants. This genetic heterogeneity may cause antigenic variation or drug resistance. We used heteroduplex analysis by temperature gradient gel electrophoresis (TGGE) to characterize genetic variants of HCV. The high resolution of TGGE was proven by comparison of DNA sequence data of different cDNA clones from the HCV 5'NCR with their corresponding migration pattern in TGGE. Using this method we were able to identify virus variants of the HCV 5'NCR even if they only differed from each other by a single base. HCV populations from three patients with chronic hepatitis C were found to consist of genetic variants, although the degree of the heterogeneity varied. In addition, we compared the genetic heterogeneity of the core and E2 regions of the HCV genome in one patient. Our results demonstrate that TGGE is a useful tool for characterization of the genetic heterogeneity of virus populations in vivo.