赵晶麻醉并发症组围术期过敏反应(CSA2012)剖析.ppt

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS) and immunoblots were performed with cassava, avocado, chestnut, banana, kiwi, and latex extracts. In SDSPAGE, the extracts were separated on 12.5% denaturing polyacrylamide gels. In immunoblot experiments, proteins were transferred to a polyvinylidene fluoride membrane (Hybond P, GE Healthcare, Uppsala, Sweden) according to the method described by Towbin et al [12]. The membrane was blocked with phosphate buffered saline (PBS) containing 0.1% Tween for 1 hour at room temperature. The patient?s serum was added to the membrane at a 1:4 dilution in blocking buffer and left under agitation overnight at 4oC. For immunoblot inhibition, the latex extract was preincubated with the patients serum for 3 hours at room temperature. The mixture was then added to the membrane onto which the cassava extract proteins had been transferred. After several washes, the membrane was incubated with horseradish-peroxidase-conjugated antihuman IgE antibody (DAKO, Barcelona, Spain) for 2 hours at room temperature. Immunodetection was performed using The cassava extract contained multiple protein bands: 96.24, 83.67, 65.52, 57.69, 48.45, 46.44, 32.85, 29.42, 26.97,19.52, 17.89, and 15.14 kd. SDSwas then performed with the avocado, chestnut, banana, kiwi, and latex extracts (Figure 1). In addition, Tricine SDSwas performed in order to show the low-molecular-weight protein bands. Immunoblotting of the cassava extract showed at least 5 IgE-binding bands of 89.75, 46.28, 26.68, 21.38, and 19.49 kd. Immunoblotting of the avocado extract revealed 7 IgE binding bands (42.44, 38.06, 24.90, 22.50, 21.00, 19.52, and 16.83 kd), the chestnut extract 6 bands (44.28, 36.37, 25.04, 21.39, 19.55, and 17.59 kd), the banana extract 3 bands (69.33, 60.48, and 30.10 kd), the kiwi extract 2 bands (33.30 and 31.50 kd), and the latex extract 2 IgE-binding bands (33.30 and 31.05 kd). Immunoblot inhibition showed 100% inhibition of the IgE-binding prote