Np30 bsa bovine serum albumin

IL-6 and IL-10 IVCCA was performed as previously described (17 ) using biotinylated capture antibodies (Invitrogen). Briefly, young and aged C57BL/6 mice were intravenously injected with 10 μg of biotinylated anti–IL-6 (MP5-32C11, Invitrogen) and anti–IL-10 (JES5-16E3, Invitrogen) capture antibodies; mice were bled within 24 hours, and serum was collected. A luminescent ELISA was performed using anti–IL-6 (MP5-20F3, Invitrogen) or anti–IL-10 (JES5-2A5, BD Biosciences) as the coating antibody. For NP-specific antibody titers, 96-well plates were coated overnight at 4°C with NP30-BSA (bovine serum albumin) (Biosearch Technologies), followed by blockade of nonspecific binding by incubation for 1 to 2 hours at 25°C with 5% BSA. Serum samples were loaded into plates with eight serial dilutions (starting from 1:100 or 1:1000), followed by incubation for 2 hours at 25°C or overnight at 4°C. After samples were washed, horseradish peroxidase–conjugated goat antibody to mouse IgG1 (PA1-74421, Thermo Fisher Scientific) was added to plates, followed by incubation for 2 hours at 25°C. The reactions were developed by incubation for 15 min at 37°C with 50 μl of trimethylboron substrate (BioLegend) and were stopped by the addition of 25 μl of 10% H₃PO₄. The plates were read at 450 and 570 nm (for correction) with an ELISA reader.

To measure NP-specific antibody titers, 96-well plates were coated with NP30-BSA (bovine serum albumin) (Biosearch Technologies) overnight at 4°C. Following blocking with 5% BSA for 1 hour at room temperature, serum samples were loaded into the plates with three dilutions per sample (1:27,000, 1:81,000, and 1:243,000) and incubated overnight at 4°C. Samples were washed and then incubated with a horseradish peroxidase-conjugated antibody reactive to mouse IgG1 (clone: PA1-74421; ThermoFisher Scientific) for 1 hour at room temperature. Samples were then washed and developed using ThermoScientific’s 1-Step Ultra TMB-ELISA solution and stopped after color change with 2N H₂SO₄. The plates were read at 450nm with an ELISA reader.