Nunc maxisorp high protein binding capacity 96 well elisa plates

Nunc MaxiSorp high protein-binding capacity 96-well ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with gangliosides GD2, GM2, GD1b, and GD3 at a concentration of 0.25 μg in 100 μL of 96% ethanol per well. GD2, GD1b, and GD3 were purchased from Sigma-Aldrich, and GM2 was obtained according to our previous work [35 (link)]. Following air drying, plate wells were blocked with 100 μL 2% BSA in PBS supplemented with 0.1% Tween-20 (PBS-T) per well for 2 h at RT. ScFv fragments, minibodies, or FDCs (100 μL solution in PBS-T per well) were added in triplicates at different concentrations, and incubation was carried out for 1.5 h. After washing with PBS-T, anti-FLAG HRP-labeled antibodies were added to the wells with scFv fragments, and anti-human Fc-specific HRP-labeled antibodies were added to the wells with minibodies (both 1:6000; Santa Cruz Biotechnology, CA, USA). After 40 min incubation and further washing, 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) was added, and the color reaction OD was measured at 450 nm by Multiscan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Percent of cross-reactivity was calculated as the ratio of TMB color reaction OD₄₅₀ in GM2-, GD1b-, or GD3-coated wells to OD₄₅₀ in GD2-coated wells.

To detect nonimmune IgG-binding activity at the surface of intact bacteria, R. massiliae was fixed with 4% PFA for 20 min at room temperature. PFA-treated Rickettsia bacteria were then washed 3 times with 1 ml of PBS and resuspended in PBS at 7.45 × 10⁸ bacteria per ml. PFA-fixed R. massiliae at 4.5 × 10⁷ bacteria per well or BSA at 1 μg/well was then applied as a coating onto Nunc MaxiSorp high-protein-binding-capacity 96-well ELISA plates (Thermo Fisher Scientific) overnight at room temperature. The wells were then washed 3 times with PBS containing 0.05% Tween (PBS-T) and blocked with PBS-T containing 3% BSA for 2 h at 37°C. The wells were washed 4 times with PBS-T and then incubated with different concentrations of HRP-labeled rabbit IgG (0 μg/ml, 10 μg/ml, and 50 μg/ml) diluted in PBS-T for 2 h at 37°C. The wells were then washed 4 times with PBS-T, and 100 μl of 1-Strep Ultra TMB ELISA substrate (Thermo Fisher Scientific) was added to each well and incubated at room temperature for 20 min, protected from light. To stop the reaction, 100 μl of a solution of 2 M sulfuric acid was added per well. The absorbance at 450 nm was measured in a BioTek PowerWave microplate spectrophotometer. Experiments were performed in triplicate, and significance was determined by an unpaired t test using GraphPad Prism v8.0.1 (GraphPad Software, Inc., CA, USA).