3 3 5 5 tetramethylbenzidine dihydrochloride

Serum hAAT expression was analysed with a standard sandwich ELISA. Wells were coated with an anti-hAAT antibody (DiaSorin, Stillwater, Minnesota, USA) in a dilution of 1∶1000 for 1 h at 37°C. Blocking was performed with 5% dry milk powder in TBS-Tween 2 for 1 h at room temperature. The serum samples were incubated in 5% dry milk powder in TBS-Tween20 in a dilution of 1∶1×10² to 1∶1×10⁶, with an incubation time of 2 h at room temperature. An antigen-specific indicator antibody (Research Diagnostics, Inc., Flanders, New Jersey, USA) linked to horseradish peroxidase was used to determine the bound antigen. After applying the substrate 3,3′,5,5′-tetramethylbenzidine-dihydrochloride (Sigma-Aldrich, Steinheim, Germany) and termination of the substrate reaction with sulphuric acid, the absorbance was measured in a fluorescent plate reader at a wavelength of 450 nm. The absorbance values were converted to µg/ml by comparison with a standard curve made from human serum.

Ab titers were assessed using a CHIKV infectious cell lysate-based enzyme-linked immunosorbent assay (ELISA). Briefly, CHIKV-infected lysate was generated by infection of primary human fibroblasts and used to coat 96 well Immulon 2 HB plates (Thermo Labsystems, Franklin, MA), with uninfected lysate used as control. Plates were blocked with PBS-0.05% Tween-20 + 5% dry nonfat milk. Serum was diluted 1:50 in the same blocking buffer, incubated for 1h at 22°C, incubated with horseradish peroxidase-labeled goat anti-mouse IgG (KPL, Gaithersburg, MD) or anti-IgM, IgG1, IgG2b or IgG2c (Southern Biotech, Birmingham, AL) and developed with 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (Sigma-Aldrich, St. Louis, MO). Reaction was terminated with 1M H₂SO₄ and absorbance measured at 450 nm. Plaque reduction neutralization test assay was done on Vero cells and neutralization titers were determined as the serum dilution with a 90% reduction in plaques (NT₉₀) compared to wells infected with CHIKV in the absence of serum.