Streptavidin alkaline phosphatase

Splenic and lymph node cells from single cell suspensions were cultured on 96-well plates bound to αCD3 (1 µg/mL) and αCD28 (1 µg/mL) (BioLegend) for 72  h at 37 °C and 5% CO₂. Following this incubation, supernatants were examined for IFN-γ, IL-2, IL-4, IL-6, IL-10, and IL-17 concentrations using cytokine ELISA. Capture and detection antibodies for these cytokines were purchased from Biolegend Streptavidin-alkaline phosphatase (BD Biosciences) and para-nitrophenyl phosphate (Thermo-Fisher Scientific) in glycine buffer were used for signal detection. Plates were read using a SpectraMax 190 plate reader (Molecular Devices; San Jose, CA, USA) at 405 nm. Concentrations of cytokines in supernatants were determined by extrapolation on the standard curve established by cytokine standard absorbance readings.

Membrane-based 96-well plates (MAIPS4510; Millipore, North Ryde, NSW, Australia) were coated with anti-mouse IFN-γ capture antibody (clone R4-6A2; Pharmingen) prior to addition of 5 × 10⁵ cells to each well followed by 50 μl NP_311–325 peptide (5 μg/well). Four wells lacking peptide were included as negative controls. Cells were cultured for 18 h at 37°C 5% CO₂ and IFN-γ detected using biotinylated mouse anti-IFN-γ detection Ab (clone XMG1.2; Pharmingen) and streptavidin–alkaline phosphatase (Pharmingen) as described elsewhere (13 (link)). Spots formed by the deposition of enzyme substrate were counted using an ELISPOT plate reader (AID Autoimmun Diagnotika, Strassberg, Germany) and analyzed using AID software. The number of spot-forming units (SFU) was calculated by subtracting the sum of the background value plus two SD and responses considered positive when the net SFU value was >20 SFU/10⁶ cells.

IL-1β cytokine was measured from the islet supernatants using sandwich ELISA following the manufacturer’s protocol (n = 4). Briefly, Nunc-Immuno 96-well plates were coated overnight (at 4°C) with purified anti-human IL-1β antibody at 1μg/mL in DPBS (BioLegend, California, USA, Cat. No. 511601). The next day, plates were blocked with DPBS + 1% BSA for 2 hours at 25°C, and then samples and standards were added to the 96-well plate in 2 replicates and incubated for 2 hours at 25°C. A dilution of 1:2 was used for the samples with the standards ranging from 20 to 5000pg/mL (BioLegend, Cat. No. 579409). Plates were subsequently incubated with biotin-conjugated antibody at 2μg/mL for 1 hour at 25°C (BioLegend, Cat. No. 511703), followed by incubation with Streptavidin-alkaline phosphatase (1:2000 dilution, BD Biosciences, Cat. No. 554065) for 1 hour at 25°C. Plates were washed 3 times with 1xDPBS containing 0.05% Tween-20 after each incubation step. Color was developed by adding PNPP substrate (Sigma-Aldrich) and absorbance was detected at 405nm using SpectraMAX 250 spectrophotometer (Molecular Devices Corp., California, USA). The amount of IL-1β in each experimental sample was determined from the absorbance value plotted against the concentration of a standard curve for the cytokine.