Fully Automated Protein Proximity Assay in Formalin-Fixed, Paraffin-Embedded Tissue Using Caged Haptens.

ABSTRACT

The ability to interrogate for the presence and distribution of protein-protein complexes (PPCs) is of high importance for the advancement of diagnostic capabilities such as determining therapeutic effects in the context of pharmaceutical development. Herein, we report a novel assay for detecting and visualizing PPCs on formalin-fixed, paraffin-embedded material using a caged hapten. To this end, we synthetically modified a nitropyrazole hapten with an alkaline phosphatase (AP)-responsive self-immolative caging group. The AP-labile caging group abrogates antibody binding; however, upon exposure to AP, the native hapten is regenerated. These caged haptens were applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the population of PPCs, was then visualized by an anti-hapten antibody conjugated to horseradish peroxidase, followed by diaminobenzidine detection. We provide proof of concept for the detection of protein proximity pairs (ß-catenin-E-cadherin and EGFR-GRB2), and confirm assay specificity through technical controls involving reagent omission experiments, and biologically by treatment with small-molecule kinase inhibitors that interrupt kinase-adaptor complexes.