858 The Immunoassay Handbook
3. Plant and animal food allergens (fruits, vegetables,
nuts, milk, eggs, shellﬁsh, and ﬁsh): lipid-transfer
proteins, proﬁlins, seed storage proteins, lactoglob-
ulins, caseins, tropomyosins, parvalbumins.
4. Injected allergens (insect venoms and some thera-
peutic proteins): phospholipases, hyaluronidases,
pathogenesis-related proteins, asparaginase.
Based on this classiﬁcation and in order to illustrate the
range and properties of allergens normally encountered in
the environment, some examples of the source, name, and
homologies of some common allergens are shown in
COMMON PROPERTIES OF ALLERGENS
Despite advances in genomics, bioinformatics and evolu-
tionary biology, and continuous effort by a large com-
munity of experts worldwide, the attributes that deﬁne
an allergen, i.e., the intrinsic characteristics that confer
allergenicity to an otherwise harmless substance remain
largely unknown. Only two mechanisms, both described
in the Dermatophagoides pteronyssinus (house dust mite
system), are currently known: a protease-dependent (Der
p 1) and a protease-independent one (Der p 2). In the
former Der p 1, protease activity has been shown to
cleave CD23, CD25, and CD40, inhibiting IL-12 secre-
tion (Ghaemmaghami et al., 2002) with the possible acti-
vation of the PAR family of G-coupled proteins in the
process (Kauffman et al., 2006). In the latter mechanism,
it has been elegantly shown that Der p 2 structurally
mimics MD2, a component of the TLR4 complex that
can co-opt other signaling components to activate its tar-
get cells (Trompette et al., 2009). Hence, the involve-
ment of complex lipids and Toll receptors has been
demonstrated. As most of the well-characterized aller-
gens are not proteases, and it is unlikely that MD2
homology could be a general mechanism, it is possible
that other mechanisms exist. The general observation
that most of the families of allergens are found in a
reduced number of families (only 2% of all sequence-
based and 5% of all structural protein families) with a
bias toward certain biochemical functions such as Ca2+,
actin, and lipid binding, most of them involved in innate
defense functions against pathogens, is interesting
(Radauer et al., 2008).
Allergens do not share common structural or functional
properties, however, in general terms, most allergens are
biological materials which, when inhaled or ingested, have
to gain access to the body via either a lipophilic barrier
(skin) or hydrophilic one by dissolving in mucosal secre-
tions. Some allergens bypass these barriers either through
stings, bites, or drug injections or by being produced
endogenously by invading parasites (Aalberse et al., 2001).
Allergens also tend to be comparatively easy to extract and
dissolve in buffered solutions. Typically, the allergy source
releases a complex mixture of potentially allergenic macro-
molecules such as carbohydrates, complex lipids, polypep-
tides, glycoproteins, and peptidoglycans. Atopic individuals
may produce IgE antibodies to one or many of the aller-
genic components, and there is strong evidence that indi-
cates that the recognition of the allergen or its epitope
proﬁle remains constant during the natural course of the
disease (Valenta et al., 2007). Thus, every allergic patient
has a range of unique antibodies to a given allergen source.
Because some allergenic components have a higher rela-
tive concentration and greater immunogenic activity than
others, it is possible to test allergic sensitivity qualitatively
using selective combinations of a limited range of
FIGURE 1 Main immunological cellular events taking place during sensitization and subsequent contact with the allergen (see text). Ag=antigen or
allergen, APC=antigen presenting cells, TH =T helper Cell. Modiﬁed from Loewenstein and Mueller, 2009. (The color version of this ﬁgure may be
viewed at www.immunoassayhandbook.com).
859CHAPTER 9.14 Allergy
DIAGNOSIS AND THERAPY
In diagnosis, atopic IgE-mediated allergy must be differ-
entiated from conditions initiated by other mechanisms,
both immunological and non-immunological (Fig. 2).
Normally, this involves testing and identifying speciﬁc IgE
in serum. A raised level generally conﬁrms a clinical diag-
nosis and acts as a basis for treatment.
Although in clinical practice, in vivo tests, such as prick
and intradermal skin testing, are still widely used methods
for demonstrating speciﬁc IgE antibody activity, the
TABLE 1 Molecular Properties of Common Allergens
Source Allergen MW (kDa) Homology
House dust mite (Dermatophagoides
Der p 1 25 Cysteine protease
Der p 2 14 Lipid-binding protein
Der p 3 30 Serine protease
Der p 5 14 Unknown
Cat (Felis domesticus) Fel d 1 38 Uteroglobin-like protein
Dog (Canis familiaris) Can f 1 25 Lipocalin
Mouse (Mus musculus) Mus m 1 21 Lipocalin
Rat (Rattus norvegicus) Rat n 1 21 Pheromone-binding lipocalin
Cockroach (Blattella germanica) Bla g 2 36 Inactive aspartic protease
Pollen/Grasses Rye (Lolium perenne) Lol p 1 28 Unknown
Timothy (Phleum pratense) Phl p 5 32 Unknown
Bermuda (Cynodon dactylon) Cyn d 1 32 Unknown
Ragweed (Artemisia vulgaris) Art v 1 28 Defensin-like protein
Birch (Betula verrucosa) Bet v 1 17 Pathogenesis-related protein
Bet v 2 15 Proﬁlin (actin binding)
Milk Bos d 5 18 β-lactoglobulin
Egg Gal d 1 28 Ovomucoid
Codﬁsh (Gadus callarias) Gad c 1 12 Ca-binding protein (parvalbumin)
Shrimp (Penaeus aztecus) Pen a 1 36 Tropomyosin
Peanut (Arachis hypogaea) Ara h l 63 Vicilin (seed storage protein)
Celery (Apium graveolens) Api g 1 16 PR-10, Bet v 1 homologous
Brazil nut (Bertolletia excelsa) Ber e 1 12 2S Albumin
Bee (Apis mellifera) Api m 1 9.5 Phospholipase A2
Wasp (Polistes annularis) Pol a 5 23 Mammalian testis proteins
Homet (Vespa crabro) Ves c 5 23 Mammalian testis proteins
Fire ant (Solenopsis invicta) Sol i 2 13 Unknown
Aspergillus fumigatus Asp f 1 18 Ribonuclease
Asp f 2 37 Fibrinogen binding
Alternaria altenata Alt a 1 29 Unknown
Hevea brasiliensis Hev b 1 15 Elongation factor
Hev b 5 16 Unknown function
Modiﬁed from Chapman, 2004, 2008.
860 The Immunoassay Handbook
advantages and great limitations of these techniques have
fueled disagreement among clinicians for decades and
hence are out of the scope of this review. One of the major
and irreplaceable advantages of in vivo methods, however,
is the strong psychological patient–doctor component
brought about by these techniques. Alternatively, current
in vitro techniques for speciﬁc IgE detection present many
advantages to clinical diagnostics such as reproducibility,
sensitivity, speciﬁcity, and stability. Most of these greatly
facilitate quality control and interlab comparisons. The
major advantages of in vitro methods, however, are
undoubtedly the avoidance of further potential sensitiza-
tions and risks of systemic reactions.
Once the eliciting allergen is known, allergy can gener-
ally be treated by eliminating exposure to the allergen,
injecting a natural or modiﬁed version of the allergen
(speciﬁc immunotherapy), or administering antihista-
mines, steroids, or other medications. The latter drugs
inhibit the release of mast cell mediators or block their
effects. As will be discussed later in the COMPONENT-
RESOLVED DIAGNOSTICS section, the success and
effectiveness of the modern and innovative immunother-
apy are directly related to the high degree of speciﬁcity
obtained by current in vitro methods.
A unifying thread linking atopic conditions (e.g., dermati-
tis, asthma, and rhinitis) is their occurrence in individuals
with markedly elevated levels of IgE antibodies. Hence,
due to their historic value, widespread use and importance,
the principles of Phadebas radioallergosorbent test
(RAST), still colloquially used to describe immunoassays
for IgE will be initially described. The theoretical concepts
of excess of antigen, developed successfully for earlier sys-
tems, are still valid for new generations of proﬁling tools
that will be discussed later.
IgE is a very particular class of immunoglobulin that is
highly catabolized with a short biological half-life of 1–5
days in peripheral blood. It does not cross the placenta or
activate classical complement pathways. However, from a
clinical perspective, the accurate assessment of sensitivity
and speciﬁcity of IgE antibodies can identify causative
allergens and aids the clinical history in the diagnosis of
allergic diseases (Plebani, 2003). In regular clinical prac-
tice, two measurements are normally carried out: total
serum IgE and speciﬁc serum IgE.
TOTAL SERUM IGE
Although their clinical role has been increasingly con-
tested (Kerkhof et al., 2003), historic habits, automation,
and good reproducibility made the immunoassays for total
serum IgE popular as an aid in the clinical diagnosis of
atopic diseases. The tests generally use a wide range of
assay principles: competitive double-antibody, solid-phase
radioimmunoassays (RIA) coexist with immunometric (sand-
wich) enzyme immunoassays. Solid phases in common use
include microparticles, polystyrene beads and tubes, and
cellulose foam. The signals measured can be radioactivity,
color, ﬂuorescence, and luminescence. Concentrations are
expressed as kU/L deﬁned by the WHO International
The serum total IgE level is generally raised in atopic dis-
ease, although an IgE concentration within the normal range
does not rule out IgE-mediated disease, especially if the dis-
ease is due to a single allergen. Total IgE measurements have
been used as an aid for clinical diagnosis to differentiate
atopic from nonatopic disease and for prediction of allergy in
children by measurement in cord blood or in newborns.
However, great care must be exercised in their interpretation
(see LIMITATIONS; Kerkhof et al., 2003). The serum IgE con-
centration is increased in atopic allergy (asthma, rhinitis),
especially in eczema patients who may exceed 20,000kU/L.
The total serum IgE concentration is normally correlated
with the number of allergens involved. IgE is also raised in
parasitosis and some immunodeﬁciencies.
The serum total IgE level in healthy individuals increases
during childhood from <1kU/L at birth to a peak at the
age of about 10 years and then declines to levels that are
FIGURE 2 Allergy-like symptoms. The majority of cases have perennial or irregular symptoms that may be IgE mediated or triggered by other
conditions. Differentiation mostly requires extensive testing.
861CHAPTER 9.14 Allergy
maintained throughout adult life. The geometric mean of
truly healthy nonsmoking adults is about 10kU/L and the
mean+2SD is slightly above 100kU/L. It is recommended
that allergy investigation is done on all children with levels
higher than the mean for the age+1SD, and in adults if the
level is higher than 100kU/L (Johansson and Yman, 1988).
The validity of the original adult reference values, geomet-
ric mean=13.2kU/L, mean+2SD=114kU/L (Zetter-
ström and Johansson, 1981) was conﬁrmed with UniCAP,
testing 63 healthy blood donors without known allergy.
Geometric mean=17.4kU/L, mean+2SD=112.9kU/L
(Persson and Yman, 1997).
Due to the low negative predictive value, total IgE levels
are in general not efﬁcient to rule out sensitization to com-
mon inhalant allergens (Kerkhof et al., 2003). High total
IgE might indicate a high probability of sensitization in
younger subjects, but identiﬁcation will still require fur-
ther speciﬁc testing. Total IgE levels are less closely linked
to allergic disease in areas with high incidence of
A wide variety of immunoassay procedures are in routine
use. The bulk of testing is performed in laboratory systems
with varying degrees of automation.
Desirable Assay Performance
An assay covering the range 0.5–5000kU/L would satisfy
the practical clinical needs for IgE determinations from
cord serum measurements on allergy risk babies to testing
of subjects with atopic dermatitis or parasitosis. To keep
imprecision (repeatability and reproducibility) as low as
possible, this range is typically split into a routine range
(preferably 2–2000kU/L) and a low range for primarily
pediatric use. UniCAP low measuring range is equivalent
to the range covered by the speciﬁc IgE assay, i.e., 0.35–
100kU/L. Although some other commercial assays include
diluents as zero calibrators, the detection limits are always
higher. An option to store calibration curves is desirable.
Types of Sample
CAP assays for total and speciﬁc IgE are generally vali-
dated for serum and plasma. Measurements in tears (Kari
et al., 1985; Sainte Laudy et al., 1994), nasal secretion
(Deuschl and Johansson, 1977; Sensi et al., 1994), and feces
(Sasai et al., 1992) have been reported. Applications out-
side those described in the manufacturer’s instructions for
use must be validated by the investigator.
ALLERGEN-SPECIFIC IGE ANTIBODY
Allergen-speciﬁc antibodies of a single immunoglobulin
class have to be measured in the presence of other antibod-
ies of the same class and antibodies of other classes speciﬁc
for the same allergen. This requires speciﬁc recognition of
the antigen-binding sites (Fab) and the class-speciﬁc epit-
opes (Fc) in the same assay. In the case of IgE, by having
the lowest concentration of any of the ﬁve immunoglobu-
lin isotypes in the sera, the concentration of speciﬁc anti-
body should be measurable down to picograms per
milliliter in samples that may contain total IgE and com-
peting antibodies of other classes well into the microgram
per milliliter range.
Early speciﬁc allergen detection tests lacked reproduc-
ibility, mainly due to large batch-to-batch variation in sta-
bility of the allergens following extensive extraction
procedures. Hence, an important advance in allergy diag-
nosis testing was the introduction of standardized allergen
extracts achieved in the late 1970s and early 1980s. The
standardization of allergens was responsible, not only for
the improvement of the accuracy of in vivo tests, but also
for the reliability of this test when coupled to the RAST
test. During the 1990s, with the availability of protein
sequences, large databases and advances in protein expres-
sion, the production of large quantities of speciﬁc well-
puriﬁed recombinant allergens became possible. Over the
past decades, many platforms have been developed for
speciﬁc IgE detection (Renault et al., 2007), however, as
previously mentioned, Phadebas RAST remained collo-
quially identiﬁed in the description of immunoassay for
speciﬁc IgE test. Hence, the basic concepts involved in the
design of the RAST test, the subsequent step with the
introduction of a solid support with higher allergen capac-
ity and automated systems, such as UniCAP, will be ini-
tially discussed (Fig. 3). Later in the section, other
platforms introduced to the market resulting from recent
advances in genomics and automation will also be brieﬂy
General Assay Characteristics
IgE assays use a solid phase coated with allergen to bind
the IgE antibody in the sample. There is a critical washing
step after the initial allergen–IgE antibody reaction and
before the incubation with labeled anti-IgE. The solid
phase may alternatively be coated with ligands that catch
allergen–IgE complexes before the washing step. A high-
capacity solid phase, like the chemically activated foam of
the Pharmacia CAP System and UniCAP, provides a large
excess of allergen that maximizes the binding of the IgE
antibody. This makes the assay less sensitive to competing
antibodies of other classes. It also means that the bound
IgE antibody can be detected with less ampliﬁcation. The
resulting lower concentration of anti-IgE, less intensive
labeling, and lower substrate concentration minimizes sev-
eral of the classic contributions to high and variable non-
Allergen in Excess
A high-capacity solid phase provides conditions for quan-
titation of the sum of all antibodies regardless of speciﬁcity
and afﬁnity. The Law of Mass Action, applied to heteroge-
neous, solid-phase immunoassays, predicts that when the
available allergen concentration is increased to a level,
where K (the afﬁnity constant) multiplied by the allergen
concentration Eqn (3) reaches ≥10, the proportion of IgE
antibody bound to the solid phase will be ≥90% and essen-
tially independent of the afﬁnity between the antibody and
the allergen (Peterman, 1991; Yman, 1994).
862 The Immunoassay Handbook
Law of Mass Action
When the assay is essentially afﬁnity independent, i.e.,
when more than approximately 90% of the IgE antibody is
bound, a further increase of the allergen concentration will
have negligible effect on the measured value (Fig. 4).
Anti-IgE preparations must be IgE Fc speciﬁc and are pref-
erentially combinations of, e.g., monoclonal antibodies with
speciﬁcity against more than one epitope on the Fc fragment
and with complementary dose–response characteristics.
Labeled anti-IgE has been prepared using several signal gen-
eration systems, in each case aiming at a high sensitivity.
Calibrators for both speciﬁc and total IgE measurements
should be traceable to the WHO International Reference
FIGURE 3 Principle of Phadebas RAST®, Pharmacia CAP System™ RAST®, and UniCAP® -speciﬁc IgE.
FIGURE 4 Diversity of IgE antibody patterns against soy bean
allergen in sera from 21 individuals. The allergenic components were
separated by SDS-g-polyacrylamide gel electrophoresis and blotted
onto nitrocellulose. Allergen-IgE complexes were detected by radiola-
beled anti-IgE and autoradiography (Perborn, 1990, unpublished data).
863CHAPTER 9.14 Allergy
Preparation for human IgE (75/502). This allows true
quantitation of the speciﬁc antibodies provided that the
allergen is in excess. The calibrator range of UniCAP-spe-
ciﬁc IgE is 0.35–100kU/L. The design of the assay should
permit storage of calibration curves up to 1 month using
curve controls in each assay to verify the validity of the
stored calibration curve.
Test Selection, Use, and Interpretation
Allergy tests should be used selectively, taking into account
the local distribution of the allergens and the clinical history
of the individual patient. Unless there is a clear link between
the appearance of symptoms and occasions of exposure to
one allergen, multisensitivity should always be suspected.
Multisensitive Patients – Phadiatop®
Before trying to identify offending allergens, patients with
allergy symptoms should ﬁrst be tested to conﬁrm that the
disease is atopic. If not, further searches for offending
allergens are rarely successful. Testing with Phadiatop
detects at least 90% of patients with atopic inhalant allergy
(Eriksson, 1990). Neither Phadiatop nor any other com-
mercial test with this type of indication gives quantitative
Phadiatop was designed to be a test for atopic allergy
with its main use for patients with respiratory symptoms.
This means that common inhaled allergens form the base.
Furthermore, it means that cases primarily sensitized by
food or insect sting, by occupational exposure, or by keep-
ing more or less exotic pets, like birds, guinea pigs, etc.,
cannot be detected unless the patients are also sensitive to
The allergens selected for Phadiatop, and the way they
are combined, are aimed at achieving an approximately
90% probability of correct classiﬁcation of atopics and
nonatopics. The combined effect of exposure, multisensi-
tization, and cross-reactivities between allergens gives a
high cumulative efﬁciency already for a relatively limited
combination of species. The order of the allergens in terms
of contribution to the cumulative efﬁciency varies between
populations depending on relative intensity of exposure,
but it has been possible to select three combinations that
cover the needs of Europe, North America, and Japan.
Allergy tests are used to help in the diagnosis and monitor-
ing of allergic patients. Assays need to be sensitive and spe-
ciﬁc and permit quantitative measurements over a wide
range (Bernstein, 1988). As with all immunoassays, there is
a need for common standards and deﬁnitions of perfor-
mance parameters. In the case of total serum IgE, the
WHO standard serves its purpose but for the measure-
ment of allergen-speciﬁc antibodies, there is no ofﬁcial
standard collection of units and there are no uniform
In reality, however, Phadebas RAST, ﬁrst available in
1974, became a working standard against which all
subsequent commercial tests were compared. Phadebas
RAST includes a reference consisting of several dilutions
of a serum containing IgE antibodies speciﬁc for birch pol-
len allergen. The dilutions were selected to permit the
construction of a calibration curve and evaluation of test
results in arbitrary units (PRU/mL) or semiquantitative
classes. This standard was internally calibrated against the
WHO IgE standard (Lundkvist, 1975). In the other gen-
erations of RAST (Pharmacia CAP System and UniCAP),
the birch reference was replaced by a direct substandard to
the WHO standard 75/502 for IgE (Yman, 1990) covering
the range 0.35–100kU/L. Concentrations lower than the
0.35kU/L calibrator are considered to be undetectable.
Tests for all allergens are evaluated against this IgE
All other commercial test kits largely adhere to this
principle. The apparent agreement in terms of classes or
units is, however, usually not supported by data deﬁning
the true concentrations of the calibrators. Direct compari-
sons are therefore difﬁcult.
Modiﬁed scoring systems are used routinely by labora-
tories and physicians. Recent instrument and micropro-
cessor technology development permit the storage of
calibration curves for each reagent batch, although con-
trol sera should always be run with the patient samples
under test. Multiallergen reagents, such as group-speciﬁc
combinations or Phadiatop, are not designed to be quan-
titative and test results should be interpreted as detect-
able or undetectable by means of a clearly deﬁned
QUANTITATIVE MEASUREMENT OF
ALLERGEN-SPECIFIC IGE ANTIBODIES
The IgE molecule has a large number of epitopes on the
Fc part. Combinations of capture–anti-IgE and conjugate–
anti-IgE can be found that make the calibrator IgE and
allergen-bound IgE appear identical. To what extent this
applies to a certain assay can only be judged on the basis of
experimental data and not on general assumptions. In the
case of the Pharmacia CAP System, it has been shown in
model experiments with several allergens that the mea-
surement of allergen-bound IgE is equivalent to IgE pro-
tein (Fig. 5).
This is possible because practically all the IgE antibod-
ies of the sample are bound to the solid phase (Borgå
et al., 1992). For instance, in the case of the timothy
ImmunoCAP, the capacity to bind IgE antibodies was
shown to be 25 times greater than is required to bind all
IgE antibodies at a concentration corresponding to the
high end of the measuring range (100kUA/L). This is a
large excess and referring to the Law of Mass Action, it is
clear that formation of allergen–IgE complex is favored.
As mentioned above, the excess is large enough to make
the antibody binding independent of the association con-
stant of the interaction (the afﬁnity), regardless of the IgE
antibody concentration. Furthermore, if all the relevant
allergenic components of the source are present on the
solid phase and shown to be capable of binding IgE anti-
bodies (Fig. 6), the system is capable of measuring the
sum of allergen-speciﬁc antibodies and satisﬁes the
requirements of a quantitative assay (Perborn et al., 1995).
864 The Immunoassay Handbook
According to the rules of WHO for establishment of stan-
dards for biological materials, an International Reference
Preparation for IgE was established in 1968. The prepara-
tion was assigned arbitrary mass units (International Units)
and submitted to collaborative trials. Later the Interna-
tional Unit was shown to correspond to 2.42ng pure IgE
by parallel chemical and immunological measurement
(Bazaral and Hamburger, 1972). The International Unit is
well established for total IgE measurements, and it is evi-
dent that measurements of speciﬁc IgE antibodies also
should move toward increasing standardization and that
the antibody concentrations ultimately should be expressed
in the same mass unit of IgE protein.
Adequate controls should be included in every assay. Uni-
versally available IgE antibody control sera with deﬁned
concentration and allergen speciﬁcity are preferred over
in-house pools. National and international quality control
programs provide regular assessment of reagent and labo-
ratory performance (Fiﬁeld et al., 1987).
The most common causes of error in RAST-type assays
are inadequate washing and failure to remove washing
solution effectively and evenly from all the reaction ves-
sels. Measurement in ﬂuids other than those speciﬁed in
the directions for use should be avoided if possible. How-
ever, if this is essential, then proper negative and positive
controls should be formulated and run, and dilution and
recovery should be conﬁrmed.
The quality documentation that should be produced for
an IgE antibody assay by the manufacturer includes:
G quality and reproducibility of allergen source
G speciﬁcity and dose–response characteristics of labeled
G capacity and reproducibility of solid-phase binding;
G linearity and parallelism of calibrator and sample dilu-
tions regardless of allergen;
G signal-to-noise ratio for the lowest calibrator;
G freedom of interference from nonspeciﬁc IgE and com-
peting IgG antibodies;
G high-dose hook data;
G stability of reagents and reagent combinations;
G correlation to reference method;
G clinical sensitivity and speciﬁcity calculated on relevant
DIAGNOSIS OF ATOPIC DISEASE AND
IDENTIFICATION OF OFFENDING
The clinical sensitivity and speciﬁcity of IgE antibody
measurements should be judged in relation to the allergy
specialist’s diagnosis of groups of consecutive patients.
The groups must be representative of the populations for
which the test is intended in terms of prevalence of disease
and degree of sensitization. Extensive clinical trials in
accordance with this principle on more than 2000 indi-
viduals showed the average diagnostic efﬁciency of the
Pharmacia CAP System to be about 90% (Yman, 1990).
FIGURE 5 Measurement of the change of concentration of IgE
antibody and IgE protein after stepwise depletion of speciﬁc IgE
antibody from serum. The allergen-speciﬁc antibody unit (UA) is
equivalent to the international IgE unit (U). Lindqvist et al., 1995.
FIGURE 6 Immunoblotting analysis of four sera with wheat-speciﬁc IgE
antibodies before (1) and after (2) contact with a wheat ImmunoCAP.
Antibodies of all subspeciﬁcities were bound to the solid-phase allergens.
865CHAPTER 9.14 Allergy
UniCAP was also evaluated in six clinical studies including
894 consecutive patients. The clinical sensitivity and speci-
ﬁcity of UniCAP-speciﬁc IgE derived from 5170 compari-
sons to clinical diagnosis were 89% and 91%, respectively
(Paganelli et al., 1998).
The term component-resolved diagnostic (CRD) has
been coined to designate a more precise diagnostic test
based on pure allergen molecules that are either produced
by recombinant (r) expression of allergen-encoding
cDNAs or by puriﬁcation from natural (n) allergen sources
(Valenta et al., 2007). CRD is recommended for identify-
ing speciﬁcally the allergenic reactivity proﬁle of a patient
along with their potential cross-reactivity interactions.
This is particularly important for the development of
strategies for allergen immunotherapy. Although some of
the immunotherapies have been known and in use since
1911, the need for identiﬁcation of the disease-eliciting
molecules was a major obstacle for prescription of allergy
vaccines. The selection of patients for immunotherapy
must take into consideration, among other factors, the
presence of an IgE-mediated allergic sensitization by the
demonstration of allergen-speciﬁc IgE antibodies.
The current success of these therapies depends on fac-
tors such as accurate diagnostics, vaccine quality, and
degree of immunoresponse to treatment. CRD as a con-
cept was greatly aided by the availability of pure proteins
in the CAP system but evolved to a higher degree of
sophistication with the development of proﬁling systems
such as protein microarrays. Interestingly, the monitoring
of success of treatment by some of the current immuno-
therapies is also associated with the development of aller-
gen-speciﬁc tests for other classes of immunoglobulins
(IgG4, IgG1, IgG2) (Valenta et al., 2007). Another impor-
tant application of CRD is related to the establishment of
geographical patterns of IgE sensitization, i.e., demo-
graphic sensitization studies where the prevalence of spe-
ciﬁc IgE responses within a particular population is able to
give important clues regarding the sensitizing allergen.
More recently, the observation that some classical speciﬁc
allergen markers do correlate with the degree of severity of
allergic diseases is also an important new development
(Nicolaou et al., 2010 – Manchester study). Altogether, the
acquired ability to carry out CRD, and the current results
in different platforms suggest that potential uses of this
technique are just beginning to be unraveled.
The move toward miniaturization of diagnostic testing is a
popular one, not just for economic but also functional rea-
sons. In the past 10 years, the ability to produce microarrays
has provided the technology for monitoring the expression
of thousands of genes, under many different conditions, on
a solid-based medium the size of a microscope slide. Ini-
tially used for DNA, the technique has evolved to other
more sophisticated applications using different attachment
methods in order to determine protein functionality, pro-
tein interactions, and enzymatic activity among others
(Renault et al., 2007).
The mechanics of protein array production are now very
well established, and a number of comprehensive reviews are
available describing this area (Harwanegg et al., 2003; Predki,
2004; Bertone and Snyder, 2005; Merkel et al., 2005; Molloy
et al., 2005). Essentially protein microarray represents a
multi-analyte, solid-phase immunoassay, where proteins are
immobilized onto the solid surface and micro-quantities of
the serum samples are then incubated under standard condi-
tions. The high concentration of proteins in the spot assures
the selection of high-afﬁnity antibodies. Hence, antibodies
to the particular protein present in the patient’s serum are
captured by the immobilized protein, and after the slides
are washed, the bound antibodies are detected with a high-
sensitivity ﬂuorescently labeled anti-isotype antibody and a
laser-based scanner (Fig. 7). The same assay format has
been further developed using different and improved solid
surfaces. Many aspects of the array technology such as the
solid support for immobilization, “direct” vs. “sandwich”
format, and methods used for signal ampliﬁcation
multi-analyte detection are reviewed elsewhere.
With the availability of a large number of well-charac-
terized recombinant proteins, arrays of different complex-
ity have been produced employing puriﬁed recombinant
allergens (Harwanegg et al., 2003), and commercial prod-
ucts such as ImmunoCAP ISAC (Phadia) are in place. The
results obtained from these arrays are very encouraging,
and a number of published examples have now shown that
the sensitivity of these tests is similar to those from RAST,
ELISA, and immunoblot tests.
Pure proteins vs. extracts: Despite the beneﬁts of the
use of pure proteins, the sheer numbers, complexity, and
logistics of producing a comprehensive assay that covers
all known protein families are sometimes overlooked. In
reality, a very small number of pure proteins are avail-
able. Hence, several groups have proposed the use of
comprehensive protein extracts. One advantage of using
heterogeneous protein mixtures from allergen extracts
on arrays instead of puriﬁed proteins should be the
detection of underrepresented proteins or protein com-
plexes in allergenic products (Kim et al., 2002). Some
promising results have been reported in studies con-
ducted with over 150 different crude extracts (Bacarese-
Hamilton et al., 2005), and complex platforms covering a
large number of extracts with simultaneous detection of
four classes of immunoglobulins have been introduced
(Renault et al., 2011).
FIGURE 7 Diagram showing the basic components of a standard
protein array. Antibodies from the patient’s serum recognize the
immobilized protein molecules on the surface of the chip. Secondary
antibodies and ﬂuorescent markers allow the target molecule to be
detected and quantiﬁed. Renault et al., 2007. (The color version of this
ﬁgure may be viewed at www.immunoassayhandbook.com).
866 The Immunoassay Handbook
The characterization of the protein epitopes recognized
by the IgE is fundamental to the understanding of the mech-
anisms leading to allergy and for the designing of safe immu-
notherapeutics. In its simple format, overlapping peptides
rather than proteins can be immobilized in slides with an
average of 10–20 amino acids in length. Cluster analysis has
shown a high degree of correlation between patient sera and
their reactivity to speciﬁc peptides (Sampson, 2004, 2005;
Shrefﬂer et al., 2005). While mapping a large number of
peptide epitopes can provide prognostic information about
the patient and some information on protein antigenicity, it
has the drawback of missing the important nonlinear epit-
opes. In a more elaborate approach, the proof-of-concept of
using engineered, properly folded, chimeric proteins con-
taining swapped, structural domains has been established in
the mapping of the three-dimensional IgE epitope of a nut
protein (Alcocer et al., 2004). With advances in the domain
swapping technologies by commercial in vitro recombina-
tion systems, it is expected that the knowledge accumulated
will further our understanding of how the body’s immune
system sees and reacts to new proteins.
Regarding the array format, whether the reaction in future
successful platforms takes place on ﬂat, solid surface slides or
on encoded beads or suspension arrays will depend more on
commercial considerations than technical constraints. One
important development in the area involves coupling the
diversity of the protein microarray with the biological output
of basophilic cell degranulation (Lin et al., 2007). High-
throughput diagnostic tests are now possible, and multiple
allergen detection marks the newest tool available to pro-
duce rapid, accurate, and immunological functional allergy
proﬁling. The progression from laboratory-based tests for
speciﬁc Ig binding to clinical practice is a long way from
being realized; however, the available molecular and cellular
technologies are closing fast the gap to a fully in vitro system.
Potentially, these in vitro biological assays might provide
unique insight into the relationship between immunoglobu-
lins and effector systems in type I hypersensitivity. This is a
signiﬁcant improvement to the reality faced by conventional
allergy clinics worldwide.
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