Clone r4 6a2

ELISPOT plates (PVDF, 96 well) were equilibrated with 70% ethanol, washed with PBS, and were coated overnight with anti-mouse IFNγ antibody (1 μg/ml, clone AN18, Mabtech). Necropsies were performed on mice eight weeks following intravenous vaccination to harvest splenocytes for growth in RPMI complete media (RPMI with L-glutamine and 10% fetal bovine serum). 96 well plates were blocked using RPMI media and 6 x 10⁵ splenocytes were combined in each well with varied agonists: MU-Ag85A peptide (100 μg/ml, FQAAYNAAGGHNAVWNFDDN), MU-Ag85B peptide (100 μg/ml, FQDAYNAAGGHNAVFNFNDN), heat killed MU (HKMU, 1 mg/ml), or whole cell lysate prepared from log phase MU liquid culture. Splenocytes were stimulated for 16 hours at 37°C. The plates were then washed with PBS + 0.05% tween 20 followed by a two hour incubation with secondary anti-mouse IFNγ antibody (1:1000, clone R46A2, Mabtech) at 37°C. After a wash and three hour room temperature incubation with VectaStain avidin peroxidase complex (Vector Labs), plates were incubated with 3-amino-9-ethylcarbazole substrate for five minutes. The reaction was ceased by submersion of plates in deionized water. Spots were visualized and quantified using a CTL Immunospot plate reader.

After peptide immunization, splenocytes were cultured with or without peptides (4 μg/ml each mCAR12 and mCDK12) overnight at 37°C in pre-coated 96-well plates (Mabtech) and cytokine secretion was detected with an anti-IFN-γ antibody (1 μg/ml, clone R4-6A2, Mabtech). Subtyping of T-cell responses was performed using purified CD3 splenocytes +/- MHC class I or class II blocking antibodies (20 µg/ml). All samples were tested in duplicates or triplicates.