INTRODUCTIONMost tests for "allergy" are actually tests for

allergic sensitization or the presence of allergen-specific

immunoglobulin E (IgE). The majority of patients who experience
symptoms upon exposure to an allergen have demonstrable allergen-
specific IgE that recognizes that allergen, making these tests
essential tools in the diagnosis of allergic disorders. This topic
provides an overview of in vitro tests for IgE-mediated allergic disease.
Skin testing for allergic disease is reviewed separately.
(See "Overview of skin testing for IgE-mediated allergic disease".)
Laboratory testing in cases of suspected anaphylaxis is reviewed in
greater detail separately. (See "Laboratory tests to support the
clinical diagnosis of anaphylaxis".)
IMMUNOASSAYS FOR ALLERGEN-SPECIFIC IgEImmunoassays
for IgE specific to an allergen of interest are widely used in the
diagnosis of allergic disease. Immunoassays are based on interactions
between antigens and antigen-specific antibodies [1,2]. In allergic
disease, the relevant antigens are usually proteins derived from other
living organisms (plants, animals, fungi, insects, microorganisms).
Immunoassays are often incorrectly referred to collectively as
"radioallergosorbent tests" (RASTs) because radioactive tests were
the earliest immunoassays to be used extensively. The term
"immunoassay" is more appropriate.

Immunoassays are available for:

●Foods (see "Diagnostic evaluation of IgE-mediated food allergy",

section on 'In vitro testing')
●Insect venoms (see "Diagnosis of Hymenoptera venom allergy")
●Environmental allergens (eg, pollens, molds, animal allergens,
dust mite, and cockroach allergens)
●Natural rubber latex (see "Latex allergy: Epidemiology, clinical
manifestations, and diagnosis")
●A small number of beta-lactam drugs (see "Penicillin allergy:
Immediate reactions")
●Some occupational allergens such as isocyanates and phthalic
anhydride (see "Occupational rhinitis", section on 'Testing for
allergen-specific IgE' and "Occupational asthma: Clinical
features, evaluation, and diagnosis", section on 'Skin and
immunologic testing')
Indications — The presence of allergen-specific IgE is correctly
interpreted as evidence that the patient is sensitized to that allergen
and may react upon exposure. Thus, this type of testing is indicated
in general terms when the clinician is assessing the likelihood that a
patient's reactions or symptoms were due to exposure to a specific
allergen.
In vitro tests are occasionally indicated to confirm negative skin tests.
As an example, a patient who experienced a systemic reaction
following a Hymenoptera sting and had a negative skin test should
have in vitro IgE testing done as well to assure that the skin was not
falsely negative, as this allergy is potentially life threatening.
(See "Diagnosis of Hymenoptera venom allergy".)
Advantages relative to skin testing — In most situations, skin testing
for IgE-mediated allergy is preferred over in vitro testing because skin
testing is more rapidly obtained, less expensive, and more sensitive
[3]. However, in vitro testing has certain advantages over skin testing:
●In vitro testing poses no risk to the patient of an allergic
reaction. In vitro testing may be preferable in older adults with
cardiovascular disease, patients with suspected sensitivities to
allergens associated with severe anaphylactic reactions (such as
latex), and patients with histories of severe reactions to minute
amounts of the allergen. The risk of an allergic reaction in
response to skin testing in such patients, although small, may not
be acceptable. (See "Overview of skin testing for IgE-mediated
allergic disease", section on 'Safety'.)
●In vitro testing is not affected by medications the patient may be
taking, with the possible exception
of omalizumab (see 'Disadvantages and limitations' below).
Patients unable to discontinue antihistamines, some
antidepressants, or other medications that may confound the
results of skin testing are good candidates for in vitro testing. In
vitro tests may also be preferred in those unable to discontinue
medications that may inhibit the management of, or physiologic
response to, anaphylaxis (ie, beta blockers and angiotensin-
converting enzyme [ACE] inhibitors). (See "Overview of skin
testing for IgE-mediated allergic disease", section on 'Medications
that should be discontinued'.)
●It is not reliant upon skin integrity or affected by skin disease:
•The skin of infants less than 12 months old may not yet fully
reflect their allergic sensitivities. In contrast, immunoassays
 are valid for infants as young as six weeks of age and can be
performed on capillary blood samples.
•Individuals who have experienced an anaphylactic reaction
may have falsely negative skin test results for several weeks
after the event. In vitro testing is not known to be affected and
can be performed in the postanaphylactic period if skin testing
cannot be reasonably postponed.
•In vitro testing may be better for patients with certain skin
conditions, such as severe and widespread atopic dermatitis or
dermographism (ie, a condition in which raised welts form upon
firmly stroking the skin and skin testing with diluent alone can
produce false-positive wheals).
●In vitro testing can be more convenient for the patient because it
only requires submitting a blood sample.
●In vitro testing can be a good alternative to skin testing when
increased risk of airborne disease is present, such as during
pandemics, as skin testing requires significant direct patient
contact time [4].
●It can be superior to skin testing in certain clinical settings.
Evolving data suggest, at least for some foods, that the level of
specific IgE as measured by one specific commercial system,
Phadia ImmunoCAP, may be more predictive than skin testing for
diagnosing true clinical reactivity upon ingestion. The studies that
demonstrated this were performed in children, and generalizability
to adults has not been confirmed. (See "Diagnostic evaluation of
IgE-mediated food allergy".)
Disadvantages and limitations
●Omalizumab, a monoclonal antibody to IgE that binds to the Fc
portion of the IgE molecule, causes both total IgE and allergen-
specific IgE to rise [5]. Omalizumab can interfere with the
performance of many immunoassays, although the ImmunoCAP
system remains accurate [6]. (See "Anti-IgE therapy", section on
'Other changes with therapy'.)
●Compared with skin testing, allergen-specific IgE tests provide
information about the current production of IgE to the allergen in
question, which can vary over time and with exposure to that
allergen. The half-life of circulating allergen-specific IgE is
approximately 48 hours, after which it becomes bound to tissue
mast cells, where it remains for at least two years and can be
detected by skin testing. Therefore, skin tests detect allergen-
 specific IgE produced over a much longer period of time than
allergen-specific IgE blood tests. Skin tests will detect the
production of allergen-specific IgE that could have occurred in the
past but is no longer being produced and is thus not detectable on
allergen-specific IgE blood tests.
Technical issues
Types — There are many methodologic variations of immunoassays
that use different methods for detection of antigen-antibody
complexes. The quality of the different detection systems is
comparable:

Enzyme-linked immunosorbent assay (ELISA)

Figure 1a
●Enzyme-linked immunosorbent assays (ELISAs) – ELISAs make
use of antibodies or antigens linked to enzymes (figure 1A). A
solution containing one reactant is placed in a well in a plastic
dish because some plastics will tightly bind many types of
macromolecules. Subsequently, the plate is washed, and a tagged
reagent is added; this reagent is linked to an enzyme. After
washing again, the amount of antigen-antibody binding is
measured based on the output of an enzymatic reaction. This is a
direct binding assay. The labeled component may be either the
antigen or the antibody.
When the substrate of the enzyme is added, the reaction
generates a colored product. Variations on the basic ELISA
technique include fluorescent enzyme immunoassays (FEIAs) and
chemiluminescence immunoassays, which also make use of
antibodies linked to enzymes, although, when the substrate of the
enzyme is added, the reaction generates a fluorescent or
chemiluminescent product.
●Radioallergosorbent tests (RASTs) – RASTs involve antibodies
that are coupled to radioactive tags [7]. This method is seldom
used today, and, as mentioned previously, the term RAST, when
applied to immunoassays in general, is an anachronism.

Immunoassays can also be solid phase or liquid phase:

Solid-phase enzyme-linked immunosorbent assay (ELISA)

Figure 1b
 ●Solid phase – Solid-phase immunoassays are more commonly
used and make use of allergens (the antigen) bound to some form
of matrix, such as a plastic plate, disc, bead, or other substrate,
which is called an "immunosorbent" or "allergosorbent." Each
system has its unique attributes. The type of matrix used also
influences assay performance. Paper discs have the lowest assay
performance, and microcellulose gels perform somewhat better.
The technique of solid-phase ELISA is described and illustrated in
the figure (figure 1B). The allergen is bound to a matrix. The
patient's serum is added, which contains antibodies of various
classes (IgE, IgG, IgM, IgA). The patient's antibodies serve as the
primary antibodies. These antibodies bind to the allergen if they
recognize epitopes on the allergenic protein, and different classes
of antibody may bind. Any unbound components of the serum are
washed away, and then bound IgE is singled out from other types
of bound immunoglobulins using anti-IgE antibodies (the
secondary antibodies). The allergen:IgE complexes are detected
by a variety of methods, as described previously.
●Liquid phase – Liquid-phase immunoassays are performed with
solutions of antigens and antibodies. Liquid-phase binding to
paramagnetic particles reduces stoichiometric and
conformational issues that can arise with binding of an allergen to
a solid-phase substrate.

Immunoassays are both qualitative and quantitative. The specific IgE

molecules from the patient's serum bind to the antigen in direct
proportion to their concentration in the serum, allowing the quantity of
specific IgE to be estimated with reference to a standard curve or by
reference to a negative and positive control. The best documented and
validated systems are those reporting quantitative results in kUA/L for
IgE directed against specific allergens (or kIU/L for total IgE
measurements), using calibrators traceable to the World Health
Organization (WHO) International Reference Preparation for human
IgE. Testing systems that report values in these units are preferable to
those reporting results in other units.

Some immunoassays will be reported in nanograms of IgE per mL. The

conversion is:

1 kIU/L = 2.4 ng IgE/mL.

Competitive binding technique — Competitive binding (or inhibition)
immunoassays are another variation on the basic immunoassay [8]. In
this technique, known quantities of well-characterized antigen
(allergen) and antibody (allergen-specific IgE) are mixed together.
Then, a sample of unknown reagent, either allergen or antibody, is
added, which competes with the known components. This technique
has several applications:
●To determine the amount of allergen (or antibody) in an
uncharacterized sample – As an example, IgE specific to a certain
dust mite allergen can be fixed to a matrix, and then a solution of
labeled, purified dust mite allergen is added. To this, a test
solution containing unknown amounts of allergen is added. The
two solutions of allergen compete for the binding sites on the IgE.
The unbound allergen is then washed away, and the amount of
label detected is used to quantify the amount of allergen in the
test solution.
●To determine the extent of cross-reactivity between different
allergens (or antibodies) – As an example, IgE specific to a
certain dust mite allergen can be fixed to a matrix, and then a
solution of labeled, purified dust mite allergen is added. To this, a
test solution containing unknown amounts of allergen from a
different type of mite (eg, storage mite) is added. The two allergen
solutions compete for binding to the IgE. If the allergens from the
two species are sufficiently similar, then binding of the labeled
allergen will be inhibited.
Accuracy — The sensitivity and specificity of immunoassays vary
with the system being used and the quality of the allergen. Overall,
sensitivity ranges from 60 to 95 percent and specificity from 30 to 95
percent [9-11]. Occasionally, patients who react to an allergen may
not have any allergen-specific IgE that is detectable with routine
testing [12,13]. In comparison, skin tests (prick/puncture method)
generally have high sensitivity and specificity (>85 percent) when
standardized inhalant extracts with high potency are used.
(See "Overview of skin testing for IgE-mediated allergic
disease" and "Overview of skin testing for IgE-mediated allergic
disease", section on 'Prick/puncture method'.)

The quality of the allergen and the manner in which it is bound to the
immobilizing matrix are the most critical aspects of solid-phase
immunoassays. Systems that make use of well-characterized and
standardized allergens, with a high level of quality control and quality
assurance, provide the most reliable clinical results:

●Values of more than 90 percent sensitivity, specificity, and

predictive values have been obtained with pollens of common
grasses and trees, dust mites, and cat allergens.
●Very high positive-predictive accuracy in children can be
achieved for some of the major food allergens with the Phadia
ImmunoCAP system [3,14-16]. The use of this in vitro test in the
diagnosis of food allergy is discussed in detail separately.
(See "Diagnostic evaluation of IgE-mediated food allergy", section
on 'Immunoassays'.)
●More problematic allergens include those from venoms, other
foods, weed pollens, latex, drugs, dogs, and molds [17,18].
Concordance among different immunoassay systems is reported to be
approximately 75 to 90 percent for well-characterized allergens, such
as birch and Timothy grass pollens [9-11,19,20], or even greater for
newer tests with well-characterized allergen extracts/molecules
[21,22] However, most molds and many other pollens are not well
characterized, and thus results may vary among different assay
systems. Generally, <15 percent intra-assay variability in the test
results is considered acceptable. When discordance is noted, it is
largely due to differences in the allergens bound on the solid-phase
matrix of the various systems, as allergen extract composition may
differ between companies. Of note, more variable results may be
obtained when using different commercial systems to measure
allergen-specific IgE to foods and less common inhalants.
Tests that have been validated according to the Clinical Laboratory
Standards Institute (formerly called the National Committee for
Clinical Laboratory Standards [NCCLS]) should be used whenever
possible [23].
Interpretation
Positive tests — A positive test is defined by the performing
laboratory. The presence of IgE to a specific allergen demonstrates
that a patient is "sensitized" to that allergen and may have a clinical
reaction to it. The definitive diagnosis of an IgE-mediated allergy
requires both sensitization and a history of allergic signs and
symptoms on exposure to that allergen. Sensitization alone is not
sufficient to diagnose an allergy, because individuals can be sensitized
to an allergen without having clinical symptoms upon exposure. The
clinical response of a sensitized individual to the suspect allergen is
best understood as a dynamic physiologic event with multiple
variables, of which the presence of allergen-specific IgE is just one.
Thus, allergy tests must be interpreted in the context of the patient's
specific clinical history, and the diagnosis of an allergic disorder
cannot be based solely on a laboratory result. This is true for in vitro
assays as well as for skin testing.
The likelihood that a positive result will correlate with clinical
reactivity is influenced by the degree of positivity, the allergen in
question, and the patient's clinical history. It is not uncommon to find
allergen-specific IgE in the serum of people who do not report allergic
symptoms. As examples, venom- and food-specific IgE have been
reported in up to 25 and 60 percent of the general population,
respectively [24-27]. In food allergy, greater serum levels of allergen-
specific IgE better predict anaphylactic reaction as determined by oral
food challenge [28].
A typical threshold for positivity is >0.35 kIU/L. The ImmunoCAP has
been shown to maintain linearity of results below this threshold and
can accurately detect minute quantities of allergen-specific IgE;
results can be reported down to 0.15 kIU/L [20]. Patients with higher
levels of antibody are more likely to experience symptoms upon
exposure to the allergen than patients with lower levels, although
strongly positive tests do not necessarily predict that a severe
reaction (eg, anaphylaxis) is more likely than a milder type of IgE-
mediated reaction (eg, urticaria) [24]. Furthermore, for most allergens,
threshold levels above which most patients will react clinically have
not been determined. A notable exception to the last statement is the
utility of Phadia ImmunoCAP results for certain foods, specifically in
pediatric patients, as mentioned previously (see 'Advantages relative
to skin testing' above). For these foods, the percentage of children
with a consistent clinical history who will react to challenge has been
determined over a range of food-specific IgE levels. For adults,
however, and for most allergens, the patient's reactivity to the
allergen must be determined by the clinical history or by a supervised
challenge procedure. (See "Diagnostic evaluation of IgE-mediated
food allergy", section on 'Immunoassays'.)
Results reported by class — Positive results of immunoassays are
often graded into classes (typically I to IV or I to VI) based upon
arbitrary divisions of a reference curve (table 1). There is some limited
clinical utility to these divisions, as levels of allergen-specific IgE at or
above class III (ie, ≥3.5 kIU/L) have been shown to be more
consistently related to symptoms upon exposure, whereas
asymptomatic sensitization may occur in some individuals having
lower than class III levels of allergen-specific IgE [14,15]. Levels of
class II or greater are generally required as proof of sensitization that
is clinically relevant when enrolling subjects in research protocols.
A specific example in which IgE immunoassays appear to augment
skin testing results is found in the selection of patients for
immunotherapy. Most modern clinical trials of allergen immunotherapy
stipulate that subjects must have a class II (ie, >0.7 kU/L) or greater
result to the allergen being studied, in addition to a positive skin test
to that allergen, to be included in the study population. This is based
upon the observation that this group of sensitized patients was most
likely to benefit in previous trials [29]. One study of sublingual tablet
immunotherapy demonstrated that, among skin test-positive
patients, only those who also had an allergen-specific IgE >0.1 kU/L
benefitted from treatment [30]. This suggests that combining allergen-
specific IgE testing with skin testing could be used to select patients
who are most likely to respond to allergen immunotherapy.

The following are examples of how the class of a result can be useful:

●If the result is markedly positive (eg, a class VI result), the

history suggests a past reaction to the allergen, and the allergen
is well characterized, then the diagnosis of an allergy can usually
be made without further evaluation.
●If the result is weakly positive (eg, a class I result), then further
evaluation is usually needed. Skin testing and possible challenge
may be indicated, based upon the patient's clinical history. Most
immunotherapy trials require that subjects have both
demonstrable allergen-specific IgE and skin test positivity.
Negative tests — A negative immunoassay result in the setting of a
strongly suggestive history does not exclude allergy. In this situation,
a skin prick test should be considered (if not contraindicated for one of
the reasons reviewed previously). (See 'Advantages relative to skin
testing' above.)
False positives — False-positive results of allergen-specific IgE can
theoretically occur in patients with extremely elevated total IgE
levels. As an example, patients with hyperimmunoglobulin E syndrome
can have a markedly elevated total IgE level (approximately 50,000
kIU/L) and test positive for IgE to allergens to which there is no history
of clinical reactivity. This situation is believed to be rare, although the
level of total IgE that could have this effect is not well defined and
may vary depending upon the immunoassay system. Failure to
thoroughly wash away unbound allergen-specific IgE in performance of
the assay can lead to false positives as the tracer anti-IgE will bind to
any residual IgE not bound to antigen. (See "Autosomal dominant
hyperimmunoglobulin E syndrome".)
TOTAL IgE LEVELSPatients with allergic conditions, such as
asthma, allergic rhinitis, or atopic dermatitis, often have higher serum
levels of total IgE compared with the general population. Although an
elevated total IgE may indicate that the patient has an atopic
condition, it provides no information about which condition or to what
allergens the patient is sensitive. Furthermore, because there is a
large degree of overlap between IgE levels in people with and without
allergic disease, the utility of total IgE in diagnosing these common
conditions is limited [31]. (See "The relationship between IgE and
allergic disease" and "The biology of IgE".)

In contrast, specific clinical situations in which the measurement of

total IgE is helpful include the following:

●Evaluation of specific disorders that are associated with very

elevated levels of total IgE, including parasitic infections, allergic
bronchopulmonary aspergillosis, and a small number of rare
immune disorders (eg, hyperimmunoglobulin E syndrome or
Wiskott-Aldrich syndrome) and malignancies (table 2) [32,33].
(See "Clinical manifestations and diagnosis of allergic
bronchopulmonary aspergillosis" and "Autosomal dominant
hyperimmunoglobulin E syndrome" and "Atopic dermatitis
(eczema): Pathogenesis, clinical manifestations, and
diagnosis" and "Wiskott-Aldrich syndrome".)
●Evaluation of patients with moderate-to-severe allergic asthma to
determine eligibility for treatment with anti-IgE therapy
(ie, omalizumab). Total IgE may also serve as a biomarker for
type 2 inflammation in asthma when considering use of
monoclonal antibodies that counter the type 2 inflammatory
process in asthma. (See "Anti-IgE therapy", section on
'Pretreatment testing'.)
Technique — Total IgE is usually measured by enzyme-linked
immunosorbent assay (ELISA) using specific anti-IgE monoclonal
antibodies. Laboratory techniques used for the assessment of
immunoglobulin concentration for other classes of immunoglobulin (ie,
IgG, IgA, IgM), such as radial immunodiffusion, cannot be used to
measure IgE concentrations because serum IgE levels are normally
too low.
TESTS FOR ANAPHYLAXISAnaphylaxis results from massive
activation of mast cells and basophils. Elevated levels of serum
tryptase and plasma histamine released from these cells can be used
to support the diagnosis of anaphylaxis. Most mediators released upon
mast cell activation, such as histamine or prostaglandins, are
characterized by a very short life; therefore, it is very difficult to
demonstrate changes in their levels in peripheral blood of patients
who experienced anaphylactic reaction. However, metabolites of
these mediators, such as N-methyl histamine and prostaglandin
compounds, can be assayed in 24-hour urine samples collected shortly
after a clinical event. They are used in the diagnosis of chronic mast
cell activation, such as that seen in mast cell activation syndrome or
mastocytosis.
Tryptase is also released from mast cells upon their activation and
stays in the blood for several hours. Its concentration can be
determined in serum using well-standardized methods such as
ImmunoCAP (form 1). It is recommended to evaluate serum tryptase in
patients suspected of anaphylactic reaction, such as those undergoing
surgical operations [34,35]. The use of laboratory tests to support the
diagnosis of anaphylaxis is reviewed in detail separately.
(See "Laboratory tests to support the clinical diagnosis of
anaphylaxis".)
COMPONENT-RESOLVED DIAGNOSTICSComponent-resolved
diagnostics (CRDs) are testing methods that allow for more specific
identification of the allergen to which a patient's specific IgE reacts.
This is a potentially important advance because standard IgE
immunoassays may detect IgE against clinically irrelevant allergens,
yielding positive results in patients who are not actually reactive to
the allergen in question. CRD involves microarray testing using
recombinant allergens and antigenic components of major allergens
and nanobiologic techniques to assess the precise antigenic epitopes
to which a patient's IgE binds.

CRDs are becoming commercially available to diagnose sensitization

to some insect venoms, animal danders, and foods and as a means of
predicting cross-reactivity between food and pollen allergens. The
more detailed information can inform diagnosis and prognosis.
Specifically, CRD is expected to assist in identifying patients who have
a higher risk of anaphylaxis because they have IgE against major
"anaphylactogenic" allergens versus patients with IgE directed against
allergens that generally do not elicit anaphylaxis.

One system uses a high-throughput, bead-based epitope assay to

analyze IgE reactivity to discrete food allergen epitopes and requires
only a few microliters of plasma. This test has gained US Food and
Drug Administration approval for detecting peanut allergy by
identifying IgE to multiple epitopes from Ara h1, Ara h2, and Ara h3
peanut proteins [36]. CRDs for foods in addition to peanut include tree
nuts, wheat, vegetables, fruits, cow's milk, and hen's egg.
(See "Diagnostic evaluation of IgE-mediated food allergy", section on
'Component testing' and "Component testing for pollen-related, plant-
derived food allergies", section on 'Peanut' and "Component testing
for pollen-related, plant-derived food allergies", section on 'Allergen-
specific use/interpretation'.)

In the case of plant foods, these techniques may help to clarify which
food-allergic patients are likely to experience "oral allergy syndrome"
and which are at risk for more serious systemic reactions to foods.
Such discrimination is not possible using only allergen skin testing or
conventional allergen-specific IgE assays.

●In the example of peanut allergy, IgE sensitization patterns can

help establish the risk of severe reactions for peanut-allergic
patients [37]. Patients sensitized only to pollen-related
components, such as the peanut allergen Ara h 8 (which is related
to a major birch pollen allergen Bet v 1), usually experience no or
very mild oral symptoms (ie, oral allergy syndrome), whereas
those who are sensitized to more stable components, such as
seed storage proteins (eg, Ara h 2), are more likely to experience
anaphylaxis to peanut. The allergen-specific IgE test alone,
without the component assessment, would erroneously assign a
risk of anaphylaxis to all of these patients. In addition, IgE
antibody profiles to 64 sequential epitopes of Ara h 1, Ara h 2, and
Ara h 3 proteins at one year of age are predictive of peanut allergy
at five years in children avoiding peanuts [38].
●For dog sensitization, reactivity to Can f 5 alone is associated
with tolerance to male dogs who are neutered or female dogs but
intolerance of non-neutered male dogs as this allergen is a
 prostatic kallikrein. Thus, for dog, CRD can assist a patient who
has a positive dog allergen-specific IgE in understanding whether
they could tolerate exposure to a dog of a specific sex.
CRD has also been explored to assess timothy grass sensitization
profiles by region. The highest mean IgE levels and largest proportion
of subjects with positivity to Phl p 1 and Phl p 5 were found in regions
where timothy grass is most prevalent. In addition, among patients
treated with timothy grass sublingual immunotherapy (SLIT) tablets,
the data suggest trends toward higher efficacy and an increased
incidence of having adverse events in subjects with detectable and
higher pretreatment Phl p 1, Phl p 5, and Phl p 6 IgE [39]. Finally, CRDs
of vespid venoms are also becoming available to assist in the
diagnostic assessment of the risk of severe allergy based on the
component to which IgE is directed [40].
A commercially available example of CRD is the ImmunoCAP ISAC,
which allows for detection of IgE against more than 100 individual
components (allergens) derived from more than 50 allergen sources.
This test allows for detection of IgE to Bet v 1, Bet v 2, and Bet v 4,
which are all derived from birch pollen. This can be applied as a
multiplex diagnostic tool, and/or the results can be searched for the
presence of IgE against a single component/allergen (eg, Bet v 2). This
tool can also be used to detect IgE against cross-reactive
carbohydrate determinants (CCDs). This is unique to in vitro methods,
as no in vivo test for detection of CCD-specific IgE is available. Tests
that provide CRD with less than 10 allergen components are also
commercially available in some countries. Euroimmun immunoblots
may serve as an example (figure 2). The test is restricted to several
components of birch and timothy grass pollens, including major
allergens such as Bet v 1, Phl p 1, and Phl p 5 and minor cross-reactive
allergens such as profilins Bet v 2/Phl p 12 and polcalcins Bet v 4/Phl p
7. This can be useful in the evaluation of patients with pollen allergy
who are being assessed for allergen immunotherapy to optimize the
constitution of their immunotherapy vaccines [41].
IgE that binds to CCDs is responsible for positive results of
immunoassays with allergen extracts containing those epitopes,
mainly pollens, foods, and venoms. The presence of CCD-specific IgE is
of little or no clinical significance and may be responsible for
discordant skin prick test and immunoassay results [42].
There are commercially available CCD inhibitors whose addition may
improve the diagnostic potential of routinely used immunoassays [43].
CCD inhibitors are not used in assays in the United States at the time
of this review.
In contrast to CCD-specific IgE, the presence of IgE binding to alpha-
gal determinant is responsible for severe anaphylaxis after ingestion
of red meat or injections of some biologics such as cetuximab. It can
be detected in serum using ImmunoCAP or other immunoenzymatic
assays. (See "Allergy to meats", section on 'Alpha-gal'.)
TESTS USED MOSTLY IN RESEARCHOther tests that are largely
used in research protocols include immunoblotting, several tests of
basophil activation and function, tests for levels of eosinophil-derived
mediators, and microarray testing [3,44-48]. In the United States,
these tests are not approved for routine clinical use, although some
are commercially available. Their use is more widespread in some
European countries.
Immunoblotting — The immunoblot technique, which is more
expensive than other assays and not quantitative, is predominantly a
research tool. This method is used to detect antibodies directed
against multiple proteins within an allergenic substance.

A mixture of allergen proteins is separated according to molecular

weight by gel electrophoresis (proteins must be within a range of
molecular weights that penetrate readily into gels). The separated
proteins are then transferred onto a nitrocellulose membrane. In
commercial diagnostic kits, nitrocellulose strips containing already
separated proteins are provided. The nitrocellulose membrane is
incubated with a test serum and then washed to remove unbound
serum components. Labeled anti-human IgE antibody is used to detect
IgE from the test serum that has bound to individual allergenic
proteins. The results are compared with reference sera.

Basophil tests — Basophils and mast cells both express the high-
affinity receptor for IgE on the cell surface and are activated when this
IgE encounters sufficient specific allergen. Mast cells reside in
tissues, while basophils circulate in the vasculature, making them
more accessible to collection and study. Several techniques have been
developed to examine basophil responses to allergens. Unfortunately,
basophils are prone to nonspecific activation by a variety of factors
and are difficult to transport and manipulate. Thus, these tests are
considered investigational. While not used commonly in th US nor
approved by the Food and Drug Administration, basophil activation
tests (BATs) are often employed in Europe and should be noted as a
rapidly advancing ex vivo method of assessing for cellular activation
by allergen resulting in increased expression of select CD markers
CD203c and CD63 [49-51]. BAT may be superior to skin testing and
routine in vitro assays in some clinical situations, such as in the
diagnostic process of patients with peanut allergy [52].
Basophil histamine release — The basophil histamine release test
measures the release of histamine from human peripheral blood
basophils incubated with allergen [3]. When well-characterized
allergens are used, this test is similar to skin testing in accuracy [44].
The test relies on living cells and thus requires that blood samples are
submitted and tested within 24 hours. Only a few laboratories perform
the test. Basophil histamine release is not standardized and is
considered an investigative tool for drug, food, and environmental
allergens.
Others — Other tests of basophil function following incubation with
allergen include release of leukotriene C4 (LTC4) and measurement of
the level of activation via expression of surface proteins (such as
CD63 or CD203c) by flow cytometry [3,45,53]. Although promising,
these tests are not as useful as skin testing and are not approved for
diagnostic use in the United States. Use of these BATs in the
diagnosis of food allergy is discussed separately. (See "Future
diagnostic tools for food allergy", section on 'Basophil activation
testing'.)
Eosinophil cationic protein levels — Eosinophils release a broad
range of biologically active mediators upon activation. Eosinophil
cationic protein (ECP) is an eosinophil-specific mediator that can be
measured in bodily fluids to estimate the extent of eosinophil
activation. Many studies have demonstrated that serum ECP levels
correlate with severity of allergic diseases such as asthma and
increase during parasitic infections [46]. It rises with increased airway
inflammation (eg, after allergen exposure) and falls during remissions,
either spontaneous or associated with therapy [47,48]. Thus, ECP is a
marker of eosinophilic inflammation, although it provides no
information about the presence of IgE-mediated allergy.

Measurement of ECP is commercially available, although this test

requires further characterization before it can be recommended for
routine clinical use.

UNVALIDATED TESTSPatients may present with allergy tests that

have been performed by alternative health care providers, including
allergen-specific IgG and IgG4 tests. These are most often performed
for the evaluation of food allergy. These methods typically yield
multiple positive results and almost always represent normal immune
responses to food. They do not predict true food hypersensitivity.
(See "Unproven and disproven tests for food allergy".)

There are validated uses of IgG and IgG4 tests:

●The presence of allergen-specific IgG4 and IgG "blocking

antibodies" is measured in studies of allergen immunotherapy.
The formation of these antibodies appears to be one of several
immunologic changes that are associated with effective
immunotherapy, although their exact functional role requires
further study. (See "Allergen immunotherapy for allergic disease:
Therapeutic mechanisms", section on 'IgG'.)
●Venom-specific IgG correlates with the adequacy of
immunotherapy in patients with venom allergy. This is discussed
in more detail separately. (See "Hymenoptera venom
immunotherapy: Technical issues, protocols, adverse effects, and
monitoring", section on 'Venom-specific IgG'.)
Other types of testing for allergy that are not supported by scientific
studies include provocation/neutralization tests, kinesiology, cytotoxic
tests, and electrodermal testing. These tests can pose risk to patients
both by potentially overlooking true allergies and by leading to
inappropriate diagnosis and advice, including dietary restrictions [54].
SOCIETY GUIDELINE LINKSLinks to society and government-
sponsored guidelines from selected countries and regions around the
world are provided separately. (See "Society guideline links: Allergy
diagnostic testing".)
SUMMARY
●Indications – In vitro allergy testing, in general terms, is
indicated when the clinician is assessing the likelihood that a
patient's reactions or symptoms were due to exposure to a
specific allergen. (See 'Indications' above.)
●Advantages over skin testing – Skin testing is usually preferred
to in vitro testing for the diagnosis of allergic disease. However, in
vitro testing has certain advantages: It poses no risk to the
patient, is not affected by medications, and requires only a simple
blood draw. In a few clinical situations, in vitro testing may be
superior to skin testing. (See 'Advantages relative to skin
testing' above.)
●Immunoassays – Immunoassays, in various forms, are the most
used in vitro tests for immunoglobulin E (IgE) mediated allergy.
These tests detect allergen-specific IgE in a patient's serum by
incubating the serum with the allergen in question, which has
been absorbed on a solid-phase medium (figure 1A-B). The bound
IgE is then detected with an anti-IgE antibody, which, in turn, has
a label attached to permit detection. (See 'Immunoassays for
allergen-specific IgE' above.)
●Importance of the clinical history – Testing for allergen-specific
IgE, like all forms of allergy testing, must be interpreted in the
context of the patient's specific clinical history. The presence of
allergen-specific IgE is correctly interpreted as evidence that the
patient is sensitized to that allergen and may react upon
exposure. Actual reactivity must be determined by history or
supervised challenge. (See 'Interpretation' above.)
●Total IgE levels – Serum levels of total IgE are of limited utility in
the diagnosis of allergic diseases. An elevated total IgE level may
indicate that the patient has an atopic condition, although it
provides no information about which allergens the patient is
sensitive to. Total IgE is used to determine the potential utility of
anti-IgE therapy (omalizumab) in patients with allergic asthma.
(See 'Total IgE levels' above.)
●Investigational tests – Tests used largely in research include
immunoblotting, basophil histamine or leukotriene release tests,
basophil activation, and levels of eosinophil mediators. These
tests are not standardized, are generally not superior to skin
testing, and cannot be recommended for routine clinical use.
(See 'Tests used mostly in research' above.)
●Unvalidated tests – Allergen-specific immunoglobulin G (IgG) and
IgG4 tests, which are believed to correlate with normal
immunologic responses to foreign substances, are not useful in
the diagnosis of allergy, except for monitoring the response to
certain forms of allergen immunotherapy. Unreliable testing
methods include provocation/neutralization tests, kinesiology,
cytotoxic tests, and electrodermal testing. (See 'Unvalidated
tests' above.)
ACKNOWLEDGMENTThe UpToDate editorial staff acknowledges
Hendrik Nolte, MD, PhD, who contributed to earlier versions of this
topic review.