Opteia reagent set

The antibody titer of SEB was quantified by ELISA. Eight mice were used per group. Sera were collected 4 weeks after first immunization with L-GUS or L-SEB-GUS. Purified recombinant SEB (0.1 μg/well) was diluted with PBS buffer and coated onto a 96-well ELISA plate (Corning, Lowell, MA) at 4°C overnight. The plate was washed with PBS containing 0.05% (w/v) Tween-20 and blocked with PBS containing 1% (w/v) bovine-serum albumin and 0.05% (w/v) Tween-20 for 2 h at room temperature. Pooled antisera obtained from eight immunized mice with L-GUS or L-SEB-GUS were serially diluted by 10-fold and separately added to the wells and incubated for 2 h. A goat anti-mouse IgG-HRP conjugate (Promega, Madison, WI) (1 : 5000 dilution) was added and incubated for 2 h before washing. HRP activity was determined with an OptEIA™ Reagent Set (BD Biosciences). The OD of each well was measured at 490 nm. The endpoint was defined as the dilution of sera producing the same OD at 490 nm as a 1/100 dilution of preimmune sera. Sera negative at the lowest dilution tested were assigned endpoint titers of 100. The data was presented as geometric mean endpoint ELISA titers as previously described [31 (link)].

Briefly, 96-well high-binding EIA/RIA plates (Costar) were coated with 50 μL/well of 1 ug/mL spike or NP proteins in phosphate-buffered saline (PBS) overnight. The coated plates were washed twice with 200 μL/well of PBS containing Tween 20 (PBS-T), blocked with blocking buffer (1% blotting-grade blocker (BIO-RAD) in PBS-T) for 30 min at 37 °C. Plasma samples were serially diluted 4-folds in blocking buffer, added to the plates, and incubated at room temperature (RT) for 2 h. The plates were washed three times with PBS-T, and HRP-conjugated goat anti-human IgG (SouthernBiotech) in blocking buffer was added, followed by incubation for 1.5 h at RT. After washing three times with PBS-T, the wells were treated with TMB substrate solution (OptEIA reagent set, BD). Finally, the reaction was stopped by the addition of a stop solution (0.5 M hydrochloric acid). Optical density at 450 nm was measured using a microplate reader (Victor 3, PerkinElmer).

Levels of NOD IAA were measured with a mouse high specificity/sensitivity competitive IAA assay in an enzyme-linked immunosorbent assay (ELISA) format and sera from NOD mice. In brief, high-binding 96-well plates (Costar) were coated with human recombinant insulin (100 U/ml; Humulin; Lilly) overnight at 4 °C. Unspecific blocking was performed with PBS containing 2% BSA for 2 h at room temperature. Preincubated NOD sera (diluted 1:10) with or without insulin competition were added and incubated for 2 h at room temperature. After four wash steps, biotinylated antimouse IgG1 (Abcam), diluted 1:10,000 in PBS/BSA was added for 30 min at room temperature. After washing horseradish peroxidase-labeled streptavidin was added for 15 min. After five additional washing steps, TMB substrate solution was added (OptEIA reagent set; BD). All samples were measured in duplicates with and without competition using human insulin.
In a second approach, to determine levels of IAA in NOD mice, a Protein A/G radiobinding assay based on ¹²⁵I-labeled recombinant human insulin, was applied as previously described^{59 (link)}. Serum from C57Bl/6 mice was used as negative control.