Monoclonal anti dnp ige

To measure β-hexosaminidase release, RBL-2H3 cells were trypsinized and centrifuged, before resuspension in DMEM-FCS and stimulated with monoclonal anti-DNP IgE (0.5 μg/ml; Sigma-Aldrich) overnight. The cells were washed with Siraganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl₂, 25 mM PIPES, 40 mM NaOH, 1 mM CaCl₂, and 0.1% BSA) and then treated with or without ST at the indicated concentrations. After incubation for 10 min at 37°C, the cells were challenged with DNP-BSA (100 ng/ml; Sigma-Aldrich) for 45 min at 37°C and then put on ice for 10 min. Culture supernatants were incubated with 1 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide (p-NAG; Sigma-Aldrich) for one hr at 37°C and then 0.1 N Na2CO3/NaHCO3 (pH 10.0) was added to stop the reaction. Optical density at 405 nm was measured. The inhibition of β-hexosaminidase granule release was calculated using the following equations: $\begin{array}{c}\%\text{\hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}\hspace{0.17em}inhibition}=\frac{\left(\text{Treated}-\text{Blank}-\text{Spontaneous}\right)}{\left(\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{B}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}-\mathrm{S}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{o}\mathrm{u}\mathrm{s}\right)},\end{array}$ where the Control was defined as the normal allergen-IgE response evoked without ST added; Treated was defined as the normal allergen-IgE response evoked with added ST; Blank was only ST and substrate added to the ELISA plate; and Spontaneous meant that the allergen-IgE response was not evoked and ST was not added.