9 lattanzio myco_key_ws_multi-mycotoxin_screening

Veronica M.T. Lattanzio, A.F. Logrieco, V. Lippolis, N.
Nivarlet, A. De Girolamo, M. Pascale, S. De Saeger
veronica.lattanzio@ispa.cnr.it
The 3rdMycoKey technological workshop :
Prevention and integrated control of mycotoxins in wheat, barley and maize
Agroscope, Zurich, June 18-19, 2019
Multi-mycotoxin screening methods:
current trends and innovative solutions

Regulation N. 178/2002/EC (General Food Law)
 the EU introduced the General Food Law (GFL) in 2002, fixing the
principles, obligations and definitions that apply in the field of food
safety.
 A general principle of the GFL is that operators in the feed and
food business have the primary responsibility for food safety at
all stages of production, processing and distribution, while
competent authorities shall monitor, enforce and verify this
responsibility through the operation of national surveillance and control
systems.
Internal control systems in production facilities, such as those
based on HACCP, may play an important role in the reduction of food
safety risks.
The demand for simplified and rapid test methods at critical control
points in the food/feed chain has never been greater
R&D trend – multiplexing

MycoKey Survey on rapid methods for mycotoxin screening
 Launched through the MycoKey web site and newsletter in February 2018
 The survey aimed at collecting and sharing current experience and future
needs on rapid methods for mycotoxin screening.
 Based on a Google form requiring 10-15 minutes to be filled
 21 questions dealing with:
- Current status of applied methods for mycotoxin analysis
- Future needs and expectations with respect to rapid methods
The state of art in operational environments

MAIN OUTCOMES (I)
- 81 filled forms collected
Target mycotoxins and commodities
- Regulated mycotoxins represent the 68% of total
analysis
- Cereals and derived products are the most widely
analyzed commodities (44%), followed by feed (21%)
Currently used methods
- 91% of the respondents use validated
(CEN/AOAC/ISO - or in house validated) methods
- laborious protocols, cost of equipment and reagents
are indicated as major drawbacks
PUBLIC
(reserach)
laboratories
PRIVATE
laboratories
Governmental/of
ficial control
Profile of the respondents
58%
25%
17%

Analysis time
(min)
% responses
< 10 22 %
10 - 30 44 %
30 - 60 17 %
No preferences 17 %
Test sample
size (g)
% responses
< 1 8 %
1 – 5 33 %
5 - 10 27 %
10 - 50 31 %
> 50 1 %
Cost per
analysis (€)
% responses
< 5 34 %
5 – 10 43 %
> 10 7 %
No preferences 16 %
LOQ % responses
~ ML 38 %
< ML 56 %
>ML 6 %
Multiplex detection
Availability of a reference material in the analysis kit
Easy calibration and verification of functionality
Validation/performances verification: 84% of respondents expressed interest in
participation in collaborative trials
MAIN OUTCOMES: desired characteristics for rapid methods for mycotoxins

MycoKey Work Package 3 – Mycotoxin Monitoring
Task 3.2 – Development of multi-mycotoxins test kits for
on-site screening
Dipstick immunoassays for rapid multi-mycotoxin analysis
DNA aptamer based strip tests for multi-mycotoxin analysis
Fluorescence polarization immunoassays for the
determination of Fusarium toxins (including modified forms)
Real-time Electrochemical Profiling for the detection of
AFB1 and OTA

MycoKey Work Package 3 – Mycotoxin Monitoring
Task 3.2 – Development of multi-mycotoxins test kits for
on-site screening
Dipstick immunoassays for rapid multi-mycotoxin analysis:
prototype multiplex strip test for the determination of
DON/ZEN/FBs in cereals (3-mycosensor)
DNA aptamer based strip tests for multi-mycotoxin analysis
Fluorescence polarization immunoassays for the
determination DON, 3-acetyl-DON, 15-acetyl-DON and DON-3-
glucoside in wheat; 2) T2, HT2, T2-glucoside and HT2-glucoside in
wheat
Real-time Electrochemical Profiling for the detection of
AFB1 and OTA

 Concept of screening method:
 Negative samples are classified as such and NOT further
analysed.
 Suspect samples need to be re-analysed using
confirmatory methods
 False suspect samples of truly negative samples have no
impact on consumer safety… BUT…a high number of false
suspect results may question the benefit of such a
screening test
SCREENING METHODS
Commission Decision (EC) No 2002/657
Commission Regulation (EU) No 519/2014
STC
 Screening test are designed to evaluate the
presence/absence of the mycotoxin of interest
against target levels (STC: screening target
concentration)
 To determine legal compliance (STC = ML)
 To achieve operational and logistical goals
(f.i. process management, STC defined by
the operator)

The validation procedure is focused on:
 the determination of specific cut-off levels ensuring a
maximum rate of false-negative results of 5%,
 the assessment of the rate of false-suspect results.
Specific guidelines for validation and verification of
mycotoxin screening methods are set in
Commission Regulation (EU) No 519/2014.
Include:
- Sensitivity
- Selectivity
- Precision
The regulation establishes that the “aim of the validation
is to demonstrate the fitness-for-purpose of the
screening method”
SCOPE: includes bioanalytical methods based on immuno-recognition or
receptor binding (ELISA, dipstick, lateral flow devices…) and direct MS or LC-MS
methods
Methods of which result of the measurement is a numerical value

Minimum
Sample Set
20 homogeneous negative
samples*
20 homogeneous samples at
STC
Additional sets of 20 homogeneous samples at other levels can be added
Intermediate
precision conditions
spread over 5
different days
Negative sample: sample known to be “free” of the mycotoxin of
interest
or sample containing up to 1/5 of the Screening Target Concentration
SINGLE LABORATORY VALIDATION
The kit responses for negative and contaminated samples are
taken as basis for the calculation of required parameters

Fluorescence polarization immunoassays for
the quantitative determination of
DON and modified forms
and T-2/HT-2 and modified forms in wheat
O
O
OH
CH3
CH2
CH3
OH
O
OH
O
O
O
CH3
CH2
CH3
OH
O
OH
CH3
O
O
O
O
CH3
CH3
OH
O
OH
O
OH
OH
OH
OH
O
O
H
OH
H
OCOCH3CH3
CH2
OCOCH3
H
CH3
H
OH(CH3)2CHCH2COO
O
O
H
H
OCOCH3
CH3
CH2
OCOCH3
H
CH3
H
O O
OH
OH
OH
OH
H(CH3)2CHCH2COO

Fluorescence Polarization Immunoassay
Concept and technology
FPIAs are homogeneous fluorescence immunoassays based on the competition in solution
of free antigen (Ag) with a fluorescently tagged antigen (Ag-FL) for an Ag-specific
monoclonal antibody
The FP measures the degree of depolarization of the emitted light
originating from a tracer excited with a plane-polarized light.
The degree of polarization (P) increases due to the reduced rotation of
the bound fluorescent trace.
P is inversely related to free antigen content in solution that competes
with the tracer.
Fast rotation
In the absence of
mycotoxin
1. Antibody in
the tube
+
+
3. Add tracer
and
incubate
2. Add sample
extract
4. Measure
fluorescence
polarization
In the presence of
mycotoxin
All the tracer binds
the antibody
Less of tracer binds
the antibody
10 100 1000
0
50
100
150
200
250
FluorescencePolarization(mP)
[Mycotoxin]
Slow rotation

 Synthesis – purification – identity confirmation of tracers for
DON, T-2, HT-2 (fluorescein derivatives)
Antibody selection (antibody-tracer binding studies): 4 monoclonal
antibodies (Mabs) tested for DON, 10 T-2-glucoside MAbs, one HT-2
MAb and 2 T-2 MAbs tested for T-2 and derivatives
Evaluating performances of competitive immunoassays in standard
solutions
Cross reactivity studies
Optimization of extraction protocols and evaluation of matrix
effects
Phases of Immunoassay Development
Lippolis V et al. (2006). Journal of Food Protection, 69, 2712-2719.
Lippolis V et al. (2011). Analytical and Bioanalytical Chemistry, 401, 2561-2571.
Lippolis V et al. (2019) Toxins, accepted for publication
Mycokey deliverable report D3.4 – available soon at EU CORDIS

Method Protocol
Sample preparation and FPIA for the determination of
DON, 3Ac-DON, 15Ac-DON and DON3G in WHEAT.
Total analysis time: 10 min
EXTRACTION
100 mL of PBS
blending 2 min
FILTRATION
WEIGHING
25 g
Measure FP
(blank)
Measure
FP
Tracer addition
(DON-FL)
DON-MAb
addition
Incubation
2 min
SamplePreparationFPIA

Method Protocol
of T-2, HT-2, T-2G and HT-2G – Protocol A
EXTRACTION
100 mL of
CH3OH:H2O
(90:10), 2 min
FILTRATION
WEIGHING
25 g
Measure FP
(blank)
Measure
FP
Tracer addition
(HT2-FL)
HT2-MAb
addition
Incubation
5 min
FILTRATION
DILUTION
(1:5 with 4% NaCL
solution)

Method Protocol
of T-2, HT-2, T-2G and HT-2G – Protocol B
EXTRACTION*
100 mL of water, blending 2 min
WEIGHING
25 g
Measure FP
(blank)
Measure
FP
Tracer addition
(HT2-FL)
HT2-MAb
addition
Incubation
5 min
FILTRATION
* Lattanzio V. et al (2009) Anal. Bioanal. Chem. 395, 1325–1334

BLANK STC
validation level (µg/kg) n.d. 1750
Mean response (µg/kg) 116 1815
SD repeatability (µg/kg) 54.0 115.1
SD intermediate precision
(µg/kg)
59.4 133.1
RSDr (%) 46.7 6.3
RSDip (%) 51.4 7.3
cut off 1585
False positive rate (%) < 0.1%
Naturally contaminated samples
Comparison of contents of DON and its
modified forms (expressed as sum) in
naturally contaminated samples analyzed by
FPIA and LC-MS reference method.
naturally contaminated samples (n=23)
blank samples (n=8)
No false positive results
Technical Specifications – Analytical Performances
Fluorescence Polarization Immunoassay for simultaneous determination of
DON, 3Ac-DON, 15Ac-DON and DON3G in WHEAT
Performance evaluation according to Regulation (EU) No 519/2014
CUT OFF level: the response, signal or concentration, obtained with the
screening method, above which the sample is classified as “suspect”.
Cut Off: response value ensuring a rate of false negative results < 5%

Protocol A (CH3OH:H2O) Protocol B (water)
BLANK STC BLANK STC
validation level (µg/kg) n.d. 100 n.d. 100
Mean response (µg/kg) 17 115 21 104
SD repeatability (µg/kg) 1.9 5.7 2.9 9.7
SD intermediate precision
(µg/kg)
2.9 10.9 3.3 13.4
RSDr (%) 16.4 4.9 13.6 9.3
RSDip (%) 24.5 9.5 15.7 13.0
cut off 96 80
False positive rate (%) < 0.1% < 0.1%
Fluorescence Polarization Immunoassay for simultaneous determination of of
T-2, HT-2, T2-glucoside and HT2-glucoside in wheat
CUT OFF level: the response, signal or concentration, obtained with the screening
method, above which the sample is classified as “suspect”.
Cut Off: response value ensuring a rate of false negative results < 5%

Multiplex dipstick immunoassays for the
semi-quantitative determination of
DON/ZEN and DON/ZEN/FB1+FB2
in wheat, barley and maize
OH
OH
O
O
O
CH3
O
O
OH
CH3
CH2
CH3
OH
O
OH
CH3
CH3
CH3
OH
CH3
NH2
OH
OH
OOH
O
O OH
O
OOH
O
O OH
O

Multiplex Dipstick Immunoassay
Concept and technology
The test is based on the indirect competitive immunoassay approach. Three test
lines (mycotoxin–protein conjugates) and one control line are located on the
strip membrane. Labelled antibodies are freeze-dried within a microwell .
Labelled antibodies
NEGATIVE
sample
ZEA
FB1+FB2
ZEA
Control
POSITIVE
sample
Test Lines darker than
CTRL line
Test Lines lighter than
CTRL line

 The starting point was by the multiplex strip test prototype developed in the
CONffIDENCE project (FP7) (qualitative assay)
New selections of antibodies (8 monoclonal and 2 polyclonal for DON, one for
FBs, one for ZEA) were tested.
Performances of the new quantitative competitive immunoassay were
evaluated in standard solutions
Cross reactivity was re-assessed
The extraction protocol was optimized
Matrix assisted calibration curves were generated.
Phases of Immunoassay Development
qualitative semi-
quantitative
Lattanzio et al, Anal. Bioanal. Chem. 2013, 405:7773
Lattanzio et al, Analytica Chimica Acta. 2012, 718:99
Mycokey deliverable report D3.2– available soon at EU CORDIS

Method Protocol
Water and methanol
extraction
Extract
dilution with
Mycobuffer
Incubation at 40°C, 10 min
Migration, 10 min
Reading
Matrix specific
calibration curve
uploaded into the
reader as QR code or
.mth file
Wheat
Barley
Maize
https://youtu.be/o6dzCPf11YY

Multiplex dipstick immunoassay for simultaneous determination of
DON and ZEN in wheat and barley
DON ZEN
0.2 STC 0.5 STC STC 0.2 STC 0.5 STC STC
validation level (µg/kg) 250 625 1250 20 50 100
Mean response
(µg/kg)
335 670 1285 22 51 110
SD repeatability (µg/kg) 77 83 207 6.9 5.5 10
SD intermediate
precision (µg/kg)
129 94 251 9.9 94 19
RSDr (%) 22 12 16 31 11 9
RSDip (%) 36 14 20 45 15 18
cut off 851 76
False positive rate
(%)
< 0.1% 3 < 0.1% 39

FB1+FB2 DON ZEN
0.2 STC STC 0.2 STC STC 0.2 STC STC
validation level (µg/kg) 800 4000 350 1750 70 350
Mean response
(µg/kg)
1297 3412 349 1777 56 371
SD repeatability (µg/kg) 168 77 143 292 12 48
SD intermediate
precision (µg/kg)
253 107 143 292 13 49
RSDr (%) 12.9 2.3 41.0 16.4 21.5 12.9
RSDip (%) 19.5 3.1 41.0 16.4 22.6 13.1
cut off 3227 1275 287
False positive rate
(%)
< 0.1% < 0.1% < 0.1%
Multiplex dipstick immunoassay for simultaneous determination of
DON, ZEN and FB1+FB2 in maize

METHOD VERIFICATION
With regards to rapid test-kits, „fitness for purpose‟ includes not only the criteria
more commonly considered when discussing laboratory-based methods
(specificity, accuracy and precision)
but also more “practical” parameters such as speed and ease to
implementation in a new operational environment.
demonstrating under local conditions that performance parameters, as
established during the validation, can be achieved by first time users: METHOD
VERIFICAION (2007 ALACC Guide, How to meet ISO 17025 requirements for
method verification)
Lattanzio et al, Toxins 2019, 11(2), 129

Screening test performances
DON in wheat
LFD
Cut off 981 µg/kg
RSDR 6.5%
False suspect 0%
ELISA
Cut off 674 µg/kg
RSDR 30%
False suspect 0%
FPIA
Cut off 1504 µg/kg
RSDR 10%
False suspect 0%
LC-HRMS
Cut off 1150 µg/kg
RSDR 10%
False suspect 0%
Test materials:
Blank: DON < 100 µg/kg
STC: DON 1600 µg/kg

Screening test performances
DON in wheat
LFD
Cut off 981 µg/kg
RSDR 6.5%
False suspect 0%
ELISA
Cut off 674 µg/kg
RSDR 30%
False suspect 0%
FPIA
Cut off 1504 µg/kg
RSDR 10%
False suspect 0%
LC-HRMS
Cut off 1150 µg/kg
RSDR 10%
False suspect 0%
Test materials:
Blank: DON < 100 µg/kg
STC: DON 1600 µg/kg
ANOVA
Between operator:
63% of the total
variance

MycoKey Round Tables
RT2. Roundtable discussion on priorities and critical issues in
chemical detection and monitoring of mycotoxins
Identify key elements that could impact when and how methods are used
to identify mycotoxins and to increase food safety

Advances in research: identify scientific and technological
innovations that are promising for method development and
implementation. Consider reliability, applicability to real word
samples, transferability to end-users
Multi-mycotoxin methodologies (highest priority score)
Methods for
rapid detection
Non organic extraction solvents
Improving rapid test performances
(alternative receptors, labels)
Mechanisms to
communicate the results
Smartphone/
on line transfer of results
Rapid and broad sharing of the results

Identify the key elements that make a method suitable to be
implemented in industries (large companies and SMEs) for auto
control, HACCP plans, and process management.
Method
application
Cost effective
Easy to use
Fast
Suitable for automation
Method
verification
Easy calibration
Traceability of the measurements
QC tools
Compliance with regulations (including sampling)
Data managment
Quick and easy interpretation of the data
Availability to every one at every time

Developing countries and rural areas: identify key elements that
make a method suitable to be implemented in developing countries
having poor resources and analytical capacity.
Antibody-based
tests
Cost effective – Easy to use
Validated
Tool Box (ready to use)
Validation - compliance with regulation and
standards (including sampling plan)
Specific challenges
Long shelf life
Minimal infrastructure needed
Water based sample preparation
Stable supply chain – availability of
spare parts
On site training
and support
Needed for analysts working in countries
having limited human and institutional
capacity building

Validation and official control: methods for official control purposes
are becoming more heavily reliant on multi-mycotoxin analysis.
Identify current QC procedures and/or metrological tools that are
efficient and up-to-date for official control and accreditated methods.
Validation and QC
tools
Certified Reference Materials
PT – collaborative trials
(multimycotoxin)
Validation
guidelines
Test kit validation programmes
Measurement Uncertainty protocol
Update and harmonization of validation criteria:
- DG SANTE
- ISO/AOAC
- LOQ/LOD
- Criteria for matrix effects
- Merging “performance criteria” and “Fitness-
for-purpose approach”

MycoKey Workshop - Training Course 18-22 November 2019
“Rapid Methods for Mycotoxin Detection
in the Food Chain”
Oil Crops Research Institute,
Chinese Academy of Agricultural Sciences (OCRI-CAAS)
Wuhan, China
THANK YOU!

9 lattanzio myco_key_ws_multi-mycotoxin_screening

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