Subjects

For radioallergosorbent assays, a serum was pooled from four sub jects with a history of allergic reactions to Brazil nuts that included oropharyngeal swelling and itching, facial swelling, laryngeal edema, and bronchospasm with wheezing. All four had positive skin-prick tests in response to extracts of Brazil nut and positive radioallergosorbent tests, with binding to Brazil-nut protein in the solid phase that was 9 to 38 times greater than binding with control serum from sub jects with no history of food allergies. For experiments using sodium dodecyl sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE), serum was obtained from five additional subjects who either indicated that they had similar symptoms or reported a history of avoiding all nuts. These five also had positive skin-prick tests and positive radioallergosorbent tests, with binding to Brazil-nut protein in the solid phase that was 4 to 64 times greater than binding with control serum from subjects with no history of food allergies. As a control, serum was pooled from eight subjects without food allergies. Skin-prick tests with transgenic material were performed on three subjects with histories of sensitivity to Brazil nuts and no history of sensitivity to soybeans and three subjects (one with atopy) with no history of sensitivity to Brazil nuts or soybeans. All subjects gave informed consent as stipulated by the University of Nebraska or the University of Wisconsin institutional review board or by Plasma Labs, International (Everett, Wash.).

Radioallergosorbent Assay

Transgenic and nontransgenic soybeans were obtained from Pioneer Hi-Bred International (Johnston, Iowa). Raw Brazil nuts were obtained from Open Harvest Grocery (Lincoln, Nebr.). The Brazil nuts, transgenic soybeans, and nontransgenic soybeans were defatted with acetone and ethyl ether.15 Extracts were prepared (1:10 wt/vol) with 0.01 M potassium phosphate–buffered saline, pH 7.4, containing 0.02 percent sodium azide and stirred at room temperature for two hours. The extracts were clarified by ultracentrifugation for one hour at 82,000×g. The protein content of the extracts was determined by the Lowry–Folin method.16 Brazil-nut protein in the solid phase was prepared according to the method of Adolphson et al.17 with microcrystalline cellulose and 10 mg of protein from defatted Brazil-nut extract.

For the radioallergosorbent assay,17 serial dilutions of Brazil-nut and soybean extracts were prepared with potassium phosphate–buffered saline containing 0.02 percent sodium azide as the diluent and incubated overnight with Brazil-nut protein in the solid phase and pooled serum from four subjects allergic to Brazil nuts. The solid phase was then washed and incubated overnight with antihuman IgE labeled with iodine-125 (Pharmacia and Upjohn, Diagnostics Division, Columbus, Ohio). Excess labeled antibody was removed by washing, and the radioactivity of the solid phase was measured with a sodium iodide scintillation detector. The percent inhibition of IgE binding was calculated with the use of values from samples without inhibitor protein as a measure of maximal binding. For the radioallergosorbent test,17 serum samples were allowed to react directly with the solid phase without inhibitor protein, and bound IgE was measured as described above. Scores for the radioallergosorbent test were calculated as a multiple of the score obtained with pooled serum from subjects without food allergies.

Electrophoresis and Immunoblotting

Extracts of Brazil nut, transgenic soybean, and nontransgenic soybean were prepared as described above, except that the pH of potassium phosphate–buffered saline containing 0.02 percent sodium azide was 7.2 and single soybean seeds were used for extraction. The Brazil nuts and soybeans were not defatted. The purified 2S albumin from the Brazil nut18 was diluted in potassium phosphate–buffered saline containing 0.02 percent sodium azide, pH 7.2. Proteins from these extracts and the 2S albumin were separated by SDS-PAGE with minigels with gradients of 10 to 20 percent (Bio-Rad Laboratories, Hercules, Calif.). The wells were loaded with 15 μg of protein from Brazil-nut and soybean extracts and 3.7 μg of 2S albumin. The proteins were transferred from gels to nitrocellulose by electroblotting as described by Towbin et al.19 Nitrocellulose blots were incubated with control serum or individual serum samples from nine subjects allergic to Brazil nuts. IgE binding was detected by autoradiography after incubation of the washed blots with 125I-labeled antihuman IgE.20 Alternatively, gels obtained with SDS-PAGE were stained with Coomassie blue to visualize the proteins.21 α-Lactalbumin (14.4 kd), soybean trypsin inhibitor (20.1 kd), carbonic anhydrase (30 kd), ovalbumin (43 kd), bovine serum albumin (67 kd), and rabbit-muscle phosphorylase b (94 kd) served as molecular-weight markers.

Skin-Prick Testing with Transgenic Material

Extracts for skin-prick (epicutaneous) testing were prepared from Brazil nuts, transgenic soybeans, and nontransgenic soybeans obtained from the sources described above. These materials were not defatted. They were prepared (1:10 wt/vol) with 0.01 M phosphate-buffered saline, pH 7.4, and rocked overnight at 4°C. The extracts were clarified by ultracentrifugation at 82,000×g for one hour and filtered through a series of 0.45-μm and 0.2-μm sterile filters. The filtrates were collected in sterile vials containing an equal volume of sterile glycerol, mixed, and stored at 4°C until use. Immediately before use, serial dilutions of sterile extracts (1:100 to 1:1,000,000 vol/vol) as well as a 1:50 dilution were prepared with a saline diluent (0.9 percent sodium chloride, 0.03 percent albumin, and 0.04 percent phenol; Miles Laboratories, Spokane, Wash.).

Skin-prick testing was performed according to the method of Norman.22 A histamine base solution (1.8 mg per milliliter and 50 percent glycerol wt/vol; Allermed, San Diego, Calif.) and saline diluent were used as positive and negative controls, respectively. Titration was conducted starting with the most dilute extracts (1:1,000,000) and progressing through serial dilutions until a positive response (3-mm wheal) was observed in subjects allergic to Brazil nuts. Subjects acting as negative controls underwent skin-prick testing with full-strength extracts and negative- and positive-control solutions as described above. The diameter of the wheal-and-flare response was measured 10 minutes after the skin was pricked. The subjects were asked to refrain from taking antihistamines or other medications that could interfere with skin-test responses for a minimum of 48 hours before testing.