Current reviews of allergy and clinical immunology

(Supported by an unrestricted educational grant from Genentech, Inc. and Novartis Pharmaceuticals Corporation)
Series editor: Harold S. Nelson, MD
Indoor allergens: Relevance of major allergen
measurements and standardization
Ronald van Ree, PhD Amsterdam, The Netherlands
This activity is available for CME credit. See page 44A for important information.
Major allergen measurements have relevance for the
standardization of allergen extracts for immunotherapy and for
epidemiologic studies into the cause of allergic diseases.
Standardization is still centered around overall IgE-binding
potencies (biological standardization). Major allergen levels
show signicant correlation with IgE-binding potencies, but
ratios of the two can differ 5- to 10-fold between individual
extracts. Major allergen quantities needed for effective and safe
subcutaneous immunotherapy are proposed to be between 5 and
20 mg per maintenance shot. Although this gure is not really
based on dose-nding studies, it has reached the status of a
guiding principle. It is necessary to add major allergen
measurements to standardization requirements to design
adequate dosage schemes and elucidate the dose-response
relationbetweenmajor allergendose andtherapeutic effect. This
will also help clarify to what extent sublingual immunotherapy
requires higher doses of major allergen. Fine specicity of
different assays toward isoforms and other variants of single
allergens often results in diverging allergen measurements.
Standardization should be based on certied major allergen
references and accompanying assays that are cross-reactive
enough to recognize all variants to facilitate comparability. This
will also ensure that primary and secondary prevention
strategies aiming at regulating allergen exposure will stay on
solid ground. (J Allergy Clin Immunol 2007;119:270-7.)
Key words: Major allergen measurements, standardization, biolog-
ical standardization, immunotherapy, optimal dose, assay differ-
ences, sensitization and tolerization thresholds
Measurement of indoor allergens has played a domi-
nant role in epidemiologic surveys that investigate envi-
ronmental risk factors for IgE sensitization and allergic
diseases, such as hay fever and asthma. Such studies have
been the driving force behind strategies for primary and
secondary prevention. With the diversication of the
supply of assays for allergen measurement, their valida-
tion becomes increasingly important. Do different assays
for a single allergen measure the same? In other words, can
results from one survey be compared with those found in
another survey if the source of a major allergen test is
different? Comparability of test results is also an issue for
standardization of allergen extracts used for diagnosis and
immunotherapy. Over the past decades, pressure to intro-
duce allergen standardization on the basis of mass units
of major allergen has increased. Regulatory agencies in
several European countries have started asking allergen
manufacturers for major allergen levels of their products,
and it is expected that this will, in the near future, become a
requirement for registration. If micrograms of company X
do not equal micrograms of company Y, major allergen
measurements will not end the current situation of com-
pany-specic units based on biological standardization
that do not facilitate comparison of potency between
products.
This review deals with the relevance of major allergen
measurements and standardization for the prevention and
treatment of allergic diseases. What is the future place of
major allergen tests in the standardization of allergen prod-
ucts? How can we guarantee comparability of allergen
measurements?
WHY ALLERGEN MEASUREMENTS ARE
IMPORTANT: STANDARDIZATION
OF ALLERGEN EXTRACTS
Allergen extracts have been used for the diagnosis and
therapy of type I allergy for about a century. They are
Abbreviations used
CRM: Certied reference material
SLIT: Sublingual immunotherapy
From the Department of Experimental Immunology, Academic Medical Center.
Disclosure of potential conict of interest: R. van Ree has consulting arrange-
ments with HALAllergy BV, Stallergenes SA, BIAL-Aristegui, and Ventria
Bioscience.
Received for publication October 17, 2006; revised October 26, 2006; accepted
for publication October 31, 2006.
Available online December 14, 2006.
Reprint requests: Ronald van Ree, PhD, Department of Experimental Immu-
nology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam,
The Netherlands. E-mail: r.vanree@amc.uva.nl.
0091-6749/$32.00
2007 American Academy of Allergy, Asthma & Immunology
doi:10.1016/j.jaci.2006.10.033
270
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biological products of high complexity, making them
prone to signicant variability. Recognition of the im-
portance of standardization of diagnostic and therapeutic
extracts has steadily gained ground over the past decades.
Supporting optimal safety of immunotherapy was origi-
nally the main driving force behind the efforts to improve
standardization. This explains why allergen standardiza-
tion is still largely centered around overall IgE-binding
potencies using skin prick tests and in vitro competitive
assays, such as RAST inhibition. European legislation is
exclusively guided by the principle of IgE-binding poten-
cies (Note for Guidance on Allergen Products [CPMP/
BWP/243/96] and European Pharmacopoeia Monograph
on Allergens [1997:1063]). In the United States licensing
by the US Food and Drug Administration of allergen
products is regulated mainly on this basis, but for some
extracts, such as cat hair and pelt, measurement of the ma-
jor allergen in mass units is required.
1
The system of IgE-
based standardization is usually referred to as biological
standardization. IgE-binding potencies reect the poly-
clonal IgE response of a population (eg, represented by
a serum pool of allergic patients) to a mixture of major
and minor allergens in an allergen extract. By denition,
IgE responses of a serum pool are dominated by IgE
against major allergens, whereas IgE against minor aller-
gens will be diluted because it is at low titer in a limited
number of sera in the pool. Consequently, only major al-
lergens make a difference in biological standardization.
For seeing relevant differences in minor allergen content,
the sensitivity of RAST inhibition assays is too low. The
often-used argument that serum pools cover all IgE spec-
icities of an allergic population and consequently cover
both major and minor allergens in standardization proto-
cols is therefore not valid.
2,3
A disadvantage of long-term
use of RAST inhibition is that the composition of serum
pools will change when they are used up and need to be
replaced.
The content of individual major allergens is not
addressed by IgE-based standardization. In the early days
of allergen standardization, knowledge about major aller-
gens was still limited. For the most important allergen
sources, major allergens have now been identied (www.
allergen.org/www.allergome.org). Major allergens are
dened on the basis of frequency of recognition by serum
IgE antibodies; that is, a frequency of greater than 50%
justies a designation as a major allergen.
4
Although the
denition of a major allergen would be improved if the titer
of specic IgE was also taken into account,
5
it is likely that
most established major allergens are of clinical impor-
tance. From this, it follows almost naturally that major
allergens in extracts are the active ingredients of immuno-
therapy, although one could say that this has never actually
been demonstrated. The rst clinical trials using (cocktails
of) recombinant allergens instead of extracts have demon-
strated clinical efcacy, thus providing convincing support
for the importance of major allergens as active ingredients
of immunotherapy.
6,7
Only quantitative data on major aller-
gens can provide insight into the dose-response relation
between active ingredients and efcacy.
BIOLOGICAL STANDARDIZATION VERSUS
MAJOR ALLERGEN MEASUREMENTS
Major allergens of the most important indoor allergen
sources (house dust mite and cat) have been identied and
characterized in detail. For house dust mite, they are the
group 1 allergens Der p 1 and Der f 1 and the group 2
allergens Der p 2 and Der f 2.
8
For cat, only 1 allergen is
considered to be a true major allergen, Fel d 1. It has been
demonstrated that IgE responses to cat correlate closely
with those to Fel d 1 and that Fel d 1 can inhibit more
than 80% of IgE binding to cat dander extract in RAST
inhibition (RI).
9
One of the arguments against standardiza-
tion in mass units of major allergen has been that there
is a poor correlation with biological activity (ie, with IgE-
binding potencies). On the basis of the dominant role of
Fel d 1 as a major allergen, the opposite would be ex-
pected. Fig 1 illustrates that there is a signicant correla-
tion between RI and mass units of Fel d 1 for a series of
different cat extracts by using Spearman rank correlation
(Rs 5 0.658, P < .01). Nevertheless, the ratio between
biological potency and mass units of Fel d 1 varied up to
a factor 5.
House dust mite extracts are far more complex with
respect to allergen composition than cat extracts. Despite
this, RAST inhibition for house dust mite correlates nicely
with mass units of Der p 1 (Rs 5 0.785, P < .01) and in
particular Der p 2 (Rs 5 0.876, P < .01), conrming the
important role of Der p 1 and Der p 2 in the IgE response
to house dust mite (Figs 2 and 3). As observed for cat, here
the ratio between biological activity and micrograms of
major allergen varies signicantly, roughly up to a factor
8 for Der p 1 and a factor 4 for Der p 2. Those in favor
of biological standardization will say that similar major
allergen concentrations can translate into signicantly dif-
ferent biological activities. Advocates of standardization
in mass units of major allergens will argue that biological
FIG 1. Correlation between biological standardization and Fel d 1
measurement. IHR, In-house reference.
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van Ree 271
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standardization does not reveal 5- to 10-fold differences in
the content of the major allergens. Both statements are cor-
rect, and therefore standardization protocols for allergen
products should preferably provide both pieces of infor-
mation (ie, IgE-binding potency and mass units of major
allergens).
2,3,10
DIFFERENCES BETWEEN ALLERGEN
PRODUCTS WARRANT MAJOR
ALLERGEN MONITORING
Is it worth the trouble to increase the burden of allergen
standardization by including major allergen measure-
ments as a requirement next to biological standardization?
A survey was performed on a series of competitor
products for sublingual immunotherapy (SLIT) to illus-
trate the relevance of establishing major allergen content.
SLIT products for the treatment of birch pollen allergy
were analyzed for their Bet v 1 content. Among the 6
products tested, signicant differences were observed
(Fig 4). Between the extremes, there was more than a
10-fold difference. When house dust mite products were
analyzed, Der p 2 concentrations varied over almost 1 or-
der of magnitude as well (Fig 5). For the other major aller-
gen, Der p 1, differences detected were up to 25-fold.
Some products had Der p 1/Der p 2 ratios close to 1,
and others were highly enriched for the group 1 allergen.
This can most likely be explained by the choice of source
material (ie, puried whole-body vs whole-culture ex-
tract). The group 1 allergen is a digestive enzyme (a cys-
teine protease) found mainly in fecal particles, a major
component of whole-culture source material.
These analyses illustrate that active ingredients of these
biopharmaceutical products vary signicantly without
being declared on the insert of the product. Because of
the lack of good dose-nding studies, especially for SLIT,
it cannot automatically be concluded that products with
the highest major allergen content are the best. Only by
measuring major allergen levels and performing dose-
nding studies can true dose-response relations be estab-
lished, both with respect to efcacy and safety.
HOW MUCH MAJOR ALLERGEN IS NEEDED
FOR EFFECTIVE TREATMENT?
The 1998 World Health Organization position paper
on allergen immunotherapy reported on optimal doses for
FIG 3. Correlation between biological standardization and Der p 2
measurement. IHR, In-house reference.
FIG 4. Bet v 1 content of 6 competitor products for SLIT.
FIG 2. Correlation between biological standardization and Der p 1
measurement. IHR, In-house reference.
FIG5. Der p 1 and Der p 2 content of 7 competitor products for SLIT.
J ALLERGY CLIN IMMUNOL
FEBRUARY 2007
272 van Ree
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injection immunotherapy.
11
The optimal dose is dened as
the dose of an allergen vaccine inducing a clinically rel-
evant effect in the majority of patients without causing
unacceptable side effects. According to the position
paper, this optimal dose should be the maintenance target
dose for all patients. On the basis of a limited number of
studies, it was concluded that doses of 5 to 20 mg of major
allergen are optimal doses for house dust mite, cat, rag-
weed, and Hymenoptera venom.
12-17
This range between
5 and 20 mg of major allergen has since then been pro-
moted as the target for efcient immunotherapy not only
for those allergens but also for other allergen sources,
such as grass and birch pollen, for which supporting clin-
ical data were not yet available. The claimed optimal dose
roughly adds up to 50 to 250 mg of major allergen per year
(on the basis of monthly maintenance shots). There are
several reasons why a designation as an optimal dose
should be interpreted with some care. The advocated
dose is an average, which implies that some patients might
respond at a lower dose, both in terms of efcacy and
safety. An optimal dose suggests maximum efcacy at
an acceptable level of side effects. Actual dose-nding
studies were only performed in a limited number of trials,
indicating that a rm conclusion on an optimal dose can-
not really be drawn.
14,17
From most studies, it can only
be concluded that, compared with placebo, a signicant
level of efcacy was reached at a certain dose that was
accompanied by an acceptable level of side effects.
Between the studies, no single method was used to moni-
tor efcacy, and the percentage improvement over placebo
needed for efcacy was not dened uniformly. Some stud-
ies were done in patients with hay fever,
13,15
and some
were done in asthmatic subjects.
12,14-17
Last but not least,
assays used to measure major allergens were not identi-
cal, were sometimes poorly validated, or were even not
described at all.
Since the publication of the 1998 position paper, many
additional clinical trials have been performed that report
concentrations of major allergen in therapeutic extracts of
pollen (birch, grass, ragweed, olive, and Parietaria spe-
cies), house dust mite, and cat.
18-30
Only 2 studies inves-
tigating immunotherapy for cat allergy
23,25
and 1 for dog
allergy
30
included a certain degree of dose nding. The
2 cat studies explored whether a dose of less than 15 mg
of Fel d 1 was as effective as 15 mg. This turned out not
to be the case. The dog study reported a similar outcome
for Can f 1. Whether higher doses would prove more effec-
tive was not investigated. In the other studies the dose of
major allergen per maintenance shot was close to or within
the range of 5 to 20 mg. In only 2 clinical trials (both for
olive pollen allergy) have higher concentrations been
used (66 and 45 mg of Ole e 1, respectively).
22,28
Generally speaking, the so-called optimal dose has seen
the light by applying major allergen measurements to
existing dosing schemes of conventional treatments and
subsequently designating these doses as optimal.
After the publication of the 1998 position paper, clinical
trials for SLIT were published that also reported major
allergen content.
31-44
Again, dose-nding studies were not
performed. Yearly doses reported ranged from 75 mg to
56 mg of major allergen, and in most cases the dose was
signicantly higher than that administered during injec-
tion immunotherapy (10- to 100-fold). Whether the doses
used were optimal cannot be answered based on the
currently available data.
On the basis of the available data for injection and
SLIT, it can only be concluded that an optimal dose is
actually still unknown. As pointed out, this is not only
because dose-nding studies were not performed but also
because major allergen measurements used were validated
poorly or not at all. Trials with (cocktails of) recombinant
allergens will provide more reliable data because quanti-
cation of major allergens can be performed simply on the
basis of validated assays for protein quantication. In case
of hypoallergenic variants (mutants/isoforms) or frag-
ments (peptides), biological standardization will not even
be possible anymore, and protein measurements are the
method of choice.
45,46
The rst published clinical trial
with nonmodied recombinant allergens used a 40-mg
cocktail of 5 major grass pollen allergens, 3 at 10 mg
and 2 at 5 mg.
6
The motivation of this choice of concentra-
tion was not described in the article, but it can safely be
assumed that decisions were at least partly made on the
basis of the 5- to 20-mg position paper statement. Again,
however, dose-nding studies were not performed.
STANDARDIZATION IN MASS UNITS:
IT SOUNDS SIMPLER THAN IT IS
New approaches for standardization of allergen ex-
tracts, such as HPLC and mass spectrometry, have been
suggested,
47
but at present, standardization in mass units
largely depends on sensitive and specic immunoassays
and on the availability of adequate standards for quanti-
cation. For standardization of allergen products, sensitiv-
ity is usually not an issue because extracts contain high
quantities of major allergen, far greater than the measuring
range of most immunoassays. Specicity is a far more
complex issue for allergen measurements. On the antibody
side, mouse mAbs and rabbit polyclonal antibodies are
available. The obvious advantage of mAbs is that they
are an innite and highly reproducible source of anti-
bodies with a high level of specicity.
48,49
Their high
degree of specicity is also their major disadvantage.
Some mAbs fail to detect specic isoforms. This has, for
example, been reported for the major house dust mite
allergen Der p 2.
50,51
A similar problem is linked to the
practice of allergen manufacturers to use mixtures of
different species, for example Dermatophagoides ptero-
nyssinus and Dermatophagoides farinae or between 4
and 10 grass species. In many cases mAbs will detect ho-
mologous major allergens from different species with dif-
ferent sensitivity. This makes it difcult to measure mass
units of major allergen in a mixture. The only possibility
is to measure each allergen source individually before
mixing by using a dedicated standard for each of them.
This is laborious and often not possible because source
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materials are mixed before extraction. Counterintuitively,
allergen standardization is usually better served with
highly cross-reactive mAbs than with extremely specic
ones. For this reason, monospecic polyclonal antibodies
might sometimes have preference over mAbs.
49
Finally,
the presence of fragments, aggregates, or both of major
allergens might also inuence the detection of major
allergens.
52
What type of standards should be used for allergen
standardization? Between 1984 and 1989, 5 extract-based
international reference standards were established under
the auspices of the World Health Organization for rag-
weed, timothy grass pollen, D pteronyssinus house dust
mite, dog, and birch pollen, respectively.
53-58
These stan-
dards were meant to be used as reference materials by
allergen manufacturers and scientists to calibrate their
in-house references used for biological standardization
and environmental allergen measurements, respectively.
Although some scientists have used these certied refer-
ence materials (CRMs) over the past 25 years, allergen
manufacturers never really implemented the system of
international units linked to the use of these CRMs. The
main reason for the limited success of these World
Health Organization references is that they did not really
facilitate the desired comparability of products on the
basis of uniform international units. This cannot actually
be expected from protocols based on IgE potency tests
that use variable serum pools to standardize allergen
extracts with variable ratios of major allergens not identi-
cal between companies and to those of the CRMs. With
the advent of recombinant major allergens, the idea was
put forward to establish puried recombinant major aller-
gens as new-generation CRMs. This idea was investigated
in the CREATE project funded by the European Union.
59
In the framework of this project, candidate reference
materials were produced for house dust mite (Der p 1, Der
p 2, Der f 1, and Der f 2) and for birch (Bet v 1), grass (Phl
p 1 and 5), and olive (Ole e 1) pollen. These recombinants
were compared physicochemically and immunologically
with their natural counterparts. The protocol also included
the evaluation and comparison of a minimum of 2
available sandwich ELISAs for the measurement of the
respective allergens. The projects outcome will be
extensively reported elsewhere. For this review, however,
several aspects are worth mentioning. Some recombinant
allergen preparations proved to be dominated by un-
folded, incorrectly folded, and/or aggregated allergen.
Such reagents turned out to be good enough to pick up
allergen-specic IgE in diagnostic tests, in which allergen
is used under saturating conditions. A variable mixture of
correctly folded and unfolded allergen, aggregated aller-
gen, or both is, however, not compatible with the use of
these materials as the CRM in allergen standardization.
Two well-folded allergens from the CREATE project
(recombinant Bet v 1 and recombinant Phl p 5a) were
selected for follow-up studies that will have to lead to
their establishment as the rst 2 new-generation CRMs
for allergen products. Der p 2 is expected to follow
shortly.
The evaluation of the sandwich ELISAs within the
CREATE project once more illustrated that differences
in the ne specicity of antibodies, in combination with
either a natural or a recombinant puried standard, can
have a great effect on the outcome of the assay. In other
words, sometimes different assays for the same allergen
produced signicantly different results, partly depending
on the choice of natural or recombinant standard. The
outcome of a sandwich ELISA depends on a complex
matrix of parameters. A recombinant standard is a single
isoform; extracts to be analyzed contain mixtures of
isoforms. Specicity or preference of antibodies for the
isoformchosen as the standard will lead to underestimation
of the allergen in extracts. Specicity or preference for
another isoform will make the standard useless or lead to
overestimation of the allergen content of extracts. If a
recombinant standard contains a signicant share of poorly
folded allergen molecules, this will also lead to overestima-
tion of allergen levels in extracts, assuming that most mAbs
have a preference for well-folded allergen. However, the
opposite is also possible because antibody reagents might
have been obtained through immunization with poorly
folded recombinant allergen. Why then not use natural
allergens as a standard instead of recombinant allergens?
They are composed of multiple isoforms and are usually
correctly folded. The presence of multiple isoforms in both
the standard and extracts only prevents incorrect calculation
of allergen levels if the isoformcomposition of the standard
and extract is (close to) identical. Purication protocols can
also inuence the isoform composition. The choice for
recombinant standards is largely motivated by the fact that
theycanbe producedinlarge quantities withlittle variability
between subsequent batches. A prerequisite, however,
for their application in standardization is that they are
correctly folded and that immunoassays for their detection
are highly cross-reactive between isoforms (ie, that they do
not measure natural and recombinant allergens differently).
For the 2 allergens chosen from the CREATE project to be
established as the rst newCRMs, these requirements were
met at an acceptable level. Finally, it is important to realize
that for the sakeof comparability, future recombinant CRMs
will need to be linked to validated reference assays.
10
WHY ALLERGEN MEASUREMENTS ARE
ALSO IMPORTANT: EPIDEMIOLOGY
OF ALLERGIC DISEASE
Measurement of major allergens is not only relevant for
quality control and standardization of diagnostic and
therapeutic allergen extracts. Measurement of environ-
mental exposure is pivotal for elucidating the relation
between allergen exposure and sensitization or tolerance.
Clearly, there is a dose-response relation between early-
life exposure to indoor allergens and the development of
respiratory allergies, such as hay fever, and asthma.
60
Not
so long ago, this relationship was thought to be a simple
linear dose-response curve, with higher exposure result-
ing in more allergy. Obviously, zero exposure excludes
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development of allergy.
61-63
Primary and secondary pre-
vention strategies have been based on this premise. At
least for cat allergen, the relation between exposure and
sensitization has now proved to be bell shaped instead of
linear.
64
In other words, the risk of development of allergy
to cat rst goes up with increasing exposure but starts de-
creasing again at higher allergen concentrations. No expo-
sure still prevents allergy, and above a certain threshold,
tolerance seems to be induced. Whether this is the result
of high allergen exposure or also of coexposure to some-
thing else, such as endotoxin, is still unclear.
65-67
The
bell-shaped relation has not yet been found for house
dust mite.
68
This all illustrates the importance of mea-
surement of environmental allergen exposure. Without
reliable epidemiologic studies addressing exposure to
allergens and other environmental factors, sound proto-
cols for prevention cannot be implemented. Obviously,
if different assays produce signicantly different results,
targets for primary and secondary prevention will be con-
icting (Fig 6). It is of the utmost importance that we all
measure the same not only for quality control and stan-
dardization of extracts used for diagnosis and therapy but
also for measurement of environmental exposure.
FOOD ALLERGENS AS INDOOR ALLERGENS?
One of the major questions in food allergy is how
sensitization at a young age occurs. A large number of rst
reactions to food seem to occur during rst exposure.
69
Which exposure then induced sensitization? Is it transpla-
cental exposure,
70
exposure to food allergens in breast
milk,
71
or exposure to food allergens in ointments,
72
or is
it the presence of food proteins in house dust. A limited
number of publications have actuallylookedat the presence
of food allergens in house dust, and some of those have
been shown to be present in concentrations similar to those
found in major inhalant allergens.
73
Fromother disciplines,
such as food safety, validated assays for the detection of
food allergens are available. It seems useful to apply these
to the analysis of house dust samples as well.
CONCLUDING REMARKS
At present, immunotherapy protocols are designed on
the basis of an array of inconsistent company-specic
units that have no direct link to major allergen content. It is
well accepted that effective immunotherapy can only be
achieved above a certain threshold of major allergen per
maintenance shot. Although support from dose-nding
studies is largely missing, between 5 and 20 mg of major
allergen is generally believed to be an optimal dose. Until
the expected replacement of extracts by puried recom-
binant allergens becomes reality, adequate and accurate
dosing of major allergens can only be achieved if these
active ingredients of therapy are quantied in the
allergen extracts used for treatment. Future regulations
for immunotherapy products should therefore include
major allergen measurements in addition to biological
standardization. A system to support this using recombi-
nant major allergens as certied references in combination
with recognized (cross-reactive) reference assays is cur-
rently under development. Measures to control indoor
allergen exposure as a strategy to prevent allergy will also
benet from such improvements in the eld of allergen
standardization by ensuring that the results from different
epidemiologic surveys are comparable.
I wish to acknowledge the partnership from the European Union
funded CREATE project (G6RD-CT-2001-00582; partners are listed
in van Ree
59
) for sharing their expertise and opinions in the eld of
allergen characterization and standardization over the past years
within and outside the project.
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FIG 6. Schematic representation of inuence of assay variation of
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Arrows indicate target exposure for prevention. B, Bell-shaped re-
lation between exposure and sensitization. Arrows indicate target
exposure for prevention.
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