Advanced Technology in Food Production

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

Citation:

Raybourne RB, Williams KM, Vogt R, Reissman DB, Winterton BS, Rubin C. Development and use of an ELISA test to detect IgE antibody to Cry9c following possible exposure to bioengineered corn. Int Arch Allergy Immunol. 2003; 132 (4): 322-328.

PubMed ID: 14707463

Study Design:

Non-Controlled Trial

Class:

D - Click here for explanation of classification scheme.

Quality Rating:

NEUTRAL: See Quality Criteria Checklist below.

Research Purpose:

To investigate the possibility of allergic reactions due to Cry9c
To determine and detect a difference in immune responsiveness to the Cry9c protein among differentially exposed groups using an ELISA method.

Inclusion Criteria:

People who self-reported allergic reactions.
Sera collected prior to the 1996 development of Starlink were used as negative controls
Included signs and symptoms consistent with systemic anaphylaxis (e.g., dizziness, weakness or loss of consciousness). Dermatological or oropharyngeal (hives, rash, pruritis, oropharyngeal tingling or swelling) and gastrointestinal symptoms (vomiting, diarrhea, abdominal cramping) were also considered provided they occurred within 12 hours of product consumption.

Exclusion Criteria:

Not described.

Description of Study Protocol:

Recruitment

Of the 28 individuals meeting the case definition, 24 completed questionnaires
A total of 10 of the 24 experienced symptoms within one hour of eating and most reported multiple symptoms
A total of 19 of the 24 were either self-treated or physician-treated for symptoms of allergic reaction
Two people were hospitalized
A total of 17 of the 24 provided serum samples for possible antibody determination. Subsequently, one additional report was discovered and serum collected, resulting in a final total of 18 sera.
Serum samples from 21 randomly selected EIS officers collected prior to the 1996 development of Starlink were used as negative controls
Other control sera were also used
- These included sera from individuals with high (3,666 IU to 11,590 IU per ml) total IgE levels (atopic sera) and sera from individuals with clinically diagnosed allergies (medical history or skin test) to cat and June Grass containing specific IgE levels of 1,100kIU per L
- These sera were provided by IBT Reference.
Clinical allergies were defined based on medical history and, in some cases, skin tests.

Design
Non-controlled trial.

Blinding Used
Implied with measurements, serum samples were collected and blind-coded by CDC personnel.

Intervention
Cry9c protein.

Statistical Analysis
Not described.

Data Collection Summary:

Timing of measurements: Blood was drawn between two days and two months following the reporting of an adverse event.
Dependent variables: IgE ELISA
Independent variables: Cry9c protein.

Description of Actual Data Sample:

Initial N

Of 51 cases:
- 28 individuals met the case definition and 24 completed questionnaires
- A total of 17 of the 24 provided serum samples for possible antibody determination. Subsequently, one additional report was discovered and serum collected, resulting in a final total of 18 sera.
For negative controls, serum samples from 21 randomly selected EIS officers collected prior to the 1996 development of Starlink were used as negative controls
Other control sera were also used, including sera from individuals with high (3,666 IU to 11,590 IU per ml) total IgE levels (atopic sera) and sera from individuals with clinically diagnosed allergies (medical history or skin test) to cat and June Grass containing specific IgE levels of 1,100kIU per L.

Attrition (Final N)
As above.

Age
Not reported.

Ethnicity
Not reported.

Other Relevant Demographics
Not reported.

Anthropometrics
Not reported.

Location
Atlanta, GA, USA.

Summary of Results:

Key Findings

SDS-PAGE analysis of r-Cry9c revealed two protein bands of approximately 66kD and 56kD MW
Both bands reacted strongly with goat antiserum to rCry9c in Western blot
The antiserum also reacted with a band of similar MW to the 66-kD band that was present in Starlink corn flour extract
No reaction was seen with an extract prepared from corn flour containing no Starlink. Duplicate blots treated with control normal goat serum were negative.
Data from this analysis indicated that tryptic peptide fragments from both bands were consistent with sequence data from Cry9c suggesting that the 56kD-band is an N-terminal truncated form of the larger molecule.
In the first two runs, a total of 17 adverse event sera were tested by ELISA for IgE directed against r-Cry9c. In addition, sera from 21 randomly selected EIS officers were included as negative controls.
Two types of positive controls were included on each ELISA plate: Sera from individuals with clinical allergy to inhalation antigens (cat and grass) or a food allergen (peanut) and a goat antiserum produced against r-Cry9c
Other controls included sera containing either high or low levels of total IgE that were used to assess the effects of high levels of total IgE on the specificity and background of the ELISA.

Author Conclusion:

While the results do not support the likely occurrence of allergic reactions to Cry9c, such reactions cannot be ruled out, nor can the possibility that sera might react with unique glycosylated epitopes of Cry9c that may be expressed in the corn plant or seed.
This study suggests that the Cry9c ELISA would likely have been able to detect IgE antibodies had they been present in serum at levels comparable to those seen in other allergic individuals. It is possible that glycosylation of Cry9c, as it is expressed in Starlink corn might affect reactivity if any IgE epitopes were to involve carbohydrate moieties, since such epitopes would not be present in bacterially expressed r-Cry9c.
The focus on IgE-specific antibodies is valid because of their demonstrated role in clinical allergy, however the presence of serum IgE alone may not correlate with clinical allergy
In addition, detection of IgG antibodies might provide evidence of exposure, but would not correlate with allergic reactions.
- The presence of specific serum IgE in this and any future study would provide participants with information that they could then use to seek further guidance from their healthcare provider to further define their personal allergy potential with respect to protein standards
- It would be useful if the producers of bioengineered products maintained sufficient stocks of the purified protein for the development of ELISA tests or other detection methods.

Funding Source:

Industry:

IBT Laboratories

Other:

University/Hospital:

University of Nebraska

Reviewer Comments:

Sample not well described
ELISA test is an acceptable method, however in some case it is difficult to detect IgE
Statistical analysis not 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?	No
3.	Were study groups comparable?		Yes
	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.)	???
	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?	N/A
	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?	No
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	N/A
	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?	No
	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