A3562 5ml

Antibody binding to CHIKV E2 was measured by an IgG enzyme linked immunosorbent assay (ELISA). Briefly, mice sera were diluted in Nunc Maxisorp Immuno ELISA plates coated with E2 diluted in PBS to a final concentration of 2 µg/mL and incubated at room temperature overnight. Plates were washed 6 times with PBS/0.05% Tween (PBS/T) and blocked with 300 µL with Pierce^TM protein-free (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at RT. Mice serum was added and serially diluted 3-fold down in PBS/T with 50 µL per well as final volume and incubated for 2 h at RT. Following washing 6 times with PBS/T, bound antibodies were detected following a 1 h incubation with 50 µL of alkaline phosphatase-conjugated antibodies specific for whole mouse IgG (A3562-5ML, Sigma Aldrich, St. Louis, MO, USA). Following additional 6 washes with PBS/T, development was achieved using 100 µL of 4-nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at OD405 were measured and analysed using a CLARIOstar instrument (BMG Labtech, Aylesbury, GB). Serum antibody endpoint titres were defined by an absorbance value three standard deviations greater than the average OD405 of the control.

Specific antibody binding to MAYV E2 or CHIKV E2 was measured by an IgG enzyme linked immunosorbent assay (ELISA) as previously described [24 (link),25 (link)]. Briefly, mice sera were diluted in Nunc Maxisorp Immuno ELISA plates coated with the MAYV E2 or CHIKV E2 diluted in PBS to a final concentration of 2 µg/mL and incubated at room temperature (RT) overnight. Plates were washed 6 times with PBS/0.05% Tween (PBS/T) and blocked with 300 µL with Pierce^TM protein-free (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at RT. Mouse serum was added and serially diluted 3-fold down in PBS/T with 50 µL per well as final volume and incubated for 2 h at RT. Following washing 6 times with PBS/T, bound antibodies were detected after a 1 h incubation with 50 µL of alkaline phosphatase-conjugated antibodies specific for whole mouse IgG (A3562-5ML, Sigma Aldrich, St. Louis, MO, USA). Development was achieved using 100 µL of 4-nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at OD₄₀₅ were measured and analysed using a CLARIOstar instrument (BMG Labtech, Aylesbury, UK). Serum antibody endpoint titers were defined by an absorbance value three standard deviations greater than the average OD₄₀₅ of the control.

Specific antibody binding to MAYV or CHIKV envelope proteins (E2 or E1) was measured by an IgG enzyme linked immunosorbent assay (ELISA) as previously described (33 (link)). Briefly, mice sera were diluted in Nunc Maxisorp Immuno ELISA plates coated with the MAYV or CHIKV envelope proteins (E2 or E1) diluted in PBS to a final concentration of 5 µg/mL and incubated at room temperature (RT) overnight. Plates were washed 6 times with PBS/0.05% Tween (PBS/T) and blocked with 300 µL with Pierce™ protein-free (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA, U.S.) for 2 h at RT. Mouse serum was added and serially diluted 3-fold down in PBS/T with 50 µL per well as final volume and incubated for 2 h at RT. Following washing 6 times with PBS/T, bound antibodies were detected following a 1 h incubation with 50 µL of alkaline phosphatase-conjugated antibodies specific for whole mouse IgG (A3562-5ML, Sigma Aldrich, SLM, U.S.). Following an additional 6 washes with PBS/T, development was achieved using 100 µL of 4-nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at OD405 were measured and analyzed using a CLARIOstar instrument (BMG Labtech, Aylesbury, GB). Serum antibody endpoint titers were defined by an absorbance value three standard deviations greater than the average OD₄₀₅ of the control.