Miniaturized and high-throughput assays for analysis of T-cell immunity specific for opportunistic pathogens and HIV.

摘要

Monitoring of antigen-specific T-cell responses is valuable in numerousconditions that include infectious diseases, vaccinations, and opportunisticinfections associated with acquired or congenital immune defects. A variety ofassays that make use of peripheral lymphocytes to test activation markers, T-cellreceptor expression, or functional responses are currently available. The lastgroup of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells maybe the limiting factor, particularly in lymphopenic subjects and in children, thegroups that more often require immune monitoring. We have developedimmunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lentitself to miniaturization and automation. Lymphoproliferation and theenzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to aminiaturized format. Here we provide examples of immune profiles and describe acomparison between miniaturized assays based on cytokine secretion orproliferation. We also demonstrate that these assays are convenient for use intesting antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells andreagents and of automation to save time and increase accuracy were demonstratedin this study using different methodological approaches valuable in the clinical immunology laboratory.