A fluorescence-based high throughput screen for the transporter associated with antigen processing

Jonathan M. Blevitt, Klaus Früh, Charlie Glass, Michael R. Jackson, Per A. Peterson, Shaoming Huang

Research output: Contribution to journalArticle

6 Scopus citations

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 The radioactive label in the traditional assays has been replaced by a fluorescent label without compromising the transport efficiency of labeled peptide or the sensitivity of the assay.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 Quadra(TM) 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.

Original languageEnglish (US)
Pages (from-to)87-91
Number of pages5
JournalJournal of Biomolecular Screening
Volume4
Issue number2
DOIs
StatePublished - Jun 26 1999

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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