take-‐home messages 2 take-‐home messages 1
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Clinical Village– Sta.on 3
Specific IgE measurement
Component-‐resolved diagnosis (CRD)
v Purified recombinant or natural allergen molecules can be produced with constant quality and are devoid of cross-‐contamina8ons from other allergen sources.
v Using components, the pa8ent’s sensi.sa.on paBern can be determined at a molecular level.
v Iden8fica8on of species-‐specific and cross-‐reac8ve marker allergens allows to differen.ate between co-‐ and cross-‐sensi.sa.on.
v CRD can improve accuracy of allergy diagnosis and AIT prescrip.on.
Take-‐home messages 2 Component-‐resolved diagnosis (CRD) Diagnosis using recombinant
allergens
Iden.fica.on of allergen molecules
CRD allows for iden.fica.on of disease-‐triggering allergen molecules
Diagnosis using allergen extracts
Iden.fica.on of allergen source
Western-‐blot of different purified recombinant (r) or natural (n) allergen molecules from 8mothy grass.
Purified allergen molecules (=components) are available as well-‐defined reagents for
allergy diagnosis5
„Species-‐specific“ marker allergens
Genuine sensi.sa.on to specific allergen sources
Cross-‐reac.ve marker allergens
Cross-‐sensi.sa.on
Marker allergens aid to differen.ate between co-‐ and cross-‐sensi.sa.on6 CRD improves accuracy of allergy
diagnosis
IgE-‐serology with components disentangles complex clinical reac8vity paJerns and improves accuracy of AIT-‐prescrip8on.7,8
1M. Focke et al., Eur J Clin Invest 2009 2M. Focke et al., Clin Exp Allergy 2008 3A. Casset et al., Int Arch Allergy Immunol 2012
4M. Curin et al., Int Arch Allergy Immunol 2011 5R. Valenta et al., Clin Exp Allergy 1999 6L. Kazemi-‐Shirazi et al., Int Arch Allergy Immunol 2002
7J. Sastre et al., Allergy 2012 8G. Stringari et al., J Allergy Clin Immunol 2014
Limita.ons of allergy diagnosis using allergen extracts
Allergen source Allergen extract
non-‐allergenic components
Allergens
Allergen extracts are mixtures of allergens and non-‐allergenic molecules
In polyreac.ve pa.ents, differen.a.on between co-‐ and cross-‐sensi.sa.on may be impossible using allergen extracts.6
B
Extract-‐based skin prick test results of 10 grass pollen allergic pa.ents2
Pa8ents differ in their molecular sensi8sa8on profiles (A). Variability of allergen composi8on and concentra8on of extracts from different producers and batches may cause considerable discrepancies of skin test results in the same pa8ent (B, example highlighted by red boxes). In addi8on, pa8ents only sensi8sed to clinically irrelevant allergens, e.g., to Phl p 4 (pa8ent 5 in panel A) may be posi8ve in extract-‐based serology.
Molecular sensi.sa.on profiles of 10 grass pollen allergic pa.ents5
A
Take-‐home messages 1 v Allergen extracts are mixtures of allergens and non-‐allergenic components. v Despite standardisa8on, extracts from different producers and batches may
vary considerably in allergen composi.on and concentra.on. v Using extracts, it may be impossible to find the correct diagnosis in pa8ents
with complex sensi.sa.on-‐ and clinical reac.vity paBerns.
Silver-‐stained gels of extracts from birch pollen1 (A), 8mothy grass pollen2 (B), house dust mite3 (C) and dog dander4 (D) from different companies.
A B C D
Allergen extracts from different companies may vary in allergen composi.on and concentra.on
Chris8an Lupinek, Nazanin Najafi, Rudolf Valenta, Ins8tute of Pathophysiology and Allergy Research, Medical University of Vienna, Austria
Clinical Village– Sta.on 3
Specific IgE measurement
Methods for IgE measurement Extract-‐based versus component-‐based serological tests
IgE-‐serology using purified allergen
molecules
Iden.fica.on of allergen molecules
IgE-‐serology using allergen extracts
Iden.fica.on of allergen source
1
Ø Allergen extracts are mixtures of allergens and non-‐allergenic molecules.
Ø IgE-‐serology using extracts allows for iden:fica:on of the allergen source against which the pa:ent is sensi:sed.
Ø Differen:a:on between co-‐ and cross-‐sensi:sa:on is not possible.
Ø Allergen molecules (=components) can be produced as recombinant proteins or purified from allergen extracts.
Ø Using components, the pa:ent’s molecular sensi:sa:on profile can be determined.
Ø This allows to differen:ate between genuine and cross-‐sensi:sa:ons.
1R. Hiller et al., FASEB J 2002 2C. Lupinek et al., Curr Treatm Op:ons Allergy 2016, in press
Take-‐home message Tests for IgE-‐serology can be classified using the following criteria: 1. Extract-‐ vs. component-‐based tests 2. Singleplex vs. mul:plex tests 3. Allergen-‐excess vs. low amount of allergen
used per test
Depending on the respec.ve clinical situa.on, the appropriate test can be selected: v Pa:ent reacts to many different allergen-‐
sources à mul:plex test v From pa:ent‘s history, only few allergen-‐
sources as possible causa:ve allergen-‐sources à singleplex test
v Detec:on of the induc:on of blocking an:bodies by AIT à test employing low amount of allergen, e.g., allergen-‐microarray
v Possible an:body boost by allergen-‐contact à test employing allergen-‐excess
v etc.
Singleplex versus mul.plex technologies
Singleplex tests for the detec.on of allergen-‐specific an.bodies
v For every an.body-‐reac.vity to be tested, one test is consumed.
v For mul.ple tes.ng, an increasing total sample volume is required.
Mul.plex systems for the detec.on of allergen-‐specific an.bodies
Allergen-‐microarray1 Purified recombinant or
natural allergen molecules
v In a single step, the pa.ent‘s an.body reac.vity paOern to a large number of allergen molecules is determined.
v Low sample consump.on (around 30µl).
2
3 Tests employing allergen excess versus tests using low amounts of allergen2
In test systems where large quan..es of allergen are immobilised (when related to levels of specific IgE in serum samples) most specific IgE is bound and can be quan.fied (like in the example shown above).
In test systems where low amounts of allergen are immobilised, like in allergen-‐microarrays, the number of specific IgE-‐molecules in the sample may exceed the number of binding sites on the test (as shown in the example above).
Ø In allergen-‐microarrays, the presence of blocking an:bodies may reduce IgE-‐l e v e l s d e t e c t e d ( B ) . Therefore, induc.on of blocking IgG an.bodies by AIT can be measured.
Ø By contrast, test systems employing an allergen excess are well suited for the quan.fica.on o f an.body responses since the l a rge number o f b i n d i n g s i t e s a l l ow s quan:ta:ve binding of IgE and IgG (D).
Ø In samples devoid of blocking an:bodies, both sys tems y ie ld s imi lar results (A and C).
Absence of blocking an.bodies
High .tre of blocking an.bodies
Allergen excess, e.g.,
ImmunoCAP
Low amount of allergen, e.g., microarray
Chris:an Lupinek, Nazanin Najafi, Rudolf Valenta, Ins:tute of Pathophysiology and Allergy Research, Medical University of Vienna, Austria
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