ABSTRACT
The transporter associated with antigen processing (TAP) is essential for antigen presentation by major histocompatibility complex (MHC) class I molecules. Traditional methods used to analyze peptide transport mediated by TAP require radioactive labeling of peptides and time-consuming manipulation of Concanavalin A-Sepharose. Drug discovery research requires rapid and reliable evaluation of large number of samples for bioactivity. To meet these requirements a nonradioactive, HTS assay for peptide transport activity of TAP has been developed. The radioactive label in the traditional assays has been replaced by a fluorescent label without compromising the transport efficiency of labeled peptide or the sensitivity of the assay. The use of multiscreen filtration plates has facilitated higher throughput and eliminated the centrifugation steps used in traditional TAP assays. The HTS assay shows similar kinetic characteristics as compared to the traditional assay. The HTS assay has been adapted on a Quadratrade mark 96-320 96-channel pipetting station (Tomtec, Hamden, CT) by optimizing time course, dose response of TAP to peptides and adenosine triphosphate (ATP), signal/noise ratio, reproducibility, and reagent stability. This HTS system has been utilized to screen a multiplexed compound library with a maximum of throughput 17,600 compounds per week.
