Horseradish peroxide conjugated goat anti mouse igg h l

Recombinant E2 protein (5 μg/ml) was immobilized onto Maxisorp ELISA plates (Thermo Fisher) overnight in sodium bicarbonate buffer, pH 9.3. Plates were washed three times with PBS/0.05% Tween-20 and blocked with 5% BSA/PBS for 1 h at 37ºC. Anti-EEEV mAbs were diluted in 2% BSA in PBS and incubated for 1 h at room temperature. After serial washing, horseradish peroxide conjugated goat anti-mouse IgG (H+L) (1:2000 dilution), Jackson ImmunoResearch) was added and incubated for 1 h at room temperature. After washing, plates were developed with 3,3′−5,5′ tetramethylbenzidine substrate (Dako), the reaction was stopped with 2 N H₂SO₄ and absorbance was read at 450 nm with a TriStar Microplate Reader (Berthold). For virus capture ELISA, ultracentrifuged SINV-EEEV virions were immobilized directly onto Maxisorp ELISA plates for 1 h at room temperature. Virus ELISA were performed similarly as above but Tween-20 detergent was omitted from the wash buffer.

Maxisorp ELISA plates were coated with anti-CHIKV mAb 4N12 (Smith et al., 2015 (link)) (2 μg/ml) overnight in sodium bicarbonate buffer, pH 9.3. Plates were washed four times with PBS and blocked with 4% BSA for 1 h at 25°C. CHIKV VLPs were diluted to 1 μg/ml in 2% BSA and added for 1 h at 25°C. Mxra8-Fc proteins were diluted in 2% BSA and incubated for 1 h at 25°C. Plates were washed with PBS and incubated with horseradish peroxide conjugated goat anti-mouse IgG (H + L) (1:2000 dilution, Jackson ImmunoResearch) for 1 h at 25°C. After washing, plates were developed with 3,3’-5,5’ tetramethylbenzidine substrate (Thermo Fisher) and 2N H₂SO₄. Plates were read at 450 nM using a TriStar Microplate Reader (Berthold). Anti-Mxra8 mAb ELISAs were performed by coating Maxisorp ELISA plates with Mxra8-Fc proteins (2 μg/ml) overnight in sodium bicarbonate buffer, pH 9.3. Plates were washed four times with PBS and 0.05% Tween-20 and blocked with 4% BSA for 1 h at 25°C. Anti-Mxra8 mAbs were diluted in 2% BSA and added for 1 h at 25°C. Plates were washed with PBS and 0.05% Tween-20 and incubated with horseradish peroxidase conjugated goat anti-Armenian hamster IgG (H + L) (1:2000 dilution, Jackson ImmunoResearch) for 1 h at 25°C. After washing, plates were developed and read as described above.