PROJECT ABSTRACT HIV remains a dangerous and prevalent disease globally contributing to millions of infections and deaths per year and tens of billions of dollars in healthcare costs. The use of Truvada as Pre Exposure Prophylaxis (PrEP) is 99% effective at preventing HIV infection if taken daily, but its impact on reducing HIV burden is limited by poor adherence. PrEP is recommended by the Centers for Disease Control (CDC) in populations known to have a high risk of HIV infection, which include men who have sex with men, people with a history of an STI, intravenous drug users, and people with a partner who is HIV positive. Monitoring of drug adherence is well known to improve drug compliance, but there are no commercial products for adherence monitoring of PrEP. Hence, there is a critical unmet need for a tool that will allow physicians to monitor adherence to PrEP in their patients. In order to be acceptable to patients on a routine basis and feasible in the physician workflow, this adherence test needs to be noninvasive, painless, inexpensive, and provide rapid, accurate results. The overall goal of this project is to develop a point-of-care (POC) test that will measure patient adherence with the PrEP regimen and ultimately prevent HIV infection in people known to be at risk of exposure. The POC test will be based on a laboratory-based mass spectrometry urine test for levels of tenofovir, one of the drugs in PrEP, which UrSure already developed. The POC assay will be faster (minutes to get a result) and therefore can be used during a clinic visit to measure PrEP adherence and, if appropriate, counsel patients on how to improve their compliance. The aims of this Phase I project are to: 1) synthesize four tenofovir derivatives and conjugate them to form two immunogens for polyclonal antibody production in Aim 2 and two HRP-conjugated proteins for ELISA use in Aim 3; 2) produce two affinity purified polyclonal antibodies against each of the two immunogens produced in Aim 1; and 3) develop a prototype ELISA using these derivatives and antibodies, which will be validated with urine samples positive and negative for tenofovir. The final deliverables of this Phase I project will be conjugated tenofovir derivatives, polyclonal antibodies to these derivatives, and an ELISA, all of which will be used to develop monoclonal antibodies and ultimately a Lateral Flow Immunoassay POC urine tenofovir test in a subsequent Phase II application. This Phase I project will be a critical step towards developing our POC urine test and bringing a powerful tool with the ability to drive PrEP adherence and reduce HIV infections to physicians worldwide.