University of Chicago MRSEC H Jaeger DMR-0213745 (IRG4) Fabrication of Antibody Arrays with SAMS

1. University of Chicago MRSECH JaegerDMR-0213745 (IRG4)Fabrication of Antibody Arrays with SAMS Human single-chain Fragments of variable regions (scFv) and engineered protein scaffolds can be used to create “chips”, in which the affinity reagents are arrayed on glass slides. In this way, the concentrations of hundreds of proteins can be measured simultaneously, much like DNA chips can measure the concentration of RNA transcripts generated from thousands of genes at one time. Not only can changes in the levels of individual proteins be measured, but phosphorylation and other post-translational modifications can be quantitated. Such “antibody chips” are potentially of great value in evaluating changes in the proteome as a consequence of growth or diseased states. Left: immobilization strategy for generating antibody arrays. Cutinase-affinity reagent fusion proteins are captured a self-assembly monolayer that contains a suicide substrate for cutinase. Right: Detection of binding of fluorescent antigens (left) to immobilized cutinase, and fusions to anti-EH scFv, anti-lysozyme VHH, fibronectin type III domain (FN3) bind to SH3 domain, and FN3 that binds streptavidin (top). The binding is specific except for the anti-SH3 scFv which also cross-reacts with GST. At the Chicago MRSEC, successful proof-of-principle experiments have been carried out in a collaboration between the Kay and Mrksich groups [1]. The figure describes the strategy employed to fabricate the antibody chips (left). We have followed binding of antigens to the first-generation chips by surface plasmon resonance and fluorescence (right). Future work will be directed to demonstrating the usefulness of the chips in evaluating cell-signaling events in microbes and human cells. [1] Antibody Arrays Prepared by Cutinase-Mediated Immobilization on Self-Assembled Monolayers, Y. Kwon, Z. Han, E. Karatan, M. Mrksich, and B.K. Kay, Anal. Chem., 76, 3923- 3929 (2004).