MAYV E2 and CHIKV E2 Antibodies ELISA

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.