Half area elisa plates

Half-area ELISA Plates (Corning) were coated with 10 μg/mL of NP2- or NP25-BSA (Biosearch technologies) and anti–mouse IgG, IgM, IgG1, IgG2b, IgG2c, or IgG3 unlabeled antibody (Southern Biotech) overnight at 4°C, or with 100–400 μg/mL sonicated filtered calf thymus DNA (Calbiochem) overnight at 37°C uncovered. Plates were washed with PBS containing 0.05% Tween-20 and blocked with 1% BSA in PBS. Diluted serum samples were incubated for 1.5 hours. Serum samples for anti-DNA IgG and IgG subclasses were diluted 1:100. Serum samples from NP-immunized mice were diluted 1:10,000 (day 7) or 1:50,000 (time course days 0, 14, 28, 42) for NP-specific IgM and IgG, and 1:2500 for NP-specific IgG subclasses. Plates were washed with wash buffer and secondary goat polyclonal alkaline phosphatase–labeled (AP-labeled) anti–mouse IgG, IgM, IgG1, IgG2b, IgG2c, or IgG3 (Southern Biotech) was added for 1 hour. After washing, plates were developed using phosphatase substrate (MilliporeSigma) dissolved in distilled water with 50 mM NaHCO₃ (MilliporeSigma) and 1 mM MgCl₂ (MilliporeSigma). Plates were read at a wavelength of 405 nM on a 1430 Multilabel Counter Spectrometer (PerkinElmer).

Half-area ELISA plates (Corning, Kennebunk, ME) were coated with 2.5 μg/mL Streptavidin in PBS for 2–3 h at RT, and blocked with 3% milk in PBS for 1 h at RT. After blocking, 1 μg/mL LIV LASV pfGP-TD was captured for 2 h at RT, followed by incubation with the indicated antibody concentration overnight at 4°C. The antibody GP solution was removed and plates were washed three times using PBS with 0.05% Tween 20, followed by treatment with PBS or 50 mM citrate/150 mM NaCl buffer adjusted to the indicated pH value for 1 h at RT. The plates were then washed three times with PBS-Tween20. Detection was performed using horseradish peroxidaseconjugated goat α-human IgG antibody reagent (KPL) for 1 h at RT. After an additional wash step, color was developed by the addition of 3,3,′5,5′ tetramethylbenzidine (TMB)-H₂O₂ (Thermo Fisher) for 5 min at RT. Development was stopped by the addition of 2N sulfuric acid. Color was read as absorbance (optical density) at 450nm.