Self-amplifying protease chain reactions for simple, sensitive, and quantitative detection

The National Institute of General Medical Sciences awarded Potomac Affinity Proteins, LLC $377,602 on July 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859, Assistance Listing R43GM159468) to develop protease chain reaction (PROCR) technology for rapid, highly sensitive, and quantitative detection of biomolecules in mix-and-read assays.

The recipient will engineer protease and inhibitor components to optimize catalytic turnover and detect three target molecules: gram-negative bacterial lipopolysaccharide (endotoxin), human interleukin-2, and human ferritin. The technology centers on a self-amplifying detection system in which polyclonal immunoglobulin G forms immune complexes around a target, concentrating proteases into catalytic clusters that activate protease-inhibitor complexes in solution. This amplification mechanism generates signal analogous to polymerase chain reaction amplification of DNA. Work is performed in Gaithersburg, Maryland, with a period of performance from July 1, 2026, through June 30, 2027.

Abstract

Our objective is to develop a protease chain reaction (PROCR) technology that will enable rapid, highly sensitive, and quantitative detection of biomolecules in simple mix and read assays. The central element of a protease chain reaction is a protease-inhibitor complex. The protease is inactive when bound to the inhibitor but, once freed, can cleave the inhibitor and release additional free protease. We are proposing to build powerful molecular detection systems from three basic components: 1) a protease conjugated to a binding molecule 2) an inhibitor protein which contains a proteolytic cleavage site for the protease IT inhibits, and 3) a protease substrate which generates a signal upon its cleavage. The key element in detection is linking protease activation to target binding. This was accomplished by fusing the protease and an IGG-binding domain. Self-amplification is triggered when polyclonal IGG forms an immune complex around the target. The immune complexes concentrate proteases resulting in a critical state and rapid self-activation of all bound proteases. The critical property that enables highly sensitive detection is that each immune complex becomes a powerful catalytic cluster that activates protease-inhibitor complexes from the solution. That is, the protease cluster becomes a multi-subunit enzyme with many catalytic centers. Inactive complexes enter the cluster and active proteases are released. This results in enormous signal amplification in response to a target molecule, analogous to the amplification of DNA by a polymerase chain reaction. In phase I, we will engineer the protease and inhibitor to further optimize catalytic turnover. This will be achieved by optimizing components for detecting three important targets: gram-negative bacterial lipopolysaccharide (ENDOTOXIN), human interleukin-2 (IL2), and human ferritin. Components and assay designs will be evaluated by their performance in no-wash, mix and read assays that will be carried out either in solution or on capture particles. PROCR materials and methods will be benchmarked against commercial assays for ENDOTOXIN (limulus amebocyte lysate assay) and standard sandwich elisa assays for IL2 and ferritin.