IgG Food Allergy Testing by ELISA/EIA
What Do They Really Tell Us?
by Sheryl B. Miller, MT (ASCP), PhD
Clinical Laboratory Director
Bastyr University Natural Health Clinic
Adverse reactions to food may initiate
a myriad of physiological effects in the body. These reactions
may be immunologically or non-immunologically mediated and
can result in signs and symptoms ranging in severity from
mild to life threatening anaphylaxis.1-9
Although the majority of severe reactions are thought to be
immunological and mediated via IgE, other immune globulins,
such as IgG and IgA, may play a role in adverse reactions
to food as well.2,10,11
The clinical laboratory has historically
played an important role in the diagnosis and management of
patients with allergy. This role has been more clearly defined
with the diagnosis of IgE mediated adverse reactions and less
well defined with the diagnosis of other immunologic etiologies
or adverse reactions of non-immunologic origin. Diagnosis
of food allergy, in particular, has classically involved the
detection of IgE antibodies with a variety of different methodologies.12-17
Of late, a number of clinical laboratories
have set up ELISA/EIA (Enzyme Immunoassays) panels to test
the presence of IgG antibodies in patients to numerous food
allergens. This is based on the findings that certain subclasses
of IgG have been associated with the in vitro degranulation
of basophils and mast cells, the activation of the complement
cascade, (both of which are important mechanisms in allergy
and anaphylaxis) and the observation that high circulating
serum concentrations of some IgG subtypes have been measured
in certain atopic individuals.18-23
The premise behind this testing is that high circulating
levels of IgG antibodies are correlated with clinical food
allergy signs and symptoms. These tests, one might extrapolate,
would help the physician pinpoint food allergies in their
patients so that patients might avoid these foods and their
associated signs and symptoms. The ELISA/EIA test itself involves
coating a 96 well plate with food antigens, adding a patient's
sera and looking for a classic antigen/antibody interaction.
In addition to the IgG antibody detected in most of the newer
commercial assays, some companies also detect IgE.
Food allergy panels have found an increasing
popularity among physicians who are looking for a reliable
method to aid in the diagnosis of an otherwise difficult diagnostic
problem. Up until now, the only methods for the detection
of food allergy included skin tests, elimination and challenge
diets, or double blind placebo controlled oral food challenges.
Skin tests, although fairly reliable for the detection of
IgE to environmental allergens, are not well correlated with
food allergy signs and symptoms.24-27
Placebo controlled food challenges and elimination/challenge
diets are extremely time consuming for the patient and practitioner
and elimination/challenge diets require a high degree of patient
motivation and compliance.
The detection of food allergies with the
use of food allergy panels, in contrast to the previously
mentioned methodologies, is easy and convenient for both patient
and physician. One need only submit a blood sample from the
patient and the laboratory returns not only the foods the
patient is allergic to but a rotation or elimination diet
for the patient. The cost is moderate to high, running on
average between $100 and $400 per panel.
The use of these food allergy panels for
the diagnosis and management of food allergies, however, is
fraught with problems. These problems include reliability
in testing, an arguable theory behind the testing and the
prevalence of treatments (food rotations or other diets) prescribed
by these testing laboratories based solely on laboratory test
results. This article will address these problems and others.
Reliability in Testing
From a laboratory point of view, there
are two essential components of any laboratory test. One is
the validity of a test. In other words, its correlation to
a disease state or condition. In laboratory statistics, this
is closely related to the positive predictive value (PPV)
of a laboratory test. This will be discussed later in the
article. Before the validity of a laboratory test can be assessed,
however, the reproducibility or reliability of the test must
be evaluated and confirmed. In the world of laboratory testing,
if a test is not reproducible, it is considered worthless.
The validity of a test or its correlation with disease states
is irrelevant if a test is not reliable.
Almost all laboratories do in-house reproducibility
checks. The majority of good laboratories not only do in-house
checks but submit to unknown reproducibility checks via testing
agencies like the CAP (College of American Pathologists).
Another option for outside reliability testing, when CAP is
not available, is for the testing laboratory to have physicians
regularly send in patient split samples (with the cost assumed
by the testing laboratory). When a sample is split, acceptable
variance between the two specimens is 10% or less, according
to universal laboratory standards If more than two split samples
are evaluated, there should not be more than 20% variance
between the high and low end values. The participation of
laboratories in outside reproducibility checks, however, is
voluntary. It remains the responsibility of the physician
using a particular laboratory to check if their laboratory
does reproducibility testing and if so, what type they do.
As part of our ongoing effort to investigate
and evaluate all laboratory tests done in-house and sent-out,
we at Bastyr University Natural Health Clinic Laboratory have
recently investigated the reproducibility of food allergy
testing panels from the three different laboratories we routinely
send samples to. These investigations are part of our normal
quality control of laboratories. The testing recently involved
sending six specimens apiece (drawn all from the same patient
at the same time) to the three labs. Three specimens were
sent at the time of the draw and three specimens were sent
frozen (according to outside laboratory processing guidelines)
a week later. Although all specimens were from the same patient,
all specimens were given different names.
Two of the three laboratories (Lab A and
Lab B) to which we send our specimens report numerical values
and interpretations for these values. High numerical values
represent high circulating levels of IgG (according to the
laboratory) and are associated with foods that should be avoided.
Low values represent lower circulating levels and are associated
with foods that may be eaten. The third laboratory (Lab C)
reports semi-quantitative numerical values (1+,2+, etc.) but
interprets all positives the same. In other words, all foods
that give even the slightest reaction (1+) should be avoided,
according to this laboratory.
Two laboratories (Lab A and B) had numerical
variances that were incredibly high. Lab A had an average
numerical variance of 73%. What that means is for any one
food (eg. American cheese), there was an average of 73% between
the high and low numerical values. Lab B had an average numerical
variance of 49%. The numerical variances, however, mean very
little to the average physician. What most doctors care about
is the interpretations. Therefore, we examined the interpretations
(clinical recommendations) from the labs as well. Lab A had
a 59% average variance in clinical interpretation. What that
means is that for any one food, the recommendations to eat
or not eat were contradicted in 59% of the foods tested in
at least two of the six samples. Lab B had an acceptable clinical
variance of 7%. Only in 7% of the foods tested were clinical
interpretations contradicted. Of special note is that Lab
A, upon learning of the results of our split samples requested
to be tested again. We complied several weeks later with three
split samples (drawn from the same patient at the same time
and sent to the lab immediately). This time there was a clinical
variance of 46%, but with only three samples!
Lab C had more reasonable variances in
its testing results. There was only an average 9% numerical
variance between all the samples. This correlated to a 9%
clinical variance because all positives by this lab were considered
significant. Both of the variances from Lab C, numerical and
clinical interpretation, were well within accepted laboratory
In conclusion, two of three labs tested
had numerical variances outside acceptable laboratory standards
and are not considered reliable. In addition, one of these
labs had clinical interpretations outside these limits as
well. It is important to note that these results have no relation
at all to the accuracy of this testing or the closeness to
the real value. Accuracy is impossible to measure for food
allergy IgG ELISA/EIA because there is no acceptable gold
standard in food allergy testing to measure this against.
This leads us to the question of validity of food allergy
testing via IgG ELISAs.
Theory Behind Testing
Second to reliability is validity when it comes to evaluating
laboratory testing. Part of the validity evaluation is to
either compare a new test to currently accepted gold standards
for the particular substance being measured or to initiate
studies that show the positive predictive value (PPV) of the
new test. In other words, what percentage of the population
with an abnormal or positive test will have a particular disease/condition/set
of defined signs and symptoms? A simple mathematical formula
exists for PPV that takes into consideration the true positives
(those correctly classified with a positive test) and false
positives (those incorrectly classified with a positive test).
This PPV is of extreme importance when no gold standard exists
for a newly measured substance like IgG for food allergy.
At this time, after extensive literature
searches and interviews with various companies offering this
test, we at Bastyr are unaware of any peer-reviewed published
study examining the positive predictive values of IgG for
the diagnosis of food allergy or the association of this test
with food allergy signs and symptoms. Only one company, (in
Florida) of all we interviewed, reports that a study examining
correlation of food IgG levels and elimination diets is currently
underway (n=50). Therefore, with regard to high serum levels
of IgG and the aforementioned in vitro work on basophils,
mast cells and complement, it is a large extrapolation that
IgG to food antigens is correlated to signs and symptoms of
food allergy. Furthermore, the clinical meaning of elevated
IgG levels in atopic individuals has caused much debate of
late, including the premise of IgG as a blocking antibody.1,28,29
What is Really Being Measured in the ELISA/EIA?
In addition to the lack of documented
correlation between IgG and food allergy, it is uncertain
if numerous companies doing this assay are even measuring
what they think they are. Upon interviewing the companies
that we send our patient samples to, we learned that all of
these companies do their own in-house ELISAs/EIA. What that
means is they designed their own EIA/ELISA tests from scratch.
The questions that arise concerning in-house ELISAs is how
and where the companies obtained the food antigens that coat
the 96 well ELISA plates. In other words, what are the circulating
antibodies in patient sera binding to?
One of the labs that we evaluated claimed
proprietary information as to the manufacture of their antigens
but the other two labs both bought the food antigens for their
ELISA panels from a company in Oklahoma. Interviewing the
chief technologist from this Oklahoma company gave some surprising
insights into their food antigen preparation. The foods to
make the antigens were obtained from a local Oklahoma market
(they tried to buy organic foods whenever they could). The
foods were then chopped finely and diluted to make the antigens.
Other than several rinses with an organic solvent (acetone),
the food antigens were not purified.
The problems that may be associated with
this preparation are enormous. For one, all food (organic
and non-organic) is coated with microorganisms. The most common
of these include bacteria and fungi but viruses and parasites
may also be found on fruits, vegetables, grains, milk and
meat products. Microorganisms have many antigens that are
highly immunogenic. It is common knowledge that most people
have high circulating levels of IgG to a number of common
microorganisms. To this likely wealth of microorganisms in
the testing wells, there is the presence of possible pesticides
and organic solvents that are not (according to the technologist
interviewed) rinsed away during preparation.
Therefore, what is really being measured
in these panels? Is it an immune reaction to certain foods
or is it a person's exposure to common bacteria and fungi?
What about a person's previous exposure to pesticides and
organic solvents? Numerous studies have shown high levels
of IgG to pesticides and organic solvents in persons with
high exposure rates. It is possible that there are many antigens
in each well. If that is true, then one would see a high number
of non-specific antigen/antibody interactions, giving a high
number of false positives in these tests.
Are there a high amount of nonspecific
binding and false positives occurring in these tests? There
is no way to test this easily, at the present time. However,
what was seen in our small study correlates with this hypothesis.
The patient whose blood was drawn for our reproducibility
studies is in very good health with no current signs and symptoms
of food allergy. This person, however, tested reactive in
76% of Lab A's test (73 positive/96 foods), in 29% (28 positive/95
foods) of Lab B's test, and reactive in 22% (22 positive/102
foods) of Lab C's test.
Last, but certainly not least, of the
problems associated with food allergy testing are the therapeutic
elimination or rotation diets that are sent back with the
test results from most of the laboratories performing IgG
food allergy testing. Although these diets are usually sent
to the physician ordering the test, they may be sent directly
to patients by certain labs via physician requests. There
are several problems with this practice. Included in these
problems are the distribution of therapeutics by a laboratory,
the prescription of therapeutics based solely on the basis
of laboratory testing and the possibility that therapy recommendations
are based on a lab test that may not be correct.
The first of these problems mentioned
above is that laboratories do not have a license to practice
medicine by prescribing treatments or therapeutics. Licensed
laboratories have the right to perform quality laboratory
testing and to provide consultation on interpretation of these
lab tests to physicians when necessary. This stops short of
prescribing therapeutics. This also applies to NDs or MDs
working for the testing laboratory. Although the laboratories
that perform food allergy testing may argue that diets are
not treatments, one may vehemently disagree with this due
to the fact that most Naturopaths and some Allopaths use dietary
changes (including elimination or rotation diets) as a major
constituent of many treatment plans. These treatment plans
are made by the doctor, often in consult with a qualified
nutritionist, after very thorough histories and physical exams
are performed with the patients. This brings me to the second
At Bastyr University, a very important
part of the ND student's clinical education is the emphasis
on the history of the patient. Medical students are taught
that the majority of diagnoses can be made from listening
to patients and asking the right questions. Another major
constituent of diagnosis is a complete physical exam of the
patient. Laboratory testing is taught to be used only as required.
That is, to confirm or rule out a diagnosis. An important
guideline taught by our chief medical officer about laboratory
testing is: If the results of a laboratory test may change
the way you treat a patient, then it is valid to order. If
not, then don't order it and waste your patient's time and
money. In this author's opinion, prescribing therapy based
solely on the results of laboratory testing is as far away
from holistic medical practice as one can get.
Another problem to consider in the practice
of therapy based solely on lab tests is what if the test is
incorrect? Although it is unlikely serious harm will come
to patients if they avoid certain foods, one has to consider
the effort, anguish, time, and cost involved in removing many
foods from a diet that may not be causing harm to patients.
In addition, there is the potentiality of promoting nutritional
deficiencies if certain food groups are removed from the diet
for long periods of time. There is also the possibility that
an allergic food may be missed from an inaccurate test. This,
however, is less likely due to the extremely high number of
foods an average person is allergic to in the typical test
reports we have received. An additional point is the cost
to the patient of a laboratory test that is neither reliable
nor valid. If a test is not reliable or valid, this cost is
excessive no matter how much it is.
In conclusion, food allergy testing by
IgG ELISA/EIA panels is a convenient and easy way to diagnose
food allergies in a patient. It is, however, a testing method
that is questionable in both its theory and validity. It is
also costly and may not be reliable, depending on which laboratory
An argument in its favor by certain physicians
is that it is extremely popular with patients because it gives
printed proof to the patient that the patient is allergic
to certain foods. This makes it easier for the doctor to convince
the patient that they need to change their diet. Is this printed
proof however, a very costly substitute for discussion with
and education of patients? Would patients insist on this test
if they knew they may not be reliable?
After preliminary investigation of food
allergy testing panels offered by three different laboratories,
it is this author's suggestion that physicians give serious
consideration to the aforementioned issues before ordering
these panels for the diagnosis and management of patients
with food allergies. If one does order these tests, it is
highly recommended that reproducibility of these tests be
investigated. At the very least, physicians should consider
the possibility of sending split samples to their testing
lab (at the cost to the lab) on a regular basis.
Sheryl B. Miller, PhD
14500 Juanita Drive NE
Bothell, Washington 98011-4995 USA
Sheryl Miller, MT (ASCP), PhD, is an Immunologist and Associate
Professor of Basic and Medical Sciences at Bastyr Univeristy
in Bothell, Washington. She is also the Laboratory Director
of the Bastyr Natural Health Clinic Laboratory.
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