Detection of antigen-specific T cells on p/MHC microarrays.
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.
Deviren
Gokhan
G
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, USA.
Gupta
Kapil
K
Paulaitis
Michael E
ME
Schneck
Jonathan P
JP
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D005452:Fluoresceins; D005453:Fluorescence; D006649:Histocompatibility Antigens / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000276:immunology*; D040081:Protein Array Analysis / Q000379:methods*; D013388:Succinimides; D013997:Time Factors
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D006649:Histocompatibility Antigens; D010455:Peptides; D013388:Succinimides