Application of in situ purified S. pneumoniae proteins to immunoassays. The overexpressed proteins were manually printed at approximately 70% humidity using the MicroCaster manual printing device (Whatman, Florham Park, NJ). Immobilized proteins were in situ purified and quantities were assessed using an Alexa Fluor 555 conjugated anti-His-tag antibody (a, c). Small white squares in a) and c) denote the position of the immobilized E. coli Lac repressor (control). The relative purity of His-tagged S. pneumoniae proteins was assessed by assays with an anti-E. coli antibody and an ATTO 550 labeled 2nd antibody (b, d) which resulted in the detection of endogenous E. coli proteins where His-tagged proteins were impure. The protein microarray on the Cu2+/IDA/PEG chip (c, d) was subjected to a detection assay with a rabbit anti-S. pneumoniae antibody followed by a 2nd antibody (labeled with ATTO 550) (e) and with a human patient serum followed by a 2nd goat anti IgG antibody (labeled with Alexa Fluor 555) (f).