A total of 20% of the hydrogel solutions were supplemented with 1 µg polyclonal rabbit anti-lysozyme antibody (Sigma-Aldrich, St. Louis, MO, USA), 20 mM N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, USA), or 0.5 mM sodium salicylate (SA) (Sigma-Aldrich, USA). Loaded hydrogel solutions were incubated at 37 °C until gel formation was completed. The gel was layered with PBS, and incubated at 37 °C while shaking. PBS was collected and replaced with an equivalent amount of fresh PBS after 1, 2, 4, 6, 8, and 24 h for polyclonal rabbit lysozyme antibody, and after 1, 2, 3, 4, 5, 6, 12, and 24 h for NAC and SA.
The amount of polyclonal rabbit anti-lysozyme antibody in the collected supernatants was determined by ELISA. Plates were coated with lysozyme, and released antibodies were detected with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich, USA).
NAC concentrations were determined by assays for sulfhydryl groups. Concentrations higher than 15 nM were detected with an assay described by Grassetti and Murray [43 (link)], and concentrations below 15 nM were analyzed by an assay described by Kukoc-Modun and Radić [44 (link)].
Released SA in the collected supernatants was analyzed by a direct UV assay (TrayCell, Hellma, Müllheim, Germany).
The amount of polyclonal rabbit anti-lysozyme antibody in the collected supernatants was determined by ELISA. Plates were coated with lysozyme, and released antibodies were detected with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich, USA).
NAC concentrations were determined by assays for sulfhydryl groups. Concentrations higher than 15 nM were detected with an assay described by Grassetti and Murray [43 (link)], and concentrations below 15 nM were analyzed by an assay described by Kukoc-Modun and Radić [44 (link)].
Released SA in the collected supernatants was analyzed by a direct UV assay (TrayCell, Hellma, Müllheim, Germany).
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