Ap substrate p nitrophenyl phosphate

To quantify antigen-specific antibodies in the mouse samples by ELISA, 96-well plates (Costar, Corning Inc.) were coated with the purified antigens (5 μg/ml; purified Hla was purchased from Sigma-Aldrich). Mouse serum was diluted 1:200 in PBS and added to the antigen-containing wells. Detection of antigen-specific IgG was performed using alkaline phosphatase (AP)–conjugated goat anti-mouse IgG (1:5000; AffiniPure, Jackson ImmunoResearch) and AP substrate p-nitrophenyl phosphate (Sigma-Aldrich) following the manufacturer’s recommendations. Absorbance was measured using a GENios spectrophotometer (Tecan). For the human samples, serum was also diluted 1:200, and the samples were processed in the same way as the mouse samples, with the secondary antibody being goat anti-human IgG. To control for plate-to-plate variability in the human studies, a set of standards was included on each plate, and the sample values were corrected on the basis of the values of the standards.