An Ad5 neutralization assay was carried out using participant plasma obtained one week previous to sigmoidoscopy as previously described38 (link).
For ELISA, 96 well plates (Thermo Scientific) were coated overnight with 1×109 viral particles of Ad5 purified by double-banding, CsCl gradients, and dialysis, and diluted in 100μl of coating buffer (Immunochemistry Technologies). Plates were then blocked for 2h with 5% non-fat milk (Fisher), 3% Inactivated Goat Serum (Invitrogen), and 0.2% Tween-20 (Sigma) in 1× PBS (Life Tech). Explant samples were assayed undiluted and at 1:10 and 1:100 dilutions in blocking buffer, and negative controls included RPMI with 20% FBS. Plasma samples were diluted in six 1:5 dilutions starting with 1:500. Samples, standard curves, and controls were incubated for 2h at 37°C.
After washing, goat anti-human IgG-HRP (Southern Biotech) or mouse anti-human IgA-HRP (Abcam) at 1:1,000 was incubated for 1h at 37°C, then detected with addition of BM Blue substrate (Roche) for 15 min. The reaction was stopped by adding 50μl of 1N H2SO4 and read within 20min using a SpectraMax M2 ELISA Plate Reader (Molecular Devices) at 450nm absorbance. Standard curves used human IgG (Bethyl) or human IgA (Sigma), at 20.0 to 0.009ng/well, and the linear ranges were used for extrapolation of the concentrations.
For ELISA, 96 well plates (Thermo Scientific) were coated overnight with 1×109 viral particles of Ad5 purified by double-banding, CsCl gradients, and dialysis, and diluted in 100μl of coating buffer (Immunochemistry Technologies). Plates were then blocked for 2h with 5% non-fat milk (Fisher), 3% Inactivated Goat Serum (Invitrogen), and 0.2% Tween-20 (Sigma) in 1× PBS (Life Tech). Explant samples were assayed undiluted and at 1:10 and 1:100 dilutions in blocking buffer, and negative controls included RPMI with 20% FBS. Plasma samples were diluted in six 1:5 dilutions starting with 1:500. Samples, standard curves, and controls were incubated for 2h at 37°C.
After washing, goat anti-human IgG-HRP (Southern Biotech) or mouse anti-human IgA-HRP (Abcam) at 1:1,000 was incubated for 1h at 37°C, then detected with addition of BM Blue substrate (Roche) for 15 min. The reaction was stopped by adding 50μl of 1N H2SO4 and read within 20min using a SpectraMax M2 ELISA Plate Reader (Molecular Devices) at 450nm absorbance. Standard curves used human IgG (Bethyl) or human IgA (Sigma), at 20.0 to 0.009ng/well, and the linear ranges were used for extrapolation of the concentrations.