Analysis of patch content by SDS PAGE and ELISA.
Epicutaneous patches were loaded with commercial cashew protein extract
(Stallergenes Greer, Lenoir, NC) prepared in PBS 1X, NaCl 0.45 % or
phosphate buffer 0.1 M and dried as described above. Dry deposition was
solubilized in distilled water and centrifugated 5 minutes at 2000 x g.
The solutions obtained before centrifugation, as well as supernatants
and pellets were analyzed by SDS-PAGE as described above or by ELISA.
For ELISA, 96-well plates were coated with proteins recovered from
patches (5µg/mL, protein equivalent) and incubated overnight at 4 °C.
Plates were saturated with PBS 1X; Tween® 0.05%; BSA
1% for 1 hour at room temperature (RT). Plates were incubated with
serially diluted plasma collected from orally sensitized mice for 2
hours at RT, then with HRP-conjugated goat anti-mouse IgG1 or IgE
(Invitrogen) diluted 1:10000 or 1:1000, respectively, for 1 hour at RT.
HRP was revealed using TMB substrate (eBioscience), for 15 minutes at
RT. The reaction was stopped with 1 N chloride acid and the optical
density (O.D.) at 450 nm was recorded.