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 99

using 26-gauge needles to the backs of the patients. The results were read 15 min
later. A positive reaction was defined as a mean wheal diameter of ≥3 mm. The
size of the wheal produced by each antigen or by the positive control histamine
was expressed in terms of maximum diameter and vertical length at the midpor-
tion of the maximal length. Skin reactivity was expressed as the ratio of the wheal
size induced by the allergen and that induced by histamine (A/H). The data were
recorded in accordance with the recommendations of the Standardization Com-
mittee of the Northern Society of Allergology [12]. An A/H of 0.1-1 and an
erythema diameter of <21 mm was assigned a reactivity of 1+. An A/H of 0.1-1
and erythema >21 mm was assigned a reactivity of 2+. An A/H in the range of
1-2 was recorded as a reactivity of 3+. For an A/H of 2-3, reactivity was 4+, and
for an A/H >3 reactivity was 5+. A positive responder was defined as a subject who
demonstra ted a response >2+ on the skin-prick test. This study was reviewed by
the institutional review board of Ajou University Medical Center, Suwon, Korea.
Table 1. Primer sequences of inserted PAT and NPT genes.
Preparation of Extracts from Wild-Type and GM Potatoes
Extracts were prepared from wild-type and GM potatoes using phosphate-buff-
ered saline (PBS; pH 7.5, 1:10 w/v), kept at 4°C overnight, and then centrifuged
at 12,000-15,000 rpm for 20 min. The supernatant was dialyzed (the cutoff mo-
lecular weight was 6000 Da; Spectrum Medical Industries, Houston, TX, USA)
against 4 l of PBS at 4°C for 72 h, and the resultant fluid was stored at -20°C un-
til ELISA, ELISA inhibition testing, and immunoblot analyses were carried out.
For the skin-prick test, extract was mixed with sterile glycerin at a ratio of 1:1.
To evaluate the effects of digestive enzymes, simulated gastric fluid (SGF; 471 U
pepsin/mg; Sigma Chemical Co., St. Louis, MO, USA) and simulated intestinal
fluid (SIF; pancreatin; Sigma) were preincubated with the extracts in the presence
or absence of heat.
ELISA for Specific IgE Antibodies to Potato Extracts
The presence of specific IgE antibodies to the two types of extract was determined
by ELISA, as previously described [13]. Microtiter plates (Corning, NY) were