The NIAID Category B priority pathogen Entamoeba histolytica is a public health threat, especially as a cause of diarrhea in travelers to and immigrants from endemic areas, men who have sex with men, and children in the developing world where it is in the top 5 causes of severe diarrhea. It is also a biodefense threat to water and food supplies due to its resistance to chlorination and low infectious dose. Here we propose to develop a point-of-care (POC) fecal antigen diagnostic test. Successful completion of the project will produce the first POC test that detects E. histolytica in both fresh and formalin-fixe specimens]. Investigators: Dr. Herbein at TechLab is an expert in developing enteric diagnostics; having directed the FDA- clearance of diagnostics for Giardia and Cryptosporidia and having served as the PI for multiple Phase I/II SBIR/STTR projects. Dr. Petri at the University of Virginia is an international leader in clinical and basic investigation of amebiasis, and in collaboration with TechLab has developed the only FDA 510(k)-cleared diagnostic test for E. histolytica. [Innovation: Innovative aspects include: (1) development and manufacture of a POC test for amebiasis that can be multiplexed with that of other category B enteric parasites; and (2) production of the first diagnostic test for E. histolytica detection in both fresh and formalin-fixed samples]. [Approach: The following two Specific Aims are proposed to accomplish our goal of a new diagnostic test for amebiasis: 1) Produce monoclonal and polyclonal antibodies against the highly abundant Jacob protein of the E. histolytica cyst. 2) Apply these antibodies to produce a POC test for E. histolytica]. Environment: The proposal builds on the longstanding partnership of Dr. Petri at the University of Virginia with Dr. Herbein of TechLab, Inc. for amebiasis diagnostic test development. These two institutions are importantly only two hours apart and that have worked together successfully for over a decade.