Binding of NGF to MOG was studied by ELISA. In brief, the MOG-Fc fusion protein was
used as the capturing reagent followed by incubation with samples containing
increasing concentrations of NGF. Bound NGF was detected using a polyclonal rabbit
anti-NGF primary antibody (Sigma-Aldrich) followed with an anti–rabbit HRP
antibody (Promega). To measure the depletion of NGF by MOG-CHO, CHO, or MOG-CHO were
cultured in medium containing increasing concentrations of NGF. NGF concentrations in
supernatant were measured after 3-h incubation by a sandwich ELISA as described
previously (Ng et al., 2007 (link)). In brief,
ELISA wells were coated with an anti-NGF capture antibody (Abcam) followed by
incubation with samples. Bound NGF was detected using a biotinylated anti-NGF
antibody (Abcam) followed by incubation with streptavidin-HRP (Abcam). All ELISAs
were performed using tetramethylbenzidine (Life Technologies) as an HRP substrate.
The substrate was incubated for 2–5 min or until adequate signal was detected.
ELISA reactions were stopped with the addition of 1 M sulfuric acid.
used as the capturing reagent followed by incubation with samples containing
increasing concentrations of NGF. Bound NGF was detected using a polyclonal rabbit
anti-NGF primary antibody (Sigma-Aldrich) followed with an anti–rabbit HRP
antibody (Promega). To measure the depletion of NGF by MOG-CHO, CHO, or MOG-CHO were
cultured in medium containing increasing concentrations of NGF. NGF concentrations in
supernatant were measured after 3-h incubation by a sandwich ELISA as described
previously (Ng et al., 2007 (link)). In brief,
ELISA wells were coated with an anti-NGF capture antibody (Abcam) followed by
incubation with samples. Bound NGF was detected using a biotinylated anti-NGF
antibody (Abcam) followed by incubation with streptavidin-HRP (Abcam). All ELISAs
were performed using tetramethylbenzidine (Life Technologies) as an HRP substrate.
The substrate was incubated for 2–5 min or until adequate signal was detected.
ELISA reactions were stopped with the addition of 1 M sulfuric acid.