Fitc conjugated mouse igg2a

For ELISA and functional assays, monoclonal antibodies against the IFN-γ clones B27 and B133.5 were obtained from ImmunoTools in Germany, and clone MD-1 was purchased from BioLegend Inc. in CA. These antibodies were IgG1-specific to IFN-γ and exhibited neutralizing capacity against IFN-γ (10 (link)–12 (link)). For the neutralization assay, mouse IgG1 (clone MOPC-21) (BioLegend Inc.) was used as the isotype-matched antibody control. For flow cytometry, FITC-conjugated anti-human HLA-DR and DP (clone HL-38) and isotype-matched control FITC-conjugated mouse IgG2a, were purchased from ImmunoTools in Germany. PE-conjugated anti-human phospho-STAT1 (pY701) antibody (BD Pharmingen) was purchased from BD Biosciences. Recombinant human IFN-γ (rIFN-γ) was purchased from R&D Systems in MN.

A cell-based assay was performed to determine the neutralizing activity of B27 and A9 mAbs. Briefly, 10 ng/mL of rIFN-γ WT was incubated with mAb at 0.1, 1, or 10 µg/mL for 1 h. The mixture was subsequently incubated with THP-1 cells (4 × 10⁵ cells) at 37 °C in 5% CO₂ incubator. After 24 h, cells were harvested to detect MHC class II surface expression by flow cytometry. Cells were washed thrice with PBS and blocked with 50 µL of 10% AB serum in PBS for 30 min. For MHC class II staining, 2.5 µL of FITC-conjugated anti-human HLA-DR and -DP (clone HL-38) or isotype-matched control (FITC-conjugated mouse IgG2a) (ImmunoTools, Friesoythe, Germany) was added and incubated for 30 min on ice. After washing, cells were resuspended in 1% paraformaldehyde-PBS. Data were collected with BD Accuri™ C6 Plus Flow Cytometer (BD Bioscience).