UNIVERSITY OF MASSACHUSETTS
One goal of our R01 supported research is to produce monoclonal antibody populations derived from immunologically humanized mice immunized with test HIV vaccine candidates . Our original strategy was to use conventional hybridization techniques employing human hybridoma cell lines as fusion partners. The ARRA funded studies were to evaluate and develop newer more efficient cloning strategies for human cells. First we propose to isolate antibody-secreting cells (ASC) from humanized mice immunized one week previously with an HIV candidate vaccine. Second, as a concurrent approach we will isolate memory B cells from humanized mice 3-4 weeks after priming and allow them to expand and differentiate to ASC in culture. Both primary and secondary ?cultured? anti-HIV ASC will be enriched by selection using a FACS or ELISPOT based single cell detection assay. Thereafter, single cell target populations will be processed using PCR cloning for the isolation of Ig VH and VL gene segments followed by expression of monoclonal antibody populations by transfection of producer cell lines. Our summer research associate has optimizing the culture of human lymphocytes and the activation of these lymphocytes for the production of antibody. She has developed methodology for the retrieval of antibody secreting B cell precursors from frozen peripheral blood patient samples. She has developed a robust ELISPOT assay for detection of antigen-specific ASC and is currently developing single cell isolation procedures for the preparation of cDNA and the cloning of Ig gene segments.