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Improvements in Diagnostic Testing for Food Allergies

By Gary DosSantos, M.Sc., Geoffrey M. Gersuk, Ph.D., and Stephen Markus, M.D.

Edited by Vivian H. Gersuk, Ph.D.

Introduction

The foods we eat have changed significantly over the past 100 years. Modern diets contain greater numbers of spices and exotic foods, increasing numbers of preservatives and additives, and foods that have been modified by elaborate processing techniques.

Concurrent with these changes in diet, there has been an increase in the incidence of eczema, irritable bowel syndrome, migraine headaches, hives, rhinitis, asthma, arthritis, and general aches and pains. Allergies have been shown to play a role in these conditions. It has been suggested that food allergies, brought on by increasingly diverse and immunogenic (immune system activating) diets, may be responsible for many of the chronic complaints that are seen today.

Food allergy, which was first reported in 1936, is an abnormal reaction by an individual’s immune system to what are ordinarily harmless foods [1] . Allergens from food proteins cause the body to produce antibodies. When the food or protein is eaten again, the body may react by producing large quantities of antibodies. Reactions to foods can occur immediately (Type I hypersensitivity) or up to a few days later (Type III hypersensitivity). Type I hypersensitivity is usually associated with IgE production, which stimulates the release of histamines, among other chemical messengers. Type III hypersensitivity is mediated by IgG production [2] . Food-specific IgG’s attach to food allergens, forming immune complexes; if not cleared by the body, these complexes can deposit at various sites in the body, where they become a source of inflammation or other irritation.

Several clinical laboratories have set up immunoglobulin assay panels consisting of many of today’s common foods and food additives [3] [4] . Concerns about the accuracy, reproducibility, and interpretation of these tests have been raised. An evaluation of laboratories conducting these tests has found that the average variance in clinical interpretation was as high as 73% or as low as 9%, depending on the laboratory conducting the testing [5] . Another report from Britain obtained similar results [6] .

We report here significant improvements in accuracy, reproducibility, sensitivity, and specificity using the solid-phase enzyme-linked immunosorbent assay (ELISA). Other methods of testing for food allergies have been discussed elsewhere [7] .

Test Overview

Food allergy testing typically consists of panels that detect circulating antibodies (most often of the IgG and IgE classes) against foods. These tests are based on scientific reports that IgE and certain classes of IgG, IgD, and IgA may participate in food intolerance [8] [9] [10] [11] [12] [13] .

In ELISA testing for food intolerance, food extracts are coated onto a multiwell polystyrene plate. Each well is coated with a different food extract. A patient’s serum is then added to the plate. If food-specific antibodies are present in a patient’s serum, they adhere to the proteins that are coated on the respective wells. A color developer is added, and the amount of bound antibodies can then be estimated by measuring the amount of color in each well. In this way, patients with elevated antibodies against specific foods can be identified, and a diet can be created to eliminate these foods.

Most laboratories performing food allergy tests identify the presence of IgE- and IgG-type antibodies. Foods that stimulate elevated IgE responses are responsible for some of the most serious clinical problems and should be avoided completely, whereas foods that stimulate IgG production may be eliminated temporarily in order to evaluate patient response [14] . Although IgE is normally associated with the allergic response, it has been suggested that up to 90% of food allergies are IgG-mediated [15] . For this reason, it is important to test for the presence of both of these antibody types.

A significant quality-control issue associated with the ELISA technique is the problem of “hot wells”: at some point during manufacturing or sample processing, defects sometimes occur in the plastic surface which can lead to false positives. Since these defects are random, normal batch production quality control methods fail to detect them.

To reduce the number of false positives and the possibility of reporting inaccurate results, a few labs run tests in duplicate and then average the numbers to get a final result. To ensure accuracy of the results, a statistical evaluation of the paired results is carried out to ensure that they fall within 20% of each other. Paired results that do not fall within 20% of each other are usually repeated if the difference would lead to different interpretations of the data.

Another process that has led to improved reliability of these tests is in the use of robotics in the lab. Robotics can dramatically improve accuracy, reproducibility, and reduce the incidence of technician error. The improved efficiency associated with the use of robotics also significantly reduces the cost of performing assays. One robot can perform the work of up to seven technicians, freeing them to perform other tasks in the laboratory.

To keep batch-to-batch variability low, reagents used in the preparation of plates should go through a final quantitative assay to verify that the concentration of each reagent being used is consistent between manufacturing runs.

At US BioTek, we routinely run low and high controls in each run for quality assurance (Figure 1). We also frequently split samples and test for reproducibility. Practitioners who wish to evaluate the reliability of a testing laboratory may wish to arrange for a split sample to be submitted. We also subscribe to an outside quality assurance program, which is provided by one of several proficiency testing organizations. These tests are used quarterly to verify our test procedures and to monitor our in-house quality control efforts. Finally, as is recommended for all laboratories conducting these types of tests, we have been issued a Clinical Laboratory Improvement Amendment (CLIA) number, which indicates that our facility is being routinely inspected.

In performing food allergy testing, there are two approaches that can be taken. The first is to assume that all of the possible antigens that can cause immune responses are known. From there, highly purified preparations of individual proteins or fragments of proteins are used to evaluate an individual’s blood serum reactivity. This approach has the advantage of being more sensitive and specific, but has the disadvantage of having a relatively small number of food proteins tested. The cost associated with this type of test tends to be significantly higher as well. This is the approach taken by many (but not all) larger commercial laboratories.

Other labs, usually those serving the alternative or complementary medical communities, try to identify foods and food groups that may be causing immune responses. These labs use partially purified food protein preparations that are similar to what a person could be expected to ingest. This approach tends to cover a greater variety of foods, and may identify individuals who react to proteins that are not generally considered to be immunogenic. Although these tests are considered to be less sensitive, they are capable of detecting antibody concentrations within the expected biological range for allergies.

One important question surrounding food allergy testing is the usefulness of IgG measurements. Since IgG antibodies to foods remain in the blood longer than IgE, it has been hypothesized that elevated IgG may be used as a predictor of allergy development. Correlations between levels of food-specific IgG, IgE, and allergies have been observed in studies of individuals with Type I (immediate onset) allergies [16] . At least one group has concluded that specific IgG and IgG4 levels should be considered in identifying offending foods [17] . Furthermore, the positive predictive value of specific IgG measurements to soybean allergies, but not milk allergies, has been reported, as has the possible role of IgG4 in anaphylaxis (severe allergic reaction) caused by soy proteins [18] [19] . The ability to predict inhalant allergy development using specific IgG measurements has also been reported [20] . A recent study involving 120 allergic and 144 non-allergic individuals found that an increased IgG antibody level to foods, especially egg white, orange, and mixtures of wheat and rice, was correlated with an increased risk of producing IgE to cat, dog, mite, egg, or milk allergens [21] . Many other reports in scientific journals point to the usefulness of IgG in food allergy testing [22] [23] [24] [25] [26] [27] [28] [29] .

How do we know that what is being measured by these tests are indeed food- specific antibodies? Careful test development and quality control has helped us to improve the specificity of these tests. At the level of test development, assays are routinely validated against a “normal” population. The results obtained for each allergen are evaluated to ensure that it yields consistent results.

Elimination of all potential food allergens followed by an oral challenge of those foods, one by one, is considered by some to be the most reliable test to identify food allergies. There are some significant difficulties in administering these tests, however, including compliance of the patient with the rigorous dietary regimen, the delayed response to allergens, and the variability of response between patients. Skin testing, which is often used to detect Type I (IgE-mediated) allergies to airborne allergens, has proven unreliable for detecting food allergies.

Naturopaths and allopaths often use dietary changes, which can include elimination and rotation diets, as part of treatment plans. Because the diet in industrialized countries is so varied, it can be a daunting task for a patient to go through an entire rotation, especially if results are not seen early on in the treatment. The identification of elevated levels of antibodies against certain food groups may help practitioners and patients to find a starting point at which to begin a rotation diet [30] .

In short, while rotation diets are a non-invasive, non-toxic way to evaluate a patient’s reactivity against many foods, food allergy testing, when performed and interpreted properly, can provide significant insights into patient complaints and can assist practitioners in determining treatment courses [31] [32] [33] [34] . By identifying foods to avoid at all times (i.e. foods to which significant quantities of IgE are present in the serum) and foods whose avoidance may help to improve a patient’s condition. (i.e. foods to which significant amounts of IgG are present), food allergy testing can provide significant benefits to practitioners and patients.

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