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Data Evaluation Record - Food Allergenicity - Sera Reactivity MRID NO: 44384405
Date: 4/8/98
Reviewed by: John L. Kough,
Ph.D., Biologist, BPPD
Secondary Reviewer: Doug Gurian-Sherman, Ph.D., Plant Pathologist,
BPPD
STUDY TYPE: Food Allergenicity - Sera Reactivity
MRID NO: 44384405
CHEMICAL NO: 006466- Bacillus thuringiensis subsp. tolworthi Cry9C toxin
TEST MATERIAL: Wild type and CBH-351 corn kernels expressing Cry9C
STUDY NO: AgrEvo Study Number Tox-97002
SPONSOR: Plant Genetic Systems (America) Inc., Des Moines, IA
TEST FACILITY: Tulane University School of Medicine, New Orleans, LA
TITLE OF REPORT: Investigation of Allergens in Wild-Type and Transgenic Corn
AUTHOR: Samuel Lehrer, Ph.D.
STUDY COMPLETED: August 19, 1997
CONCLUSION: The 21 sera samples from suspected corn-sensitive individuals all tested positive in the RAST assay by having ≥3% reactivity. The transgenic and wild-type aqueous corn extracts were not obviously different in responsiveness for individuals and a t-test of the RAST % reactivity did not reveal any significant differences. The RAST inhibition assay gave results indicating that both wild type and transgenic corn extracts gave substantial inhibition of the wild type corn RAST. Statistical analysis of the inhibition curves generated for RAST inhibition from wild type versus transgenic corn extracts did not indicate significantly different 50% inhibition values, slopes or y-intercepts. The type of extract, aqueous or alcoholic, utilized in the inhibition assays was never specified. Both the wild type and transgenic aqueous corn extracts gave higher levels of reactivity in the immunoblot assay than the alcoholic extracts. A comparison of the IgE reactions for specific corn atopic individuals indicated that there were similar reactive banding patterns in both transgenic and wild type corn. In some individuals there were a greater number of reactive bands ranging in molecular weight whereas in others there were only one or two bands, generally of lower molecular weight, which had very significant staining. There was no identification of individuals in the SDS-PAGE lanes so no correlation between the intensity of the % reactivity in RAST and the number or intensity of staining in the immunoblot assay could be made. A two-fold dilution series with a pool of 10 RAST positive corn atopic sera was tested against the wild type and transgenic corn extracts. The pattern of reactivity was very similar between the transgenic and wild type extracts with the intensity of the reaction again being higher for the aqueous versus alcoholic extracts. There were some unique bands present in either the wild type or transgenic extracts but since these bands did not show detectable effects on the serum reactivity kinetics in the RAST or RAST inhibition assays it is difficult to judge the importance of their presence.
CLASSIFICATION: SUPPLEMENTAL. This study does not address the potential for inducing food allergy from a novel protein lacking a history of dietary exposure. An additional control testing purified Cry9C protein against corn atopic sera should have been included to establish the negative reactivity background. The study does establish a baseline of corn allergen reactivity for subsequent comparisons if such an allergic response does occur over time.
STUDY DESIGN
A comparison of the reactivity of corn sensitive individuals' serum to extracts of Cry9C containing corn hybrids and their "wild-type" corn ancestors was done to establish if an alteration in the corn reactivity had occurred. This study was not done according to GLP standards.
TEST METHODS
Test Substance:
Extract of Cry9C and wild-type corn kernels was done by grinding 40 gm of corn kernels to a fine meal texture and extracting the meal in two solutions. The first extraction was done by adding a 4EC phosphate buffered saline solution (0.5M NaCl, 10µM EDTA, pH 7.8), extracting the meal for 1 hour with stirring followed by a 20,000g centrifugation and collecting the aqueous supernatant which was stored at -20ºC. The pellet was subsequently extracted with 4EC 55% isopropanol containing 0.5% 2-mercaptoethanol, 0.5% sodium acetate for 1 hour with stirring. The slurry was centrifuged at 13,000 g with the alcoholic supernatant retained and stored at -20ºC. The dry weight yields of these extracts was determined to be 3.0 to 3.7% for the aqueous extracts and 2.0 to 2.7% for the alcoholic extracts.
Test Sera:
Sera from 21 purported corn sensitive individuals were collected from three sera banks with cooperating allergists: Tulane University (3), Kaiser-Permanente, Los Angeles, CA (8) and Plasma Lab International, Everett, WA (10). The corn allergy of the individuals was determined by having any two of the following: a history of corn allergy or food allergy compatible with corn reactivity; a positive skin prick test to corn extract and/or a positive Radio Allergo Sorbent Test (RAST) that was at least three fold greater than negative control sera. No sera from people who have been confirmed by the positive double-blind placebo controlled food challenge is available at this time. Control sera was obtained from five non-atopic individuals who can tolerate corn.
Test System:
The sera were confirmed by RAST using aqueous extracts bound to paper discs. The coupling to the Whatman #50 paper discs was done by cyanogen bromide activation using 1mg dry weight of aqueous corn extract. Twenty-five µl of serum was diluted with 75µl of 0.001M PBS (pH 7.2), added to the discs, incubated overnight at room temperature and washed three times with 0.9% saline to remove unbound serum. The discs were reacted with equine anti-human IgE labeled with I125 (15,000 cpm, Sanofi Diagnostics Pasteur, Inc.) and washed three times with 0.9% saline to remove unbound equine reagent. The discs were then read in a Beckman 5500 gamma counter to determine bound I125. The assays were performed in duplicate and expressed as a percent of the added I125 with a value of =3% considered positive.
Another RAST format was used to judge the response utilizing a pooled sample of 17 RAST positive sera. These sera were tested against the bound antigen in a RAST inhibition format wherein the pooled sera are exposed to both a RAST disc with corn allergens and a 50µl sample of corn allergen solution. The corn allergen solution consisted of either wild type or transgenic corn allergen extract as described above in a dilution series (0.1µg to 1mg in 10-fold dilutions). The experiment was repeated three times. The results of the inhibition assay were analyzed by linear regression after a logit-log transformation to determine if there was a difference in the response between the two extracts.
The extracts were also assayed by an immunoblot format whereby the constituent proteins of the corn extracts were separated by SDS-PAGE, blotted and bound to a cyanogen activated nitrocellulose membrane (0.45μm pore size, BAS85, Schleicher & Schüll), then reacted with sera. The SDS-PAGE was a typical 6cm high12.5% separating gel. The samples were denatured by boiling for 5 minutes prior to loading onto the gel. The SDS-PAGE gels were transferred by semi-dry blotting. For detection the blots were reacted overnight with either individual sera or a pooled sample of 10 RAST positive sera. The sera were diluted 1:2 with TBS-Tween (100mM Tris-HCl; 100mM NaCl; 2.5mM MgCl2; 0.2l w/v NaN3; 0.05% v/v Tween 20, pH 7.4). This same buffer solution was used for dilution and washing steps throughout the immunoblot tests. After reaction with the sera and washing, the binding was visualized by the binding of a 1:1,000 dilution of an alkaline phosphatase conjugated anti-human IgE monoclonal antibody (Southern Biotechnology Associates, Birmingham, AL). The blot was then washed with freshly made assay buffer (100mM diethanolamine/HCl; 1.0mM MgCl2, pH 10.0), reacted for five minutes with a 1:20 dilution of Nitroblock7 chemiluminescence enhancer (Tropix, Bedford, MA), washed with buffer and then incubated for 5 minutes with 250μM CSPD( disodium 3-(4-methoxyspiro{dioxetane-3,2'-(5'chloro)tricyclo{3.3.1.1.3,7]decan}-4-yl)phenyl phosphate; Tropix, Bedford, MA). Excessive liquid was drained and the strips were exposed to autoradiography film for 15, 30, 60 or 120 seconds.
The strips were also visually assessed, after washing with TBS-Tween and TSB-AP (100mM Tris/HCl; 100mM NaCl, 5mM MgCl2, pH 9.5), by a colorimetric assay employing 450μM BCIP (5-bromo-4-chloro-indolyl-phosphate disodium salt, SIGMA, St. Louis, MO) and 400μM NBT (nitroblue tetrazolium chloride, SIGMA, St. Louis, MO) dissolved in TBS-AP and incubated at 37EC.
RESULTS AND DISCUSSION
The 21 sera samples from suspected corn-sensitive individuals all tested positive in the RAST assay by having =3% reactivity. The transgenic and wild-type aqueous corn extracts were not obviously different in responsiveness for individuals and a t-test of the RAST % reactivity did not reveal any significant differences. However, the % reactivity ranged from 3.91 & 4.40% to 39.39% & 39.90% for individuals against wild type and transgenic corn extracts, respectively. Ten of the sera had above 20% reactivity, a level considered substantial sensitivity.
The RAST inhibition assay was run three times and gave results indicating that both wild type and transgenic corn extracts gave substantial inhibition of the wild type corn RAST. Statistical analysis of the inhibition curves generated for RAST inhibition from wild type versus transgenic corn extracts did not indicate significantly different 50% inhibition values or y-intercepts. The first two analyses indicated that there was a significantly different slope between the transgenic and wild type corn extracts. However, a repeat RAST inhibition assay including four replicates for each data point rather than two used for the first two assays indicated that the slopes were not significantly different between the extracts. The type of extract, aqueous or alcoholic, utilized in the inhibition assays was never specified.
Both the wild type and transgenic aqueous corn extracts gave higher levels of reactivity in the immunoblot assay than the alcoholic extracts. A comparison of the IgE reactions for specific corn atopic individuals indicated that there were similar reactive banding patterns in both transgenic and wild type corn. In some individuals there were a greater number of reactive bands ranging in molecular weight whereas in others there were only one or two bands, generally of lower molecular weight, which had very significant staining. There was no identification of individuals in the SDS-PAGE lanes so no correlation between the intensity of the % reactivity in RAST and the number or intensity of staining in the immunoblot assay could be made.
A two-fold dilution series with a pool of 10 RAST positive corn atopic sera was tested against the wild type and transgenic corn extracts. The pattern of reactivity was very similar between the transgenic and wild type extracts with the intensity of the reaction again being higher for the aqueous versus alcoholic extracts. There were some unique bands present in either the wild type or transgenic extracts but since these bands did not show detectable effects on the serum reactivity kinetics in the RAST or RAST inhibition assays it is difficult to judge the importance of their presence. Overall, this study does not address the potential for inducing food allergy from a novel protein lacking a wide history of dietary exposure. The study does establish a baseline of corn allergen reactivity for subsequent comparisons if such an allergic response does occur over time.