title: A sensitive ELISPOT assay to detect low-frequency human T lymphocytes.
abstract: We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 x 10(5) PBMC with the standard assay compared to 19 SFC per 2 x 10(5) PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocyte cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.
authors.affiliation: Anergen Incorporated, Redwood City, CA 94063, USA.
authors.forename: M
authors.initials: M
authors.lastname: McCutcheon
authors.forename: N
authors.initials: N
authors.lastname: Wehner
authors.forename: A
authors.initials: A
authors.lastname: Wensky
authors.forename: M
authors.initials: M
authors.lastname: Kushner
authors.forename: S
authors.initials: S
authors.lastname: Doan
authors.forename: L
authors.initials: L
authors.lastname: Hsiao
authors.forename: P
authors.initials: P
authors.lastname: Calabresi
authors.forename: T
authors.initials: T
authors.lastname: Ha
authors.forename: T V
authors.initials: TV
authors.lastname: Tran
authors.forename: K M
authors.initials: KM
authors.lastname: Tate
authors.forename: J
authors.initials: J
authors.lastname: Winkelhake
authors.forename: E G
authors.initials: EG
authors.lastname: Spack
mesh_terms: D000595:Amino Acid Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D008222:Lymphokines / Q000032:analysis; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D007376:Interleukin-2; D008222:Lymphokines