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.