PROJECT ABSTRACT Tuberculosis (TB) remains a major world-wide health problem and the development of a sensitive point-of-care (POC) diagnostic for active TB infection is a global public health priority. Lateral flow (LF) assays based on the detection of mycobacterial lipoarabinomannan (LAM) antigen in patient urines are potential POC tests for TB, but current assays have suboptimal sensitivity, and are not suitable for all populations. An early form of this assay, the Alere Determine™ TB LAM Ag test, was strongly recommended by the WHO for diagnosis of active TB in HIV-positive patients with a CD4 cell count of <200 cells/mm and advanced HIV disease. A newer assay (the Fujifilm SILVAMP TB LAM assay) based on novel monoclonal antibodies (mAbs) isolated or characterized in my lab was found to be considerably more sensitive for both HIV-positive and HIV-negative populations, but still did meet the WHO’s Target Product Profile for wide-spread utility. The FujiLAM assay utilizes a unique combination of a mAb (S4-20) that is highly specific for M.tb LAM and a second mAb (A194-01) that is widely reactive with LAM from many mycobacterial species. More recent studies in my lab using a standard panel of 25 large-scale urine samples collected from Ugandan patients preselected for a range of HIV clinical status, CD4 cell count and LAM urinary concentrations have identified profound differences in the immunochemical properties between ManLAM and uLAM, and have shown significant diversity in the immunoreactivity of uLAM present in different patient urine samples. These studies have included a novel combination of mAbs (FDX01/A194-01) that possesses increased sensitivity and breadth of detection of uLAM in our standard urine panel, but may have lower specificity for M.tb. All of the mAbs described above were isolated using ManLAM antigen purified from cultured M.tb. Based on these results we now propose to characterize antigenic diversity of uLAM across a range of TB phenotypes, including pulmonary and extrapulmonary TB, HIV+ and HIV- TB, and determine the specificity of these different forms for TB against other respiratory diseases, latent TB, and TB-uninfected cohorts. In parallel we will isolate mAbs that are highly sensitive and specific for different forms of uLAM by B cell sorting and antibody cloning. We will then select and validate the highest performing mAb combinations for TB disease detection across a large panel of diverse TB and non-TB clinical samples, and compare their sensitivity and specificity with that ofthe novel FDX01/A194-01 assay. These studies will address the hypotheses that different LAM immunotypes detected in patient urines reflect important bacteriological and host features integral to a generalizable diagnostic test, and that exploiting these forms will lead to the enhanced detection of uLAM that will improve the sensitivity and specificity of urine assays for detection of active TB infection.