Streptavidin horseradish peroxidase conjugate

A NHP-specific IFN-γ ELISpot assay was used on PBMC, according to the manufacturer protocol (U-CyTech, Utrecht), to determine the frequency of antigen-specific IFN-γ producing cells both in the vaccination phase (38 weeks) and infection phase (12 weeks). In brief, 200.000 freshly isolated PBMC were incubated in triplicate for 24 h with specified antigens or control stimuli. Subsequently, supernatant was collected and stored (−80 °C), and cells were transferred to specific anti-IFN-γ coated filter plates (PVDF, Millipore) for an additional overnight (18 h) incubation. Cells were discarded and membrane-bound IFN-γ was detected using biotinylated anti-IFN-γ antibody, streptavidin-horseradish peroxidase conjugate, and tetramethylbenzidine (TMB) substrate (the latter from MAbTech, Stockholm). Spots were quantified using an automated reader (AELVIS, Hannover).

Maxisorp 96-well plates (Nunc, Roskilde, Denmark) were coated at 4 °C overnight with 2 μg/ml of mAbs to IFN-γ or IL-2 in 100 μl PBS/well. Other assay steps were performed at RT. Five washes with 200 μl/well of PBS with 0.1% Tween 20 were made between all assay steps. After coating, wells were blocked for 1 h with incubation buffer (PBS with 0.05% Tween 20 and 0.1% bovine serum albumin; 200 μl/well). HEK supernatants containing IFN-γ or IL-2 with semi-quantified concentrations were added at serial dilutions in incubation buffer (100 μl/well) and incubated 2 h. Next, biotinylated anti-BAM tag mAb (Mabtech) at 1 μg/ml was added (100 μl/well) and incubated for 1 h. Following that, streptavidin-horse radish peroxidase conjugate (Mabtech) diluted 1:1000 in incubation buffer was added (100 μl/well) and incubated for 1 h at RT. The assay was developed with TMB substrate (Mabtech) and stopped with 0.18 M H₂SO₄ followed by absor-bance measurement at 450 nm–650 nm with an ELISA reader (Labsystems, Helsinki, Finland).