A critical aspect of this program project includes evaluating antibodies for their potency against HIV and affinities to gpl 20s and Fc receptors. The Cell/Biochemical Assay Automation Core will design and carry out cell-based and biochemical assays to characterize the specificity, efficacy, and potency of HIV-neutralizing antibodies. We have adapted the standard in vitro pseudovirus neutralization assay, which measures the reduction in luciferase reporter gene expression in the presence of a potential inhibitor following pseudovirus infection in TZM-bl cells, for execution by a liquid handing robot. Core A personnel will validate, refine, and trouble-shoot protocols for the automated in vitro neutralization assays and then perform automated in vitro neutralization assays for evaluating antibodies for Drs. Nussenzweig, Ravetch, and Bjorkman. In addition. Core A will perform rapid and high-throughput binding assays to assess the affinities of antibodies for gpl20s and/or gp140s, as required for Drs. Nussenzweig and Bjorkman, and the affinities of modified Fc regions for Fc receptors, as required by Dr. Ravetch. The core will train and assist students, postdoctoral fellows, research assistants, and investigators in the analysis and interpretation of data from automated assays. In addition, the core will maintain two custom-equipped Evo Freedom Liquid handling stations, including daily calibration and care, and annual preventative maintenance.
HIV/AIDS is a global public health problem. Efforts to develop a vaccine or improve antibodies for passive immunization require screening large numbers of potential reagents: using in vitro neutralization assays to evaluate antibody potencies and protein-protein binding assays to identify interactions. We have automated these techniques in order to efficiently evaluate large numbers of reagents for the investigators in this collaborative project.
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