Streptavidin peroxidase

IgM, IgGs and IgA were measured in the NLF supernatants by ELISA. Briefly, 96-well plates (Nunc, Rochester NJ, USA) were coated with unlabeled goat-anti human Igs (10 µg/ml: Southern Biotechnology Birmingham AL, USA) and they were then blocked with Phosphate buffered saline (PBS, BioWhittaker, Lonza Group Basilea Switzerland) + 0.5% gelatin. The supernatants were thawed and serial dilutions in the same buffer were added to the wells in duplicate. The plates were then incubated with biotinylated goat anti-human-IgM, -IgG or -IgA (Southern Biotechnology), and subsequently with streptavidin-peroxidase (Southern Biotechnology). The ELISA plates were revealed with 0.5M O-phenylenediamine (Sigma-Aldrich St. Louis MI, USA) and the reaction was stopped with 3N SO₄H₂. OD values were obtained at 450 nm and standard curves were generated using purified myeloma proteins for human IgM, IgA (Southern Biotechnology) and IgG1 (a generous gift from Dr E. Fernández, Hospital La Princesa, Madrid). The Ig concentrations were calculated using the GraphPad Prism 5.0 software.

IgM, IgG and IgA were measured by ELISA in the supernatants obtained from B cell splenic cultures. Briefly, flat-bottom 96-well plates (Nunc, Rochester, NJ, USA) were coated with unlabeled goat-anti mouse total Ig (10 µg/ml; SouthernBiotech, Birmingham, AL, USA) and they were then blocked with 0.5% gelatin in phosphate buffered saline (PBS, BioWhittaker™, Lonza Group). Frozen supernatants were thawed and serial dilutions were prepared in the ELISA buffer (see below) and were plated in duplicate (at 50 μL/well). After overnight (4ºC) incubation, the plates were washed in PBS, 50 μL/well of biotinylated goat anti-mouse-IgM, -IgG or -IgA (SouthernBiotech) were added and the plates were incubated for 1 h. Subsequently, 50 μL/well of streptavidin-peroxidase (SouthernBiotech) were added and incubated for 30 min at RT. All the regents were previously tested and were diluted in 0.1% Tween-20 0.5% gelatin PBS. The ELISA plates were then revealed with 0.5 M O-phenylenediamine (Sigma-Aldrich) and the reaction was stopped with 3 N H₂SO₄. OD values were obtained at 450 nm in a Multiskan FC ELISA-reader (Thermo Fisher Scientific). Standard curves were generated using purified mouse IgM, IgG and IgA (SouthernBiotech), and the Ig concentrations were calculated using the GraphPad Prism 8.0 software.