Protein−protein interactions (PPI) are a ubiquitous mode of transmitting signals in cells and tissues. We are testing a stepwise, generic, structure-driven approach for finding low molecular weight inhibitors of protein−protein interactions. The approach requires development of a high-affinity, single chain antibody directed specifically against the interaction surface of one of the proteins to obtain structural information on the interface. To this end, we developed a single chain antibody (sc1E3) against hIL-1β that exhibited the equivalent affinity of the soluble IL-1 receptor type I (sIL-1R) for hIL-1β and competitively blocked the sIL-1R from binding to the cytokine. The antibody proved to be more specific for hIL-1β than the sIL-1R in that it failed to bind to either murine IL-1β or human/murine IL-1α proteins. Additionally, failure of sc1E3 to bind to several hIL-1β mutant proteins, altered at receptor site B, indicated that the antibody interacted preferentially with this site. This, coupled with ...