Undecapeptide amide sublibraries

The peptides and peptide mixtures were cleaved from the resins and the side chains were deprotected with trifluoroacetic acid phenol ethanedithiol thioanisole water (96 2 1 2 1, v/w/v/v/v) (1 ml) within 3 h. The products were filtered from the resins, which were washed once with acetic acid (0.3 ml), and were precipitated at — 20 °C by the addition of cold n-heptane diethylether (1 1, v/v) (5 ml). The precipitates were washed twice by sonication with n-heptane diethylether (1 1, v/v) and were lyophilized from acetic acid water tert.-butyl alcohol (1 10 50, v/v/v), yielding individual peptides, the completely randomized peptide amide libraries Xn and 220 undecapeptide amide sublibraries O/X 0-NH2. The biotinylated peptide sequence (see below) was obtained by elongating the ApoB 100 peptide 2877-2894 with two spacer amino acids (Ahx), followed by biotin using the coupling method described above. 

Screening results from 220 sublibraries were clustered to an Activity Pattern of the peptide library. Six sequence positions have strong influence on DR1-binding (positions 2,5,7,8,10,11) (Fig. 11.4). In these positions the most potent amino acids (relative competition >2.80) as well as unfavorable amino acids were selected to design new undecapeptide amides with expected high or low DR1 -affinity. In positions 1,3,4,6 and 9, which are more tolerant with respect to amino acid exchanges, one amino acid with a relative competition value near 1 was introduced. 

Based on the proliferative response of TCC to 220 peptide amide sublibraries, individual (defined) undecapeptide amides potentially acting as synthetic epitopes were synthesized. From investigation of these peptides in proliferation assays it became evident that TCC 5G7 responded to all selected peptides at much lower antigen concentration than MBP (86-96). The synthetic epitopes induced maximum stimulation at a concentration of 1 ng/ml, whereas 100 pg/ml of the natural epitope MBP(86-96) was necessary to induce comparable proliferation. 

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