In response to the findings of the EPI-AID investigation, CDC developed a protocol (IRB approval #3019) for collecting serum from groups of people with the potential for being differentially exposed to Cry9c. Simultaneously, FDA laboratories developed a test method for detecting Cry9c-specific IgE antibodies in serum. The goal of this research was to determine if we could detect a difference in immune responsiveness to the Cry9c protein among differentially exposed groups using an ELISA method. Results of the serum test would indicate if individuals had specific IgE antibodies to Cry9c, suggestive of hypersensitivity.
For this study, CDC secured serum samples from three distinct groups of people: people who reported the adverse health effects, people identified as being highly sensitive to a variety of allergens, and people who had historically banked serum samples collected before Cry9c entered the food supply. CDC sent these as coded serum samples without any personal identifiers or category identifiers to FDA for analysis. After consultation with scientists and physicians at other federal agencies, CDC decided to request that serum specimens be tested only for IgE reactivity with Cry9c. Since IgE is the only type of antibody that causes immediate hypersensitivity in humans, any other antibody reactivity (IgG or IgA) would be irrelevant to the immediate-type allergic reactions specified in the case definition. All serum specimens in this study were tested using the Cry9c-specific-IgE-ELISA method (Cry9c-ELISA) that FDA developed.
SERUM GROUP A
This group of 17 samples was from the people who provided serum as part of EPI-AID #2001-13, described above. These people had contacted FDA to report an adverse health event after consuming food products containing corn and met the case definition set by CDC to be included in this study.
SERUM GROUP B
This group of serum samples was from six people with documented multiple allergies. These people are referred to as “atopic” because they have high levels of IgE circulating in their serum. An atopic person is more likely than the average person to have an immune response if exposed to an allergenic ingredient in food. Investigations by both the USDA and FDA revealed that Cry9c was widely dispersed in corn supplies in small amounts. Serum from Group B was used to assess for reactivity against Cry9c. A positive test result among serum samples in Group B might suggest that either the Cry9c protein was allergenic or that there was nonspecific IgE binding in this new assay (a lack of specificity).
SERUM GROUP C
Group C consisted of 21 banked serum samples drawn prior to1996 (before StarLink™ corn was ever grown) from Epidemic Intelligence Service Officers at CDC. Serum from Group C was used to verify the absence of cross-reactivity with proteins that were already in the food supply before Cry9c was released. This group was determined, a priori, to be the comparison group (”negative control”) for Group A (“cases”).
To avoid bias in the laboratory analysis, CDC sent serum samples from Groups A, B, and C to FDA as a batch (except for 1 case and 1 atopic control which were late arrivals to CDC), and labeled the specimens with just a simple code number. CDC sent a second batch to FDA containing the last case and atopic control serum specimens, along with replicas of cases previously sent to avoid analytical bias. FDA tested the batches on separate days using the new Cry9c-ELISA (see Appendix B, FDA Procedure for Detection of Antibodies to Cry9c). All specimens in the first batch were tested at the same time within the same analytical run on two different days, along with internal controls. The same was done for the second batch. Internal controls included a reagent blank (wells to which sample diluent without serum was added) and goat serum both from normal goats (nonimmunized) and from goats that were purposefully sensitized against the Cry9c protein (“positive control” for the assay). Wells coated with cat, grass, or peanut allergen were also used to ensure performance of the assay. Serum from people known to be allergic to cat, grass, or peanuts were applied to wells coated with the respective allergen to assess if the assay was able to detect allergen-specific IgE. The results consisted of optical density values that define the level of light absorbance in a specific well. The value of a specific coded sample cannot be directly compared to a second run of that sample; all of the values in a particular run are interpreted with regard to their relation to each other and to the blank samples and the positive serum samples from sensitized goats.