Advanced Technology in Food Production

ATFP: Human Consumption of Plant Foods Produced Using Genetic Engineering (GE) Technologies (2015)

Citation:

Nakajima O, Teshima R, Takagi K, Okunuki H, Sawada J. ELISA method for monitoring human serum IgE specific for Cry1Ab introduced into genetically modified corn. Regul Toxicol Pharmacol. 2007; 47(1): 90-95.

PubMed ID: 16982119

Study Design:

Non-Randomized Crossover Trial

Class:

C - Click here for explanation of classification scheme.

Quality Rating:

NEUTRAL: See Quality Criteria Checklist below.

Research Purpose:

To evaluate effects of genetic modification (GM) on potential allergenicity by testing serum samples from an allergic patient population for IgE specific for Cry1Ab introduced into genetically modified corn using the ELISA method.

Inclusion Criteria:

Patients

Serum samples positive for allergen-specific IgE, when examined using the Immuno-CAP method
Clinically exhibiting allergic symptoms
Commercially available sera were from corn-allergic patients.

Controls

Specific IgE-negative.

Corn

GM corn (MON810) and non-GM corn purchased at the same time
Both corns were grown in Ohio, US, and were closely related genetically.

Exclusion Criteria:

Not described.

Description of Study Protocol:

Recruitment

Serum samples were obtained from 36 Japanese patients with food allergies and from 10 healthy volunteers in Japan
Recruitment process was not described.

Design
Non-randomized crossover trial.

Blinding Used
Implied with measurements

Intervention

The levels of IgE specific for a recombinant protein, Cry1Ab, were determined using an improved ELISA method
A soluble from the Cry1Ab protein purified from pCold1 vector-transformed Escherichia coli pTf16/BL21 was used as the ELISA coating antigen and 1 M NaCl was used as the washing buffer to remove IgE non-specifically bound to the coated antigen
Sera from patients allergic to major food allergens were obtained, diluted 20-fold and tested
Sera from patients with corn allergy were also tested against whole extracts of non-GM and GM-corn (MON 810), using immunoblotting.

Statistical Analysis

The cut-off level for ELISA-positive was set as absorbance for a patient's serum sample greater than the value of the average plus five standard deviations of the control sera.

Data Collection Summary:

Timing of Measurements

Serum samples from Japanese patients were obtained between 2003 and 2004. Commercial serum samples were purchased in 2000.

Dependent Variables

Detection of IgE specific to Cry1Ab (by using ELISA to measure colorimetric intensity and compare with controls)
Reactivity of IgE from corn-allergic patients with non-GM and GM-corn extracts (by using Western blot to examine immunoblotting staining patterns of both corn extracts using sera from corn-allergic patients)
Pepsin digestibility of Cry1Ab (by digestibility test in simulated gastric fluid [SGF])

Independent Variables

Corn allergic patients vs. controls.

Description of Actual Data Sample:

Initial N: 54 serum samples (36 from patients with food allergies; eight commercially purchased; 10 from healthy volunteers)
Attrition (final N): As above
Age: Not reported
Ethnicity: 46 serum samples were from Japanese patients and volunteers; eight commercially available sera were from patients in the United States
Other relevant demographics: Not reported
Anthropometrics: Not reported
Location: Japan and United States.

Summary of Results:

Key Findings

Sera from 44 patients allergic to major food allergens found no identifiable IgE above background levels
ELISA method showed that all the values for Cry1Ab-specific IgE for the 44 serum samples from allergic patients were less than the average plus five standard deviations of the values from 10 healthy subjects. The distributions of the Control Group and the Patients Group were similar.
The three samples with the highest ELISA values were further examined using ELISA inhibition and almost the same amount of IgE was bound to the coating antigen. IgE specific to Cry1Ab was not found in the sera from food-allergic patients.
When sera from two patients with corn allergy were tested against extracts of non-GM and GM-corn (MON810), the immunoblotting staining patterns were similar for the two types of corn
When Cry1Ab in corn extracts from MON810 or Bt11 was examined in digestibility test, it was no longer detected after 0.5 minutes of digestion. Cry1Ab degraded very rapidly. When Cry1Ab in E. coli was tested, it also was rapidly digested, with a half-life of 0.3 minutes.

Author Conclusion:

Significant levels of IgE antibodies specific to Cry1Ab were not found in the sera of Japanese patients with food allergies
An ELISA method using soluble Cry1Ab antigen is a convenient screening method that can be used prior to confirmatory methods, such as Western blotting or ELISA inhibition tests.

Funding Source:

Government:

Ministry of Health, Labor and Welfare, Japan; Japan Science and Technology Agency

Reviewer Comments:

Purchased serum samples from patients with corn allergy and the serum sample from a Japanese patient with corn allergy were both obtained after GM corns started to sell. Because GM corns containing Cry1Ab had been consumed largely in the corn market in the US and in Japan, it is possible to assume that the patients who provided the sera had eaten GM corn containing Cry1Ab.
Monitoring the occurrence of IgE reactivity with Cry1Ab is an important component of post-marketing surveillance to assure the safety of GM food. This study is considered one of the pilot post-marketing studies for GM-food and a large number of serum samples is recommended to be examined in the future.

Quality Criteria Checklist: Primary Research
Relevance Questions
	1.	Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies)	Yes
	2.	Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about?	Yes
	3.	Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice?	Yes
	4.	Is the intervention or procedure feasible? (NA for some epidemiological studies)	Yes

Validity Questions
1.	Was the research question clearly stated?		Yes
	1.1.	Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified?	Yes
	1.2.	Was (were) the outcome(s) [dependent variable(s)] clearly indicated?	Yes
	1.3.	Were the target population and setting specified?	Yes
2.	Was the selection of study subjects/patients free from bias?		???
	2.1.	Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study?	Yes
	2.2.	Were criteria applied equally to all study groups?	Yes
	2.3.	Were health, demographics, and other characteristics of subjects described?	No
	2.4.	Were the subjects/patients a representative sample of the relevant population?	???
3.	Were study groups comparable?		???
	3.1.	Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)	Yes
	3.2.	Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?	???
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	Yes
	3.4.	If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis?	N/A
	3.5.	If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.)	N/A
	3.6.	If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")?	N/A
4.	Was method of handling withdrawals described?		Yes
	4.1.	Were follow-up methods described and the same for all groups?	Yes
	4.2.	Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.)	Yes
	4.3.	Were all enrolled subjects/patients (in the original sample) accounted for?	Yes
	4.4.	Were reasons for withdrawals similar across groups?	N/A
	4.5.	If diagnostic test, was decision to perform reference test not dependent on results of test under study?	N/A
5.	Was blinding used to prevent introduction of bias?		Yes
	5.1.	In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate?	N/A
	5.2.	Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.)	Yes
	5.3.	In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded?	N/A
	5.4.	In case control study, was case definition explicit and case ascertainment not influenced by exposure status?	N/A
	5.5.	In diagnostic study, were test results blinded to patient history and other test results?	N/A
6.	Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described?		Yes
	6.1.	In RCT or other intervention trial, were protocols described for all regimens studied?	Yes
	6.2.	In observational study, were interventions, study settings, and clinicians/provider described?	N/A
	6.3.	Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect?	Yes
	6.4.	Was the amount of exposure and, if relevant, subject/patient compliance measured?	Yes
	6.5.	Were co-interventions (e.g., ancillary treatments, other therapies) described?	N/A
	6.6.	Were extra or unplanned treatments described?	N/A
	6.7.	Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?	Yes
	6.8.	In diagnostic study, were details of test administration and replication sufficient?	N/A
7.	Were outcomes clearly defined and the measurements valid and reliable?		Yes
	7.1.	Were primary and secondary endpoints described and relevant to the question?	N/A
	7.2.	Were nutrition measures appropriate to question and outcomes of concern?	Yes
	7.3.	Was the period of follow-up long enough for important outcome(s) to occur?	N/A
	7.4.	Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures?	Yes
	7.5.	Was the measurement of effect at an appropriate level of precision?	Yes
	7.6.	Were other factors accounted for (measured) that could affect outcomes?	Yes
	7.7.	Were the measurements conducted consistently across groups?	Yes
8.	Was the statistical analysis appropriate for the study design and type of outcome indicators?		Yes
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	Yes
	8.2.	Were correct statistical tests used and assumptions of test not violated?	Yes
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	No
	8.4.	Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)?	N/A
	8.5.	Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)?	N/A
	8.6.	Was clinical significance as well as statistical significance reported?	N/A
	8.7.	If negative findings, was a power calculation reported to address type 2 error?	N/A
9.	Are conclusions supported by results with biases and limitations taken into consideration?		Yes
	9.1.	Is there a discussion of findings?	Yes
	9.2.	Are biases and study limitations identified and discussed?	Yes
10.	Is bias due to study's funding or sponsorship unlikely?		Yes
	10.1.	Were sources of funding and investigators' affiliations described?	Yes
	10.2.	Was the study free from apparent conflict of interest?	Yes