Evaluation of the Sensitization Rates and Identification of IgE-Binding Components in Wild and Genetically Modified Potatoes in Patients with Allergic Disorders - Genetic Engineering: Recent Developments in Applications - page 100

coated with 100 µ l of extract (GM or wild-type, 10 µ g/ml)/well and kept over-
night at 4°C. Each well was washed three times with 0.05% PBS-Tween (PBST),
and the remaining binding sites were blocked by incubation with 10% fetal bovine
serum (FBS)-PBS for 1 h at room temperature. The wells were then incubated for
2 h at room temperature with either 50 µ l of the patients' sera or the control sera,
both at 50% dilution. The control sera were from the 38 healthy controls who had
tested negative in the skin-prick tests to the common inhalant and food allergens
and to potatoes. The wells were washed three times with PBST, 1:1000 v/v biotin-
labeled goat anti-human IgE antibody (Vector Lab, Burlingame, CA, USA) was
added to each well, and the plates were incubated for 1 h at room temperature.
After another washing, 100 µ l of 1:1000 v/v streptavidin-peroxidase (Sigma) was
added, the plates were incubated for 30 min, and the wells were washed again.
The colorimetric reaction was developed with a TMB (3,3',5,5'-tetramethyl-
benzidine) substrate solution for 15 min at room temperature. The reaction was
stopped by the addition of 100 µl of 2 N sulfuric acid, and absorbance was read
at 450 nm using an automated microplate reader (Benchmark; Bio-Rad Labo-
ratories, Hercules, CA, USA). All assays were carried out in duplicate. Positive
cutoff values were determined from the mean plus two standard deviations of the
absorbance values of the 38 healthy controls.
eLisA inhibition test
The specificity of IgE binding to the potato extracts was tested and the allergenic
potency of the GM and wild-type potato extracts was analyzed by a competitive
ELISA inhibition test. Sera from four patients with high levels of specific IgE
binding were pooled, preincubated overnight at 4°C with five concentrations
(1-100 µ g/ml) of either Dermatophagoides pteronyssinus or extract from GM
or wild-type potatoes, and then incubated for 12 h in microtiter plates coated
with extracts from the two types of potato. The same steps were followed as in the
ELISA. As a control, samples were preincubated with equal volumes of PBS (pH
7.5) instead of house dust mite or potato extracts. The percentage of inhibition
of serum IgE binding was expressed as: 100 - (absorbance of the samples preincu-
bated with allergens/absorbance of the samples preincubated with PBS) × 100.
sds-PAGe and immunoblot Analysis
SDS-PAGE and immunoblot analysis were carried out under reducing condi-
tions according to previously described methods [13]. Extracts from the two
potatoes were mixed with sample buffer (Tris-HCl 31 mmol/l, 10% glycerol,