Secondary screening and evaluation of peptides for inclusion in the pathogen binding 100-peptide microarray. (A) Heat map of pathogens screened against 275 peptide array. Each pathogen was labeled with AF555 and AF647 and incubated on the array. (B) Representative binding curves from secondary screening of 275 peptide library by ELISA. 96-well plates were coated with Vaccinia virus (blue squares), A/PR/8/34 H1N1(black circles), F. tularensis (green diamonds), or R. prowazekii Madrid (red triangles) and each peptide was incubated at the indicated concentrations. (C) In vitro cytotoxicity screen of peptide library. HEK293 cells (1 × 106 cells) were incubated for 1 hour with 25 µM of each peptide in replicate wells. Few peptides decreased cell viability >10% (dotted line).