Advanced Technology in Food Production

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


Takagi K, Teshima R, Nakajima O, Okunuki H, Sawada J. Improved ELISA method for screening human antigen-specific IgE and its application for monitoring specific IgE for novel proteins in genetically modified foods. Regul Toxicol Pharmacol. 2006; 44 (2): 182-188.

PubMed ID: 16364525
Study Design:
Non-Randomized Crossover Trial
C - Click here for explanation of classification scheme.
Quality Rating:
Neutral NEUTRAL: See Quality Criteria Checklist below.
Research Purpose:
The purpose of this study was to improve enzyme-linked immunosorbent assay (ELISA) methods for screening human antigen-specific IgE and its application for monitoring specific IgE for novel proteins in genetically modified foods.
Inclusion Criteria:
  • Japanese patient positive for allergen-specific IgE in the Immuno-CAP method
  • Has clinical allergic symptoms
  • Samples collected between 1990 and 1992
  • Provided written informed consent.
Exclusion Criteria:
Not described.
Description of Study Protocol:


  • 193 serum samples collected
  • The allergic serum samples were collected either between 1990 and 1992 (Group A, 29 samples) or between 1999 and 2004 (Group B, 132 samples)
  • Some sera commercially available from patients in the United States (Plasma Lab International, Everett, WA) were also used (Group C, 23 samples)
  • The sera collected between 1990 and 1992 were used as the sera of the pre-GM food period
  • Sera from healthy volunteers (specific IgE-negative) in Japan were used as a control group (Group D, nine samples)
  • A total of 19 Group B patients (14.4%) were allergic to soybeans and two (1.5%) were allergic to corn.


Non-randomized crossover trial.

Blinding Used

Implied with measurements.


Novel proteins in genetically modified foods.

Statistical Analysis

Not described.
Data Collection Summary:

Timing of Measurements

Measurements made in all subjects.

Dependent Variables

  • ELISA was judged to be positive when the fluorescence or intensity or absorbance for a patients serum was greater than the value of the average +5SD of control sera
  • Assay plates (96) wells were coated with each protein (0.2mcg per 50mcl of 50mM sodium carbonate buffer (pH, 9.6 per well) and incubated at 4° Celsius, covered overnight
  • For fluorometric detection, the wells were incubated with rabbit antihuman IgE antibodies
  • After one hour incubation at room temperature and washing with PBS-T, a 50mcl of B-galactosidase-conjugated anti-rabbit Ig was added to each well and plates were incubated for one hour at room temperature and washed
  • Extract of GM or non-GM soybeans was prepared as previously described, each extract was loaded on a SDS-Page 2D gel and separated by electrophoresis.
Independent Variables
Novel proteins in genetically modified foods.
Description of Actual Data Sample:

Initial N

193 serum samples collected:

  • The allergic serum samples were collected either between 1990 and 1992 (Group A, 29 samples) or between 1999 and 2004 (Group B, 132 samples)
  • Some sera commercially available from patients in the United States (Plasma Lab International, Everett, WA) were also used (Group C, 23 samples)
  • The sera collected between 1990 and 1992 were used as the sera of the pre-GM food period
  • Sera from healthy volunteers (specific IgE-negative) in Japan were used as a control group (Group D, nine samples)
  • A total of 19 of the Group B patients (14.4%) were allergic to soybeans and two (1.5%) were allergic to corn.

Attrition (Final N)

As above.


Not reported.


Not reported.

Other Relevant Demographics

Not reported.


Not reported.



Summary of Results:

Key Findings

  • The data indicated that the values of absorbance or fluorescence intensity in Group B sera for Cry 9C or CP4-EPSPS was comparable to the average or less than the average +5SD of that of Group D (healthy subjects)
  • On the other hand, the values for PAT in three sera (P1, P2 and P3) of Group B were apparently higher than the Group D average
  • The fluorescence intensity was remarkably and dose-dependently decreased by the pre-incubation of the P1 serum with OVA
  • The binding to the PAT-coated wells was clearly reduced by NaCl washing in sera P1 and P2, whereas both sera P1 and P2 were still reactive with OVA coated well even after NaCl washing
  • No statistically significant differences were seen among the four groups (one-way ANOVA). All the absorbance values were within the range of the average ±5SD of Group D.
Author Conclusion:
  • The improved ELISA method might be a convenient screening method prior to the confirmatory methods such as Western blotting or ELISA-inhibition
  • When specific IgE was detected, further study is needed like basophil degranulation test, skin test or oral challenge
  • Serum IgE test is located in the first step of allergenicity evaluation.
Funding Source:
Government: Ministry of Health, Labor and Welfare and by the Cooperative System for Supporting Priority Research of the Japan Science and Technology Agency
Reviewer Comments:
  • Large number of sera samples from United States and Japan, but subjects were not well described
  • No statistical analysis described.
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? No
  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? No
  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? Yes
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? No
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) N/A
  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? Yes
  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? No
  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? Yes
  6.6. Were extra or unplanned treatments described? Yes
  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? Yes
  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? Yes
  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? No
  8.1. Were statistical analyses adequately described and the results reported appropriately? No
  8.2. Were correct statistical tests used and assumptions of test not violated? No
  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)? No
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? No
  8.6. Was clinical significance as well as statistical significance reported? No
  8.7. If negative findings, was a power calculation reported to address type 2 error? No
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