Emax plus reader

Cytokine concentrations in culture supernatants were determined by ELISA, using cytokine-specific coating and biotinylated detection antibodies diluted per manufacturer’s protocol (eBioscience/Invitrogen/Thermo Fisher Scientific). Streptavidin-HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) binding to biotinylated detection antibodies was visualized with TMB substrate (KPL/Seracare, Milford, MA) and the reaction was stopped with 0.18 M H₂SO₄. Plates were read at 450 nm via Emax Plus Reader (Molecular Devices, San Jose, CA). Data were analyzed with SoftMax Pro software (Molecular Devices); unknowns were compared with a standard curve containing at least five to seven dilution points of the relevant recombinant cytokine (eBioscience/Invitrogen/Thermo Fisher Scientific) on each assay plate. In all cases, the coefficient of determination for the standard curve (r²) was ≥0.98. ELISA unknowns were diluted to fall within the range of standard values.

Standard solid-phase assays were used to measure the antibody reactivity in mouse sera, as described previously (86 (link)). One µg of Sm (Immunovision, Springdale, AR) or EBNA-1 mosaic (BiosPacific) was coated per well in each of 96 polystyrene wells/plate. Mouse sera at a dilution of 1:100 (Sm) or 1:1000 (EBNA-1) were incubated in each well for 3 hrs. After incubation, plates were washed and incubated with anti-mouse alkaline phosphatase-conjugated γ-chain-specific goat IgG (Sigma-Aldrich/MilliporeSigma) at 1/10,000 dilution. Para nitrophenyl phosphate disodium (PNPP, Sigma-Aldrich/MilliporeSigma) was used as a substrate for alkaline phosphatase, and plates were read at 405 nm via Emax Plus reader (Molecular Devices). ELISA tests were considered positive if the optical density (OD) was at least two standard deviations above the naïve/adjuvant control mean.

Sera were assessed for anti-nuclear antibodies (ANA; Alpha Diagnostic International, San Antonio, TX), anti-dsDNA antibodies (Alpha Diagnostic Int’l), blood urea nitrogen (BUN; Arbor Assays, Ann Arbor, MI), and serum creatinine (Arbor Assays) per manufacturers’ protocols. For ANA and anti-dsDNA assays, sera were measured in duplicate at a 1:100 dilution in a 96-well plate format, and the HRP-coupled secondary Ab was goat anti mouse IgG (H and L). Negative and positive control sera, as well as 5 point calibration curve samples, provided by the manufacturer, were run concurrently with the unknown samples. Sera were diluted 1:10 for BUN assays and 1:30 for creatinine assays, per manufacturers’ protocols. Sera were run in duplicate alongside a 5 (creatinine) or 7 (BUN) point standard curve. All assays were read at 450 nm using an Emax Plus Reader (Molecular Devices). Unknowns were compared with a calibration curve containing five dilution points on each assay plate. In all cases, the coefficient of determination for the standard curve (r²) was ≥0.98.