ELISPOT assay. The number of cytokine-producing cells that recognize specific antigens in PBMC from type 1 diabetic patients and control subjects was quantified by the ELISPOT assay as described . Fresh blood was drawn from each patient directly into Vacutainer CPT tubes (Becton Dickinson, Inc., Franklin Lakes, NJ, USA) at each of six centers in the US and shipped immediately to the central laboratory at the University of Colorado Health Sciences Center, Barbara Davis Center for Childhood Diabetes. Blood samples were received no later than 24 h after the blood was drawn and PBMC were isolated by centrifugation via Vacutainer CPT Ficoll–Paque density gradient immediately upon receipt, and lymphocytes were washed and stored frozen in a dimethyl sulfoxide solution in liquid nitrogen. 3 × 105 PBMC obtained from frozen samples were seeded in triplicate wells of 96-well Unifilter 350 filter-backed plates (Polyfiltronics; Fisher, Tustin, CA, USA) coated with antihuman IFN-γ monoclonal antibody (clone 2G1; Endogen, Inc., Cambridge, MA, USA) or anti-IL-5 monoclonal antibody (clone TRFK5; BD PharMingen, Inc., San Diego, CA, USA). Cells were cultured in triplicate in the presence or absence of 5 µg/ml Tetanus toxoid (Accurate Chemicals, Westbury, NY, USA), 10 µg/ml phytohaemagglutinin (PHA-M; Sigma, St. Louis, MO, USA), 1 µg/ml Staphylococcus enterotoxin B (SEB; BD PharMingen), insulin B(9−23) (50 µm) or the APL, NBI-6024 (10 and 50 µm). These peptides were synthesized by a solid-phase method as described ; human insulin B-chain (9–23) amide (B(9−23)) [SHLVEALYLVCGERG] and NBI-6024 [SHLVEALALVAGERG]. RPMI 1640 medium containing 1% N-2-Hydroxyethylpiperazine-N′-2-ethane (HEPES), l-glutamine, Na Pyruvate, Non-essential amino acid NEAA, Penicillin/Streptomycin, 10−5mβ-mercaptoethanol and 1% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA; endotoxin <10 pg/ml) was used. After 24 h (for IFN-γ assessment) or 48 h (for IL-5 assessment) of incubation at 37 °C in the presence of 5% CO2, cells were washed away and cytokines were detected with antihuman IFN-γ (clone B133.5; Endogen) or antihuman IL-5 (clone JES1-5A10; PharMingen) secondary biotinylated monoclonal antibody plus avidin-peroxidase (DAKO, Philadelphia, PA, USA). 3-Amino-9-ethyl carbazole substrate solution (Pierce, Inc., Dallas, TX, USA) was used to develop the reaction which was stopped by washing the plate with water. Spots derived from cytokine-producing cells were quantified using the Series-1 Immunospot and Satellite Analyzers (Autoimmun Diagnostika, Inc., Strassberg, Germany). We demonstrated that this ELISPOT assay showed good sensitivity and reproducibility by participating in a workshop that compared five different assay formats . At least three of the seven longitudinal samples collected per patient were analysed simultaneously to reduce interassay variability. The majority of samples showed strong responses to the positive control mitogens, SEB and PHA (see Results). Note that the analytical and statistical approach for defining positive ELISPOT responses is described in the Results.