Akp streptavidin

HA1-2-specific IFN-γ- or IL-4-producing cells were quantified in splenic lymphocyte cultures using the BD^™ ELISPOT set (BD Biosciences) following the manufacturer’s protocol. Briefly, 96-well ELISPOT PVDF microplates were coated with a capture antibody in sterile PBS, incubated overnight at 4°C, and blocked (2 h at room temperature) with complete RPMI medium. Splenic lymphocytes were counted and plated in ELISPOT plates at 2 × 10⁵ cells/well (in triplicate) in complete RPMI medium. Cells were mock stimulated with RPMI or stimulated with HA1-2 (5 μg/mL) for 24 h incubation at 37°C and 5% CO₂. After the incubation period, ELISPOT plates were processed following the manufacturer’s specifications using a biotinylated antibody as a detection antibody, streptavidin-AKP (BD Pharmingen) and a BCIP/NBT Liquid Substrate System (Sigma-Aldrich) for assay development (20 min at room temperature). All assay plates were scanned and analyzed using an automated ELISPOT reader system (Bioreader 5000-Vβ, BioSys, Karben, Germany).

We evaluated SIVmac239-specific immune responses through the IFN-γ-mediated ELISpot assay as we previously reported (52 (link)). Briefly, a 96-well PVDF plate (Merck Millipore, Cat. No. MSBVS1210) was coated with monoclonal coating antibodies (U-CyTech, Cat. No. CT605-10) overnight at 4°C. PBMCs were isolated through density gradient centrifugation by Lymphoprep (Axis-Shield, Cat. No. 1114547). The plate was washed three times with DPBS (BasalMedia, Cat. No. B210KJ) and 200 µL per well of R10 medium (RPMI 1640, 10% fetal bovine serum, 1% penicillin‒streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, and 10 mM HEPES) were added to block the plate for 2 h. PBMCs were diluted with R10 medium, and 300,000 PBMCs were added to each well. The SIVmac239 peptide stocks were diluted 20 times by R10, and 10 µL of each peptide was added to the corresponding well. After incubation for 24 h, the plate was washed, and biotinylated monoclonal detection antibodies (U-CyTech, Cat. No. CT605-10) were added. The next morning, streptavidin-AKP (BD PharMingen, Cat. No. 554065) was added and incubated for 2 h at 37°C. The 1-STEP NBT/BCIP (Thermo Scientific, Cat. No. 34042) was then added and incubated for 10 min at 37°C. The plate was washed and dried before being read by an enzyme-linked immunospot imager (Bioreader6000, BIOSYS, Germany).