HPSEC-Competition Assay

High-performance size-exclusion chromatography (HPSEC) was used for competition studies. Solubilized HLA-DR1 (0.13 pM) was incubated for 48 h at 37 °C with the N-ter-minally 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled IM-(19-31)-peptide dissolved in 150 mM sodium phosphate, pH 5.5, containing 15 % (v/v) acetonitrile, 0.1 % (w/v) Zwittergent-12 (Calbiochem) and a cocktail of protease inhibitors (0.2 mM PMSF, 5 pM leupeptin, 10 pM pepstatin, and 1 pM chymostatin). Competition assays were performed in a 1.5 pM solution of AMCA-peptide as competitors, different peptides, the peptide library or peptide sublibraries were added in concentrations ranging from 

3 pM to 41.3 pM. All samples were analyzed on a Pharmacia Superdex 75 HR 5/20 high-performance gel filtration column, essentially as described previously [63]. The column was operated at a flow rate of 0.4 ml/min (250 p.s.i.) using the HPSEC buffer, pH 6.0. The effluent passed through a Merck fluorescence spectrophotometer (350/450 nm) and a Merck UV detector (214 nm) set up in series. Fluorescence and UV signals eluting with HLA-DR1 dimers were recorded by a model D-2500 integrator (Merck-Hitachi). 

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