Several distinct properties of the IgE repertoire determine
effector cell degranulation in response to allergen challenge
Lars Harder Christensen, PhD,a,bJens Holm, PhD,aGitte Lund,aErik Riise, PhD,band Kaare Lund, PhDa
Background: On cross-linking of receptor-bound IgE antibodies
by allergens, effector cells (basophils and mast cells) involved in
type I allergic reactions degranulate and release the potent
chemical mediators stored inside their granules. Total and
allergen-specific IgE concentrations, IgE affinity for allergen,
and IgE clonality are all distinct properties of allergic patients?
IgE repertoires. However, the inability to isolate individual IgE
antibodies from allergic patients? sera presents a major barrier
to understanding the importance of patient-specific IgE
repertoires for the manifestation and severity of allergic
Objective: We sought to investigate how individual properties of
an IgE repertoire affect effector cell degranulation.
Methods: A panel of recombinant IgE (rIgE) antibodies specific
for the major house dust mite allergen Der p 2 was developed
and characterized in regard to Der p 2 affinity, as well as Der p
2 epitope specificity, by using surface plasmon resonance
technology. Human basophils were sensitized with different
combinations of rIgEs, and degranulation responses were
measured by means of flow cytometry after challenge with Der
Results: A total of 31 Der p 2–specific rIgEs were produced.
They bound a total of 9 different Der p 2 epitopes in the affinity
range (KDvalue) of 0.0358 to 291 nM.
Factors increasing human basophil degranulation were
increased total IgE concentrations, increased concentrations of
allergen-specific IgE relative to non–allergen-specific IgE, more
even concentration of individual allergen-specific IgE clones,
increased IgE affinity for allergen, and increased number of
allergen epitopes recognized by the IgE repertoire (increased
Conclusion: This study demonstrates how distinct properties of
the IgE repertoire, such as total and allergen-specific IgE
antibody concentration, IgE affinity, and IgE clonality, affect
effector cell degranulation. (J Allergy Clin Immunol
Key words: Allergy, recombinant IgE, human IgE, chimeric IgE, an-
tibody, affinity, epitope, allergen, Der p 2, effector cell degranula-
tion, basophil activation test, basophils, diagnosis
Effector cells (basophils and mast cells) involved in type I
allergic reactions contain high-affinity IgE receptors (FceRI)
embedded in their plasma membranes.1On cross-linking of
receptor-bound IgE by allergens, effector cells degranulate
and release the potent chemical mediators (eg, histamine)
stored inside their granules, leading to immediate allergic
IgE clonality are all distinct properties of an allergic patient’s IgE
repertoire. Most commonly seen are patient-specific variations in
tle and less accessible parameters, such as overall IgE affinity for
allergens and the number of allergen epitopes recognized by a
patient’s IgE repertoire (clonality), have been connected with
toms.4-7Still, theextent towhichthese individualproperties ofan
allergic patient’s IgE repertoire contribute to effector cell degran-
ulation remains to be established in detail. However, the inability
to isolate individual IgE antibodies from allergic patients? sera
presents a major barrier to understanding the importance of
patient-specific IgE repertoires for the manifestation and severity
of allergic symptoms.
Inthis study we developed apanelof31fully human ormouse/
human chimeric recombinant IgE (rIgE) antibodies specific for
the major house dust mite (HDM) allergen Der p 2, which is a
monomeric, 14-kDa protein recognized by 80% of all individuals
with HDM allergy.8The rIgE antibodies were characterized with
regard to individual affinity for Der p 2, as well as to their Der p 2
epitope specificity. Human basophils were sensitized with differ-
compositions, to assess the contribution of IgE concentration,
IgE affinity, and IgE clonality on allergen-mediated effector cell
Cloning and expression of rIgE antibodies
Previously, a set of 2 mammalian expression vectors encoding the human
IgG3heavy chain (pLNOH2) and the human k light chain (pLNOK) was con-
structed by Norderhaug et al.9Forexpression of the rIgE antibodies described
here, the human constant IgG3region in pLNOH2 was replaced with the
human constant IgE region (CeH1-CeH4), resulting in the new vector pLNO-
H2IgE. CeH1-CeH4 was PCR amplified from humangenomic DNA to include
introns for enhanced recombinant expression yields.10
Ten hybridoma cell lines expressing murine anti-Der p 2 antibodies (IgG
isotype) were made, as previously described,11from mice immunized with
Fromathe Department of Experimental Immunology, ALK-Abello ´ A/S, Hørsholm, and
bthe Antibody Technology Group, the Faculty of Pharmaceutical Sciences, University
Supported by the University of Copenhagen, Denmark, and by ALK-Abello ´ A/S,
Disclosure of potential conflict of interest: L. H. Christensen is employed by ALK-
Abello ´. J. Holm is employed by and owns stock in ALK-Abello ´. G. Lund is employed
by and owns stock in ALK-Abello ´. E. Riise has declared that he has no conflict of
interest. K. Lund is employed by and owns stock in ALK-Abello ´.
May 19, 2008.
Available online June 24, 2008.
Reprint requests: Lars Harder Christensen, PhD, ALK-Abello ´, Department of Experi-
? 2008 American Academy of Allergy, Asthma & Immunology
HDM: House dust mite
natural Der p 2. Variable antibody regions (VHDJH/VkJk) from these hybrid-
oma cell lines were RT-PCR amplified and cloned into pLNOH2IgE and
pLNOK in frame with CeH1-CeH4 and Ck, respectively.
Recombinant IgE antibodies were produced with the Freestyle 293
Expression System (Invitrogen, Carlsbad, Calif). The concentration of rIgE
ranged from 150 mg/L to 15 mg/L (mean, 6.8 mg/L), as determined in an
Advia Centaur assay (Bayer, Leverkusen, Germany), by using biotinylated
anti-IgE as the detector antibody.
Generation of Der p 2–specific chimeric rIgE
antibodies by shuffling of antibody chains
HEK293 cells were transfected with all possible combinations of heavy-
and light-chain vectors encoding the 10 hybridoma-derived chimeric rIgE
antibodies mentioned above. The chain-shuffled, chimeric rIgE antibodies
were screened for their ability to bind Der p 2 in ELISA: Maxisorp microtiter
wells (Nunc, Roskilde, Denmark) coated with recombinant Der p 2 (rDer p 2;
1 mg/mL) or with BSA (1 mg/mL) as a negative control were blocked (PBS
containing 2 wt/vol% skim milk) for 2 hours and incubated with rIgE-
containing cell supernatant for 1 hour. Bound rIgEs were detected with
DAKO, Glostrup, Denmark), followed by addition of TMB One developing
agent (Kem-En-Tec Diagnostics, Taastrup, Denmark). Absorbance was
measured at 450 nm.
Mapping of relative positions of Der p 2 epitopes
Surface plasmon resonance mapping experiments were performed on a
Biacore 2000 (Biacore, Uppsala, Sweden). Monoclonal anti-human IgE
(developed in-house at ALK-Abello ´, Hørsholm, Denmark) was immobilized
on a CM5 chip (Biacore) by using an Amine Coupling Kit (Biacore). The
cycles were run as follows. The first rDer p 2–specific rIgE clonewas injected
for 4 minutes, followed by injection of a blocking rIgE (anti–tetanus toxoid)
for 5 minutes. This latter step was included to block free anti-human IgE sites
at the chip surface not bound by the first rDer p 2–specific rIgE to avoid false-
positive responses. Then rDer p 2 (10 mg/mL) was injected for 1 minute,
immediately followed by injection of the second rDer p 2–specific rIgE clone
for 4 minutes.
Mapping of Der p 2 epitopes with rDer p 2 variants
Six rDer p 2 variants were developed at ALK-Abello ´ (Henmar et al,
unpublished data). Briefly, Der p 2 (isoform 2.0101) was mutated in one of 6
surface-exposed positions: K6A, K15E, H30N, E62S, H74N, or K82N. The
rDer p 2 variants were expressed in Pichia pastoris. Surface plasmon reso-
nance experiments were carried out with the same anti-human IgE CM5
chip as described above. Cycles were run as follows. A Der p 2–specific
rIgE clone was injected for 4 minutes, followed by injection of an rDer p 2
variant (10 mg/mL) for 1.5 minutes with a dissociation time of 5 minutes.
Controls included injection of rDer p 2 wild-type (positive control) or super-
natant from P pastoris transfected with an empty vector (pGAPz, negative
Determination of rIgE affinities for rDer p 2
Surface plasmon resonance affinity measurements were carried out with
a CM5 chip immobilized with anti-human IgE, as described above. Cycles
were run as follows. A rIgE clone was injected for 5 minutes, followed by
injection of rDer p 2 for 5 minutes with a dissociation time of 10 minutes.
The rDer p 2 concentration was varied by 2-fold dilutions between each
cycle in a range from 0.349 to 5714 nM, for a complete coverage of rDer
p 2 concentrations from zero response to saturation level (Rmax). The
affinity constant KD(kd/ka) was determined with BIAevaluation software
Basophil activation test
PBMCs were isolated on a density gradient (Lymphoprep; Nycomed,
Zurich,Switzerland) from nonatopicdonors whose basophilswere previously
tested to give equally high maximal degranulation responses (>80%). Native
IgE antibodies bound to the basophil cell surface were stripped off with ice-
cold lactic acid buffer, pH 3.9 (Bie & Berntsen, Herlev, Denmark), for 5
at 378C. Sensitized cells were challenged with rDer p 2 in a concentration
range of 0.1 pg/mL to 1 mg/mL in RPMI medium plus 0.5% human serum
albumin plus 2 ng/mL IL-3. Degranulation was carried out for 1 hour at 378C.
After degranulation, cells were labeled with a cocktail consisting of
anti-CD63–fluorescein isothiocyanate, anti-CD123–phycoerythrin, and anti-
HLA-DR–peridinin-chlorophyll-protein complex (catalog no. 341068; BD
Biosciences, Franklin Lakes, NJ) in addition to a non–cross-linking,
biotinylated anti-IgE (developed in house at ALK-Abello ´), followed by
addition of 10 mL of streptavidin-allophycocyanin (catalog no. 349024, BD
Basophil degranulation was measured with a FACSAria cell sorter (BD
and monocytes), followed by gating on HLA-DR2cells (basophils but not
monocytes). Nondegranulated and degranulated basophils were counted
with the CD63 marker. Two hundred thousand PBMCs were counted from
each sample, corresponding to 1000 to 6000 basophils.
Controls wereincludedtoconfirm thatnativeIgEwasproperlystrippedoff
during the stripping step and to ensure the absence of unspecific background
Cloning and expression of Der p 2–specific
Ten murine anti-Der p 2 IgG antibodies were converted into
mouse/human chimeric IgE antibodies named H1 through H8,
H10, and H12. In addition, 9 fully human Der p 2–specific rIgE
B cells of a patient allergic to HDM by using the phage display
technique, as described in detail elsewhere (Christensen et al,
Mapping of antibody epitopes on Der p 2
By using surface plasmon resonance, 2 different approaches
were applied to map antibody epitopes on Der p 2. First, relative
positions of Der p 2 epitopes recognized by the rIgE clones were
simultaneously, as shown in Fig 1, A and B. Seven individual
binding patterns (epitopes) were identified (Fig 1, C).
In the second mapping approach, a rough estimate of Der p 2
surface areas involved in each epitope was determined by testing
the capability of individual rIgE clones to bind a panel of
recombinant Der p 2 variants, as shown in Fig 1, D and E. Nine
different binding patterns were observed (Fig 1, F). Mutation of
Der p 2 in position K15, H74, or K82 each led to abolished anti-
tral position of these amino acids in the respective epitopes.12
Contrarily, mutation of Der p 2 in position K6, H30, or E62
only weakly affected binding of some rIgE clones (Fig 1, F), in-
dicating a peripheral position of these amino acids in the respec-
J ALLERGY CLIN IMMUNOL
VOLUME 122, NUMBER 2
CHRISTENSEN ET AL 299
map of epitopes recognized by the various rIgE clones was
constructed (Fig 1, G and H). As can be seen from the models,
several combinations of up to 3 different rIgE clones were able
to bind rDer p 2 simultaneously.
Generation of additional Der p 2–specific chimeric
rIgE antibodies by means of light-chain shuffling
By shuffling the light chains of the 10 hybridoma-derived rIgE
clones, 12 additional Der p 2–specific rIgE clones were obtained
(Table I). The chain-shuffled chimeric rIgE clones were named
Hx:Hy, where Hx and Hy denote the respective heavy- and
light-chain clones from which they were derived.
mapped analogous to the 2 approaches shown in Fig 1, A and D
(data not shown). In all cases, each individual chain-shuffled
rIgE clones exclusively bound the same Der p 2 epitope as the
parent clone from which the heavy chain was derived, indicating
Determination of rIgE affinities for rDer p 2
by means of surface plasmon resonance experiments. Represen-
tative sensorgrams of rIgE clones binding rDer p 2 with high,
medium, and low affinity (arbitrarily assigned categories) are
shown in Figure 2, A through C, and a summary of all results is
FIG 1. Der p 2 epitope mapping. Principle (A), examples (B), and summary (C) of results obtained by means
of relative epitope-mapping experiments by using surface plasmon resonance are shown. Principle (D),
example (E), and summary (F) of results obtained by means of epitope-mapping experiments with rDer p
2 variants by using surface plasmon resonance are shown. G and H, Three- and 2-dimensional maps of
Der p 2 epitopes bound by the rIgE clones. Source of Der p 2 structure: Derewenda et al13; PDB ID, 1KTJ.
J ALLERGY CLIN IMMUNOL
300 CHRISTENSEN ET AL
shown in Fig 2, D. Measured affinities ranged from 0.0358 to 291
Although chain-shuffled rIgE clones were all binding the same
epitope as the parent rIgE clone from which the heavy chain was
derived, their affinity for rDer p 2 was generally greatly altered,
with most chain-shuffled rIgE clones binding rDer p 2 with lower
affinity than their parent heavy-chain rIgE clone.
Higher total IgE concentrations increase both
basophil sensitivity and maximal degranulation
We used the basophil activation test (a flow cytometric method
based on the CD63 marker) to assess basophil degranulation, a
method now widely used as an alternative to histamine release
anchored in the granule membrane, fuses with and becomes ex-
posed on the outside of the basophil plasma membrane,15which
perfectly correlates with histamine release, as examined by
others15,16and seen our own observations.
Humanbasophils weresensitized withdifferentconcentrations
of a mixture consisting of equimolar quantities of 3 rIgE clones
recognizing nonoverlapping Der p 2 epitopes. An irrelevant non–
Der p 2–specific rIgE antibody (anti-tox, binding tetanus toxoid)
allergic IgE repertoire that contains both allergen-specific and
non–allergen-specific IgE. Sensitization of basophils with in-
creasing total rIgE concentrations led to increased basophil de-
granulation responses observed as both increased maximal
degranulation responses and increased sensitivity (the latter
term was defined as the concentration of rDer p 2 triggering a
Higher concentrations of allergen-specific IgE
relative to non–allergen-specific IgE increase both
basophil sensitivity and maximal degranulation
The effect of changing the relative concentration between
allergen-specific and non–allergen-specific IgE was examined
by sensitizing human basophils with a fixed concentration of
total IgE but different ratios of Der p 2–specific to non–Der p
2–specific rIgE (Fig 4).
As was also seen when increasing the total rIgE concentration,
basophils sensitized with increasing relative concentrations of
Der p 2–specific rIgE led to both increased maximal degranula-
tion response and sensitivity (Fig 4).
Equimolar concentrations of individual allergen-
specific IgEs result in the highest maximal
We examined the consequences of changing the relative
configuration. Human basophils were sensitized with different
ratios of 2 rIgE clones binding nonoverlapping Der p 2 epitopes
with equal affinity (Fig 5). The highest level of basophil degran-
ulation was seen when the 2 rIgE clones were present in equimo-
lar amounts (ratio of 50:50). In the case of uneven antibody
concentrations, maximal basophil degranulation decreased
when the ratio between the 2 Der p 2–specific rIgE clones was
95:5 and even more pronounced with a ratio of 99:1. Identical
results were obtained with the reciprocal ratios (Fig 5). Changing
the ratio of the 2 nonoverlapping IgEs affected maximal degran-
100:0/0:100) showed no degranulation, as expected, because of
epitope specificities for productive cross-linking.
Higher affinity of individual allergen-specific IgEs
increases basophil sensitivity
Human basophils were sensitized with different combinations
of 2 rIgE clones having different affinities for rDer p 2, still
binding the same 2 epitope clusters (Fig 6), to investigatehow the
affinity of individual IgEs affects effector cell degranulation.
Generally, basophil sensitivity increased with increasing affin-
ityofindividual rIgEclones involved inthe combinations(Fig 6).
Basophils sensitized with 2 low-affinity rIgE clones (LL combi-
tration than basophils sensitized with 2 high-affinity rIgE clones
In contrast to the low sensitivity of basophils sensitized with 2
low-affinity rIgE clones, a dramatic effect was seen when a low-
affinity rIgE clone was combined with a high-affinity rIgE clone
(HL combination, Fig 6). This latter combination showed a baso-
affinity rIgE clones (HH combination, Fig 6).
All rIgE affinity combinations resulted in similar maximal
basophil degranulation levels (approximately 80%), except for
the combination of 2 low-affinity antibodies (LL combination,
Fig 6). It was not tested whether this combination reached the
same maximal response at higher rDer p 2 concentrations,
Higher IgE clonality increases basophil sensitivity
The effect of IgE clonality (ie, the number of different IgE
sensitizing human basophils with combinations of 1, 2, or 3 rIgE
clones directed toward nonoverlapping Der p 2 epitopes (Fig 7).
Basophils sensitized with3 differentrIgE clones (brown curve,
Fig 7) showed 5- to 20-fold higher sensitivity than basophils
TABLE I. Chain-shuffled rIgE clones and their ability to bind rDer
p 2, as determined by means of ELISA
Light chain clone
H1 H2H3 H4H5 H6H7H8H10 H12
Heavy chain clone
1, Chain-shuffled rIgE clone able to bind rDer p 2; 2, chain-shuffled rIgE clone
unable to bind rDer p 2.
J ALLERGY CLIN IMMUNOL
VOLUME 122, NUMBER 2
CHRISTENSEN ET AL 301
sensitized with only 2 different rIgE clones (purple, grey, and or-
ange curves; Fig 7). The maximal degranulation level seemed
largely unaffected by changes in IgE clonality (Fig 7). Again, ba-
sophils monosensitized with a single rIgE clone showed no de-
granulation, as expected.
Using a panel of well-characterized recombinant IgE anti-
bodies, we have demonstrated how individual components of a
complex IgE repertoire affect effector cell degranulation. We
show that the composition of the allergen-specific IgE repertoire
FIG 3. Basophil degranulation at different concentrations of total rIgE.
FACS results of human basophils sensitized with total rIgE concentrations
of 3, 1, 0.2, 0.04, 0.008, and 0.0016 mg/mL, respectively, are shown. The
composition of the rIgE mixture was kept constant with 20% rDer p 2–
specific rIgE consisting of equimolar quantities (6.67% each) of 3 rIgE
clones (G, H10, and H12) recognizing nonoverlapping Der p 2 epitopes and
80% non–Der p 2–specific rIgE (anti-tox). Upper left, Schematic representa-
tion of Der p 2 epitope specificity and affinity of rIgE clones G, H10, and H12;
analog to Figs 1, H, and 2, D. H, High affinity; M, medium affinity; L, low
FIG 4. Basophil degranulation at different concentrations of Der p 2–
specific rIgE relative to non–Der p 2–specific rIgE. FACS results of human
basophils sensitized with different concentrations of Der p 2–specific rIgE
(srIgE) relative to non–Der p 2–specific rIgE (non-srIgE) are shown. The
composition of the rIgE mixtures consisted of equimolar quantities of 3
rIgE clones (E, H10, and H12) recognizing nonoverlapping Der p 2
epitopes and variable concentrations of non–Der p 2–specific rIgE (anti-
tox). The total rIgE concentration was kept constant at 1 mg/mL.
FIG 2. IgE affinities for rDer p 2. A-C, Examples of affinity determinations of rIgE clones binding rDer p 2 with
high, medium, and low affinity, respectively, as obtained by using surface plasmon resonance experiments.
Only the relevant part from where rDer p 2 is injected is shown. D, Summary of affinities obtained of all rIgE
clones (color coded according to the epitope specificities marked in Fig 1, G and H).
J ALLERGY CLIN IMMUNOL
302 CHRISTENSEN ET AL
is of major importance for basophil degranulation and that in
particular basophil reactivity in response to allergen challenge
strongly depends on the interplay between individual IgE
The effect of FceRI-bound allergen-specific IgE concentration
on basophil degranulation was investigated both with regard to
variations in the total IgE concentration at a fixed relative
concentration of allergen-specific IgE and at a fixed total IgE
IgE. Both these scenarios represent naturally occurring variations
in the IgE levels of allergic patients.3Not surprisingly, these ex-
periments showed that changes in the concentration of equimolar
degranulation levels. A novel finding, however, is that when we
changed the relativeconcentrations ofindividual IgE specificities
to levels other than equimolar, which is likely to be the case in
vivo, the maximal basophil degranulation levels were reduced
while leaving basophil sensitivity unchanged. We take this result
to indicate a purely quantitative effect in that the IgE specificity
present in the lowest concentration is the limiting factor in deter-
mining the maximal obtainable number of cross-linking events
during allergen challenge. A direct consequence of this finding
is that although allergic patients? sera might present similar titers
of allergen-specific IgE, they might differ with regard to their
ability to mediate effector cell degranulation in the presence of
In contrast, changes in affinity and/or clonality of the IgEs,
which are both factors affecting the overall binding strength
(avidity) of the IgE/allergen complexes, led almost exclusively to
changes in basophil sensitivity, leaving the maximal degranula-
tion levels largely unaffected. Interestingly, we found that the
presence of only a single antibody of high affinity in the allergen-
specific IgE repertoire is required for the recruitment of an IgE of
even very low affinity into productive FceRI/IgE/allergen com-
plexes. In our experiments basophil degranulation, mediated by
the high-affinity rIgE clone H10 and the low-affinity rIgE clone
H7:H12, was nearly as efficient in terms of basophil sensitivity as
2 high-affinity IgEs, despite the approximately 1000-fold differ-
ence in affinity between these 2 nonoverlapping IgE antibodies.
This experimental observation is in agreement with a model
allergen initially becomes captured by a high-affinity FceRI-
this complex encounters other, even low-affinity, FceRI-bound
IgEs causing efficient cross-linking and effector cell degranula-
tion. For the allergic patient, and in particular the procedures in-
volved in allergy diagnostics, this has the direct implication that
FIG 7. Basophil degranulation at different rIgE clonalities. FACS results of
human basophils sensitized with combinations of 1, 2, or 3 rIgE clones
binding nonoverlapping Der p 2 epitopes are shown. All other parameters
were kept constant; that is, the total IgE concentration was 1 mg/mL, the
relative concentrations were 20% Der p 2–specific to 80% non–Der p 2–
specific rIgE, and equimolar concentrations of the rDer p 2–specific rIgE
clones were used.
FIG 6. Basophil degranulation at different rIgE affinity combinations. FACS
results of human basophils sensitized with different combinations of 2 rIgE
clones having different affinities for rDer p 2 are shown. All other param-
eters were kept constant; that is, rIgE clones were directed against the same
2 epitope clusters, the total IgE concentration was 1 mg/mL, the relative
concentrations were 20% Der p 2–specific to 80% non–Der p 2–specific rIgE,
and equimolar concentrations of the 2 Der p 2–specific rIgE clones were
used. H, High affinity; M, medium affinity; L, low affinity.
FIG 5. Basophil degranulation at different ratios between 2 Der p 2–specific
rIgE clones. FACS results of human basophils sensitized with different
ratios of 2 Der p 2–specific rIgE clones (E and H12) are shown. All other
parameters were kept constant; that is, the total concentration of rIgE was
1 mg/mL, and the relative concentrations were 20% Der p 2–specific to
80% non–Der p 2–specific rIgE.
J ALLERGY CLIN IMMUNOL
VOLUME 122, NUMBER 2
CHRISTENSEN ET AL 303
the presence of low-affinity allergen-specific IgEs in a patient’s Download full-text
serum should not be neglected. A prominent role of low-affinity
IgE antibodies for allergic symptoms could very well be in the
lergic symptoms when challenged with homologous allergen
molecules from related species.18
The requirements for degranulation of effector cells in vitro
have previously been addressed by different means, including
sensitization with complex sera,7,19,20cross-linking with poly-
lymerized IgE,25,26cross-linking with allergen extracts,7and use
of rat effector cell lines,21-25,27in addition to a variety of other
model systems. To our knowledge, the present study is the first
in which factors governing human effector cell degranulation
of well-characterized, allergen-specific IgE antibodies directly
mimicking allergic patients? sera of different compositions.
In current practice the IgE response in allergic patients is
typically measured against complex allergen extracts at the level
of polyclonal serum, either by means of skin prick tests or IgE
titer measurements.28A much more detailed picture of the aller-
gic status of individual patients would emerge if more detailed
the composition of the IgE repertoire. More detailed knowledge
about the development over time of IgE clonality, individual
IgE antibody affinity, and concentration might furthermore lead
to a better understanding of the factors determining the severity
and development (ie, the atopic march29) of allergic diseases.
In memory of Professor Jan Engberg, who was a great contributor and was
supposed to be a coauthor of this article.
We thank Jette Skovsgaard for excellent technical assistance. We thank
Oscar Duffort for making the hybridoma cell lines, Caroline Bolwig for
and Annette Giselsson for making the rIgE concentration determinations.
Finally, we are grateful to Lars Norderhaug for kindly providing the pLNOK
and pLNOH2 vectors.
Clinical implications: Understanding the effect of IgE/allergen
interactions inrelationtoeffectorcell degranulationmight con-
tribute to the improvement of future diagnostic tests based on
individual IgE repertoire compositions.
1. Turner H, Kinet JP. Signalling through the high-affinity IgE receptor Fc epsilonRI.
2. Schroeder JT, MacGlashan DW Jr, Lichtenstein LM. Human basophils: mediator
release and cytokine production. Adv Immunol 2001;77:93-122.
3. Johansson SG, Oman H, Nopp A, Pettersson S. The importance of IgE antibody
levels in anti-IgE treatment. Allergy 2006;61:1216-9.
4. Beyer K, Ellman-Grunther L, Jarvinen KM, Wood RA, Hourihane J, Sampson HA.
Measurement of peptide-specific IgE as an additional tool in identifying patients
with clinical reactivity to peanuts. J Allergy Clin Immunol 2003;112:202-7.
5. Jarvinen KM, Beyer K, Vila L, Chatchatee P, Busse PJ, Sampson HA. B-cell ep-
itopes as a screening instrument for persistent cow’s milk allergy. J Allergy Clin
6. Pierson-Mullany LK, Jackola DR, Blumenthal MN, Rosenberg A. Evidence of an
affinity threshold for IgE-allergen binding in the percutaneous skin test reaction.
Clin Exp Allergy 2002;32:107-16.
7. Shreffler WG, Beyer K, Chu TH, Burks AW, Sampson HA. Microarray immuno-
assay: association of clinical history, in vitro IgE function, and heterogeneity of
allergenic peanut epitopes. J Allergy Clin Immunol 2004;113:776-82.
8. Chua KY, Doyle CR, Simpson RJ, Turner KJ, Stewart GA, Thomas WR. Isolation
of cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay.
Int Arch Allergy Appl Immunol 1990;91:118-23.
9. Norderhaug L, Olafsen T, Michaelsen TE, Sandlie I. Versatile vectors for transient
and stable expression of recombinant antibody molecules in mammalian cells.
J Immunol Methods 1997;204:77-87.
10. Nott A, Meislin SH, Moore MJ. A quantitative analysis of intron effects on mam-
malian gene expression. RNA 2003;9:607-17.
11. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of pre-
defined specificity. Nature 1975;256:495-7.
12. Persson H, Ohlin M. Exploring central and peripheral diversity in antibody evolu-
tion. Mol Immunol 2007;44:2729-36.
13. Derewenda U, Li J, Derewenda Z, Dauter Z, Mueller GA, Rule GS, et al. The crys-
tal structure of a major dust mite allergen Der p2, and its biological implications.
J Mol Biol 2002;318:189-97.
14. Kleine-Tebbe J, Erdmann S, Knol EF, MacGlashan DW Jr, Poulsen LK, Gibbs BF.
Diagnostic tests based on human basophils: potentials, pitfalls and perspectives. Int
Arch Allergy Immunol 2006;141:79-90.
15. Knol EF, Mul FP, Jansen H, Calafat J, Roos D. Monitoring human basophil ac-
tivation via CD63 monoclonal antibody 435. J Allergy Clin Immunol 1991;88:
16. Sainte-Laudy J, Sabbah A, Vallon C, Guerin JC. Analysis of anti-IgE and allergen
induced human basophil activation by flow cytometry. Comparison with histamine
release. Inflamm Res 1998;47:401-8.
17. Aalberse RC, Kleine B, Stapel SO, van Ree R. Structural aspects of cross-reactivity
and its relation to antibody affinity. Allergy 2001;56(suppl):27-9.
18. Aalberse RC, Akkerdaas J, van Ree R. Cross-reactivity of IgE antibodies to aller-
gens. Allergy 2001;56:478-90.
19. Pruzansky JJ, Patterson R. Limiting concentrations of human basophil-bound IgE
antibody required for histamine release. Immunology 1988;64:307-10.
20. Mita H, Yasueda H, Akiyama K. Affinity of IgE antibody to antigen influences
allergen-induced histamine release. Clin Exp Allergy 2000;30:1583-9.
21. Collins AM, Thelian D, Basil M. Antigen valency as a determinant of the respon-
siveness of IgE-sensitised rat basophil leukemia cells. Int Arch Allergy Immunol
22. Collins AM, Basil M, Nguyen K, Thelian D. Rat basophil leukaemia (RBL) cells
sensitized with low affinity IgE respond to high valency antigen. Clin Exp Allergy
23. Gieras A, Focke-Tejkl M, Ball T, Verdino P, Hartl A, Thalhamer J, et al. Molecular
determinants of allergen-induced effector cell degranulation. J Allergy Clin Immu-
24. Marchand F, Mecheri S, Guilloux L, Iannascoli B, Weyer A, Blank U. Human
serum IgE-mediated mast cell degranulation shows poor correlation to allergen-
specific IgE content. Allergy 2003;58:1037-43.
25. Fewtrell C, Metzger H. Larger oligomers of IgE are more effective than dimers in
stimulating rat basophilic leukemia cells. J Immunol 1980;125:701-10.
26. Kagey-Sobotka A, Dembo M, Goldstein B, Metzger H, Lichtenstein LM. Qualita-
tive characteristics of histamine release from human basophils by covalently cross-
linked IgE. J Immunol 1981;127:2285-91.
27. Healicon RM, Foreman JC. Characteristics of histamine release from rat mast
cells in relation to the valency of the stimulating ligand. Immunology 1986;
28. Romano A, Demoly P. Recent advances in the diagnosis of drug allergy. Curr Opin
Allergy Clin Immunol 2007;7:299-303.
29. Hahn EL, Bacharier LB. The atopic march: the pattern of allergic disease develop-
ment in childhood. Immunol Allergy Clin North Am 2005;25:231-46.
J ALLERGY CLIN IMMUNOL
304 CHRISTENSEN ET AL