Abts 2 component microwell peroxidase substrate

MorHap and TT ELISAs were performed as described earlier [27 (link)]. HIV capture antigens (0.1 µg in 0.1 ml DPBS) were added to Immulon 2HB round bottom plates (CN54 gp70 or gp120), or to Nunc MaxiSorp flat bottom plates (CN54 gp140, V3) (Thermo Fisher Scientific), followed by incubation at 4°C, overnight. Unbound antigen was removed and 200 µl of DPBS (pH 7.4) with 0.5% milk-0.1% Tween 20 (blocking buffer) was added at RT overnight. Serum samples were diluted in blocking buffer starting at 1:400 in serial 2-fold dilutions. One hundred µl of each sample was added to the plate in triplicate and the plate was incubated at RT for 1 h; washed 4 times with DPBS/0.5% Tween 20. HRP-conjugated anti-mouse secondary IgG (0.1 µg in 0.1 ml blocking buffer) was added and the plates were incubated for 1 h, followed by washing. Substrate (ABTS^® 2-Component Microwell Peroxidase Substrate, Seracare, Gaithersburg, MD, USA) (0.1 ml/well) was added and the plate was incubated for 1 h. Color development was stopped by adding 100 µl/well of 1% sodium dodecyl sulfate. Absorbance was read at 405 nm on a Spectramax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). End point titer was defined as the dilution at which the absorbance was twice the background.