Научно-практический семинар

по молекулярной аллергологии, 31 октября

2019 г.:

„ALEX – новая тест-система для

молекулярной аллергодиагностики“

Томас Хорн

www.macroarraydx.com 1
 MADx – Macro Array Diagnostics

▪ Foundation 2016

▪ Based in Vienna, Austria

▪ Private company with public

funding

▪ 20 employees, 150 years of allergy know how

▪ IVD Manufacturing (CE products), focus on allergy

▪ ISO 13485 & 9001 certification since June 2017

▪ Market entry with CE marked IVD product in August 2017

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What is ALEX?
• ALEX – Allergy Explorer
• Multiarray, chip-based diagnostic system to
detect specific IgE against 282 different allergens
in one single run out of one single patient
sample (100 µl)

• Includes both - allergen extracts and allergen

components

• Blocking of CCD antibodies is included in the test

procedure

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Why ALEX?
• Many (adult) patients are sensitized to multiple allergenic sources

• Use of allergen components reveals information which cannot be

obtained with SPT or extract based IVD

• Chip-based screening reduces risk of „missed“ sensitizations

• Comprehensive allergen portfolio supports risk assessment of patient

(Ara h 8 versus Ara h 2/Ara h 6)

• CCD blockage improves clinical specificity of IgE testing

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Where is ALEX sold?

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Example patient result with ALEX
• Patient is sensitized to grass pollen major allergens, also Cynodon,
and to the cross reacting Profilin

• The patient has a genuine sensitization against the birch tree family
(Bet v 1) and might react against birch, elder, hazel etc.
• Patient also has a genuine sensitization against Cypress tree and
Olive tree (Cup a 1, Ole e 1)
• Patient is sensitized to Parietaria and Artemisia, but not to Ambrosia
• The peanut and soy sensitizations are related to the PR-10, and not
to storage proteins
• Same goes for Latex – no genuine latex sensitization

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Principle of ALEX

• ALEX is a solid-phase immunoassay

• 283 test results
• Allergen extracts or molecular allergens
• Balanced allergy panel
• Inhalant allergens
• Food allergens
• Insect venoms
• Latex
• Comprehensive view of a patient’s
sensitization status

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ALEX Technology / IP
• EP Patent was filed March 2016
• PCT/EP2017/057481 was renewed this year
• The patent was published September 2017
• Nationalization phase from 2018

1 mm

Mite

Birch

Cat
Two-step test manufacturing

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Cross-reactive carbohydrate
determinants (CCD)
• CCDs are sugars located on allergens (very similar between species).
• Plant, insects and molluscs
• CCDs are able to induce production of IgE
• Clinically irrelevant, but lead to results of extract based tests that are falsely elevate!

9
CCD antibody blocking efficiency
Serum A Serum A Serum B Serum B

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Why reducing CCD antibodies?

“We conclude that the use of a CCD inhibitor in in vitro assays is a viable option to mitigate the influence of
anti-CCD antibodies on the measured level of asIgE immunoglobulin, which increase the reliability of
testing particularly in cases displaying multiple allergies.”

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Why reducing CCD antibodies?

“Elimination of CCD-specific IgE antibodies by means of a synthetic CCD-blocker drastically reduced the
number of false-positive in vitro test results without compromising the sensitivity for relevant IgE
interactions. Thus, the herein described CCD-blocker constitutes a valuable tool for increasing the test
specificity of routine in vitro allergy diagnosis.”

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What do you need to run ALEX?

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Assay principle
Preparation Step 1 Washing

Serum Wash unbound

Blood collection and incubation antibodies away
serum preparation 120 min 3x 5 min

Step 2 Washing Step Step 3 Analysis

Detection- Wash unbound Enzymatic Image acquisition,

antibody detection antibodies (color)-reaction analysis and report
30 min away 5 x 5 min 5-10 min ˂ 1 min

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After Test Procedure: Read Image from ALEX array

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Approve results, export reports (CSV, ASTM, PDF…)

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Big Question:
• How does ALEX perform in comparison with other allergy test
systems?

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We cannot expect equal results with different
testing systems…

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We cannot expect equal results with different
systems…

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We cannot expect equal results with different
systems…

“From a practical point of view and to avoid major errors in the diagnosis and treatment of
allergic disease, it is important to bear in mind that the results obtained with different
specific IgE detection methods are not equivalent.”

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Reason for discrepant Results with different
methods

• method specific reasons (allergens/isoforms, detection system,

calibration system, etc.)
• statistical reasons (important for results near the cut-off)
• specimen related reasons (different blood samples, t1 ≠ t2)
• Clinically false positive results due to irrelevant sensitizations (CCD)
• errors related to the assay handling

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Different Ways to describe Assay Performance
• Analytical Performance:
• Precision
• Accuracy
• LoD, LoB, LoQ
• Carry over, stability...
• comparison with reference methods

• Clinical Performance:
• Sensitivity, Specificity in clinical trials
• PPV, NPV

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How does ALEX compare with
ImmunoCAP

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in-house comparison data ALEX vs. ImmunoCAP

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Comparatibilty in published studies

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Statistical errors at cut-Off level

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effect of statistics on results near Cut-Off

Due to statistical reasons, some

results will be below cut-off

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Effect of anti-CCD Antibodies
• Nut Study (Data on File)
• 19 patients with sensitizations to different nuts/peanut
• data from ImmunoCAP, ISAC, ALEX, DBPCFC

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...Are positive results always clinically relevant?

zoomed-in on low values

…many positive results with

ImmunoCAP could not be
confirmed by provocation test
- green dots

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Can CCD‘s also occur in ImmunoCAP Allergen
Components?

CONCLUSIONS:
Cellulose used as a solid-phase allergen in ImmunoCAP carrier can contain varying amounts
of CCDs sufficient to cause false-positive test results up to 2 kUA/L with nonglycosylated
recombinant allergens in patients with high levels of anti-CCD IgE antibodies.

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External Quality Scheme UKNEQAS

ALEX
ImmunoCAP

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How does ALEX peform in comparison with
ImmunoCAP ISAC?

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ALEX – ISAC (1)

n = 198

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ALEX – ISAC (1)

- Kappa values around 0.90

“In conclusion, the results of

ALEX correlated well with ISAC
assay.“
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ALEX – ISAC (2)

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ALEX – ISAC (2)

“The comparison between ISAC

and ALEX sIgE detection against
common allergen components
showed high overall agreement.”

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Development roadmap

End of 2019 End of 2019 Mid 2020

Raptor Cloud Server ALEX Version 2 (CE) ALEX Automation System

▪ Cloud based data storage ▪ Improved allergen panel ▪ Fully automatic

(optional) processing
▪ Fixing of few existing
▪ Improved intelligence for gaps in molecular ▪ Batch mode
image analysis allergen panel
▪ 50 samples per run
▪ App store for ▪ Improved assay design
interpretation, ▪ 2 runs per day
visualization and decision ▪ Higher sensitivity and
support dynamic range ▪ 30.000 sIgE results per
day

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Thank you for your attention!

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