Abstract
The development of high‐throughput protein microarrays for rapidly determining antigen‐specific T‐cell receptor repertoires of diverse T‐cell populations can enable comprehensive, broad‐based analyses of T‐cell responses. Promising applications include medical diagnostics, vaccine development, treatment of autoimmune diseases and detection of potential agents of bioterrorism. In this study, we examined the feasibility of using peptide/major histocompatibility complex (p/MHC) microarrays to selectively capture and enumerate antigen‐specific T cells. Results are presented for p/MHC microarrays consisting of a dimeric MHC‐immunoglobulin complex, K^b^‐Ig, loaded with either a cognate or non‐cognate peptide for binding CD8^+^ T cells. We quantified the sensitivity of these K^b^‐Ig microarrays by measuring a lower detection limit of 0.05% antigen‐specific CD8^+^ T cells mixed with splenocytes from C57BL/6J mouse. A fivefold increase in this lower detection limit (0.01%) was achieved using a secondary capture anti‐Ig antibody to coat the microarray surface. This higher sensitivity is comparable to that obtained using standard state‐of‐the‐art fluorescence activated cell sorting (FACS) instruments. We also found that contacting the T‐cell suspension with the K^b^‐Ig microarrays under mild shear flow conditions produced more uniform distributions of captured T cells on the individual spots and better spot‐to‐spot reproducibility across the entire microarray. Copyright © 2006 John Wiley & Sons, Ltd.