Figure 1

Configuration of Collaborative Enzyme Enhanced Reactive-immunoassay (CEER). When target proteins are bound to specific capture antibodies printed on nitrocellulose surface after incubating with cell lysate, unbound non-target proteins are removed from the slide. One of the detector antibodies against an alternate epitope on captured target-protein is conjugated with GO. Binding of another detector antibody specific to phosphorylated sites (P) on target protein (a) or another non-overlapping epitope (b) conjugated with HRP completes the formation of immuno-complex necessary for signal generation and subsequent tyramide mediated signal amplification through GO-HRP enzyme channeling in the presence of glucose. The capture and detection antibodies were selected to minimize competition between them (i.e., all antibodies can simultaneously bind their corresponding epitope on the signal transduction protein). (c) A slide configuration for CEER is shown. Capture antibodies for each specific target protein are printed in triplicates in serial dilution. Each slide contains cell line controls for standard curve generation for accurate quantitation of samples on each slide run. Internal quality control samples are run on each slide to ensure the quality of data generated from each array-slide.