Peptide array

Initial optimization experiments were conducted using pooled, overlapping
15- or 16-mer peptides corresponding to the sequences of CFP-10, ESAT-6, and
TB10.4 (BEI Resources, NIAID, NIH: Peptide Array, M.
tuberculosis CFP-10 Protein, NR-34825; Peptide Array, M.
tuberculosis ESAT-6 Protein, NR-34824; Peptide Array, M.
tuberculosis TB10.4 Protein, NR-34826). 18-mer peptides
(overlapping by 11 amino acids) corresponding to the 60 Mtb Ags listed in Table 1 were synthesized on a 10mg scale
using Fmoc chemistry (Synthetic Biomolecules, San Diego, CA) according to the
H37Rv genome sequence. Peptides were pooled by Ag (10 – 121 peptides per
pool, with a median of 39 peptides per pool); peptide pools were numbered
consecutively according to Rv gene numbers (Table 1). After pooling the peptides for each Ag, pools were
lyophilized, followed by reconstitution in DMSO at a final centration of 1mg/ml
per peptide. Peptide pools were further diluted in RPMI-1640 medium supplemented
with 2mM L-glutamine, 100U/ml penicillin, and 100µg/ml streptomycin, to
prepare working stocks for use in the stimulation assays described below.
Phytohemagglutinin-M (PHA; Sigma-Aldrich) was used a positive control.

Genotype 1a or 1b HCV 15 to 18-mer peptides with 11 or 12 amino acid overlaps spanning the entire HCV polyprotein (BEI Resources, NIAID, NIH: Peptide Array, Hepatitis C Virus) were reconstituted in 5% sterile dimethylsulphoxide (DMSO) and pooled consecutively into twenty-one groups. Peptides were aliquoted and stored at −80°C until use.