Advanced Technology in Food Production

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

Citation:

Ladics GS, Bardina L, Cressman RF, Mattsson JL, Sampson HA. Lack of cross-reactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust mite Der p7 protein with human sera positive for Der p7-IgE. Regul Toxicol Pharmacol. 2006; 44 (2): 136-143.

PubMed ID: 16406630

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 investigate the ability of serum from dust mite allergic subjects to recognize the Cry1F protein in maize grain using an IgE sera-screening study.

Inclusion Criteria:

Dust mite allergy based on clinical histories and skin prick testing
Demonstrated Der p7-specific antibodies via immunoblot using subjects' sera.

Exclusion Criteria:

No history of dust mite allergy based on clinical histories and skin prick testing
No demonstrated Der p7-specific antibodies via immunoblot using subjects' sera.

Description of Study Protocol:

Recruitment

Not discussed; serum samples were provided by Dr. Tsai of Kuo-Tai Hospital, Tapei, Taiwan and Dr. High A Sampson of the United States.

Design

Non-randomized crossover trial. The amino acid sequence of Cry1F protein (expressed by maize line 1507) was compared to a database of known allergens using criteria established by the ILSI-IFBC and FAO/WHO Expert Consultation of Allergenicity of Foods Derived from Biotechnology. The Cry1F protein did not show significant similarity or a match of eight contiguous identical amino acids with any allergen, but a single six contiguous amino acid match was identified between Cry1F and Der p7 protein of the dust mite. To investigate whether Cry1F was cross-reactive with Der p7, sera from 10 dust mite allergic patients containing Der p7 specific IgE antibody were used to compare IgE specific binding. SDS-PAGE analyses, probing immunoblots with Cry1F monoclonal antibodies and dust mite allergic patient sera were conducted. Normal human serum was used as a negative control for immunoblots for Der p7-specific monoclonal antibody and immunoblots for detection of IgE binding to the the separated maize protein.

Blinding Used

Implied with measurements.

Intervention

Cross-reactivity with Der p7 and Cry1F in dust mite-allergic patients.

Data Collection Summary:

Timing of Measurements

Immunoblotting done with Der p7 specific monoclonal antibody and dust mite allergic patient sera completed once during screening process
Completed at one point during investigation:
- SDS analyses
- Probing immunoblots with Cry1F monoclonal antibodies
- Probing immunoblots with dust mite allergic patient sera.

Dependent Variables

IgE binding study results regarding reactivity of Cry1F and Der p7 proteins.

Independent Variables

Presence of dust mite allergy.

Description of Actual Data Sample:

Initial N: Sera of 20 subjects screened; sera of 10 subjects entered into study
Attrition (final N): N=10 subjects (gender not described)
Ethnicity: Not described; sera samples were obtained from Taiwan and United States
Location: Sera samples from Taiwan and United States.

Summary of Results:

Key Findings

No evidence of cross-reactivity was observed between Cry1F and Der p7
Amino acid sequence comparisons:
- No significant similarity as defined by the PAO/WHO was demonstrated with any of the allergens contained in the Pioneer Allergen Database
- No contiguous identical eight amino acid matches were observed between Cry1F protein and known allergens
- One single amino acid match was identified between the Cry1F protein and Der p7 protein of the dust mite.
IgE binding study with Cry1F containing maize grain and sera from dust mite-allergic patients containing IgE to the Der p7 protein:
- Sera from 10 dust mite allergic subjects had IgE antibodies that bound to Der p7, protein band in lane three
- None of these subjects had IgE antibodies recognizing Cry1F, a 61kDa protein expressed in maize
- Sufficient Cry1F protein in the maize extract to be readily identified by Cry1f-specific antibody and sufficient Der p7-specific IgE antibody in the human sera to cause an identifiable reaction with the Der p7 protein
- No evidence that dust mite-allergic individuals possess cross-reactive IgE antibodies to corn transfected with the Cry1F protein.

Author Conclusion:

This study provides in vitro IgE sera screening data, that when considered in the context of other bioinformatic data, adds further evidence arguing against the use of a six contiguous identical amino acid search to identify potential cross-reactive allergens. Cry1F is heat labile, rapidly hydrolyzed in an in vitro pepsin resistance assay, not glycosylated and not from an allergenic source. Taken together, these data indicate a lack of allergenic concern for Cry1F. The six contiguous amino acid match that was observed between the Cry1F and Der p7 proteins was also found to occur in proteins from a number of other species, including humans. IgE sera screening study results confirmed the conclusion based on the previous weight of the evidence data and assessment that Cry1F lacks allergenic potential.

Funding Source:

Other:	Funding source not described
In-Kind support reported by Industry:	Yes

Reviewer Comments:

Recruitment of patient sera was not discussed. Small number of samples. No statistical analysis were 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?		???
	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?		N/A
	3.1.	Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)	N/A
	3.2.	Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?	N/A
	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?	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?	Yes
	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?	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?	???
	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)?	No
	8.6.	Was clinical significance as well as statistical significance reported?	Yes
	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?		???
	10.1.	Were sources of funding and investigators' affiliations described?	No
	10.2.	Was the study free from apparent conflict of interest?	Yes