(548c) Development of Peptide and Protein Assays Based on Enzyme-Multiplied Assay Technique

Enzyme-multiplied assay technique (EMAT) is a homogeneous method widely used in urine and blood testing due its simplicity, sensitivity, and fast response time. We are currently aiming to extend EMAT for protein and peptide detection. With this capability, EMAT can potentially be used in areas such as bioterrorism defense and infectious disease detection.

Conceptually, EMAT consists of an analyte-reporter enzyme conjugate and an anti-analyte antibody. When the antibody binds to the analyte-enzyme conjugate, enzyme activity is repressed due to physical blockage or conformational change of the active site. When introduced, free analyte competes for antibody binding sites and at least partially prevents repression, thus the reporter enzyme can more readily turn over substrate, and a signal related to the quantity of free analyte present is generated.

As a proof-of-the-concept study, a peptide assay based on EMAT was prepared by conjugating a peptide sequence from influenza hemagglutinin A (HA) to glucose-6-phosphate dehydrogenase (G6PDH) via a crosslinker. Matrix-assisted laser desorption/ionization mass spectrometry shows that an average of 14 peptides attach to each G6PDH. 70% repression is achieved upon antibody binding. Preliminary data shows that repression can be relieved with ≥100 nM of free HA. Lower detection limits can be achieved by optimizing the antibody to HA-G6PDH ratio. With a similar approach, assays that are useful for biotoxin detection can be developed.