2.6 Detection of S1-specific antibody and S1-responsive Th cytokine secreting cells in spleen and lungs by ELISPOT assay
Two weeks after triple vaccination, the spleen and lungs were dissected out carefully from the control untreated mice and immunized mice. Lymphocytes were isolated from the spleen using the method described in detail previously,21 while lung lymphocytes were isolated by collagenase digestion followed by Percoll (GE Healthcare, Buckinghamshire, UK) density gradient centrifugation, as described previously.21
S1-specific antibody secreting cells (ASCs) were detected by enzyme-linked immunospot (ELISPOT) assay as described previously.19 Briefly, the isolated lymphocytes of lung and spleen were seeded at 2 × 105 cells (lung) or 1 × 106 cells (spleen)/well onto MultiScreen 96-well plates MSIPS4510 (Millipore, Bedford, MA) that had been coated with 1 μg S1 protein/well, and incubated with fresh complete RPMI (cRPMI) medium (containing 10 mM HEPES, pH 7.2, 1 mM sodium pyruvate, 1% MEM non-essential amino acids solution, 14.3 μM 2-mercaptoethanol, 10 μg/mL gentamycin and 10% heat-inactivated fetal bovine serum) for 4 h (lung lymphocytes) or 24 h (splenocytes). After incubation, the S1-specific ASCs spots were detected by using goat anti-mouse IgG or anti-mouse IgA antibody conjugated with horseradish peroxidase (Sigma-Aldrich) and 3-amino-9-ethylcarbazole (Sigma-Aldrich). The number of ASC spots was counted by ELISPOT counter ImmunoSpot S6 (CTL, Cleveland, OH).
The S1-responsive Th cytokine secreting cells (CSCs) were detected by the ELISPOT assay using Mouse IFN-γ, IL-4 and IL-17A ELISpot BASIC kit (HRP) (MABTECH, Nacka Strand, Sweden), according to the protocols provided by the manufacturers. The splenocytes and lung lymphocytes were seeded at 1 × 105 cells (lung) or 1 × 106 cells (spleen)/well onto MultiScreen 96-well plates MSIPS4510 that had been coated with each cytokine captor antibody, and incubated with fresh cRPMI for 24 h with or without 20 μg/mL S1 protein. After incubation, the S1-responsive Th CSC spots were detected by each biotin conjugated cytokine detection antibody, streptavidin-horseradish peroxidase and 3-amino-9-ethylcarbazole. The number of CSC spots was counted by ELISPOT counter ImmunoSpot S6.