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South African Society of Dairy Technology Symposium 24-26/04

UNISENSOR

Unisensor is a Belgian company researching,

developing and manufacturing diagnostic tools for the detection of contaminants present in

food products.

EU RESEARCH PROGRAMS

2015

“ GOOD FOOD ”

“ CONFFIDENCE ”

CompanyTimeline milestones

2005

Twinsensor

PATENTED

CONCEPT

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BEADYPLEX

R&D EFFORT

EXTENSO

2017

REVOLUTIONARY MILK ANALYSISPLATFORM

MULTIPLE: + 100 molecules in a single testDetection level at EU MRL

X1

7

Our Product Development StrategyIn two words…

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Multiple tests in one (antibiotics)• Reliable & Usable everywhere• Rapid & Cost-effective• Validated by external parties

MULTIPLEXING + CONTAMINANTS1 x

17 x

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BEADYPLEXScheme of analytical methods for antibiotic residues analysis

Confirmation

Screening

IMMUNOAssaysStructureELISA

LFIA

LC-MSPHYSICOCHEMICAL

Composition

TESTINGPRINCIPLES

METHODSAVAILABLE

MICROBIOLOGICALActivity Inhibition

Complexity

Price

http://www.google.be/url?sa=i&rct=j&q=LC-MS&source=images&cd=&cad=rja&docid=I5fRxKItkFXBKM&tbnid=o_3ewSAWT9cT_M:&ved=0CAUQjRw&url=http://www.agilent.com/about/newsroom/lsca/imagelibrary/index_2003.html&ei=EbhmUca3G6OP0AXO-oGYCw&bvm=bv.45107431,d.d2k&psig=AFQjCNEuYLOCq2kxkQ_vbnYZuj2TSqWZCg&ust=1365772686067446http://www.google.be/url?sa=i&rct=j&q=lateral+flow+chromatography&source=images&cd=&cad=rja&docid=o8IBVpS_pYmSoM&tbnid=QEPoAWNDCq_ArM:&ved=0CAUQjRw&url=http://www.biomerica.com/products/product_detail.asp?ProductID=70&ei=ALpmUeTBLuPE0QW7lYHgDQ&bvm=bv.45107431,d.d2k&psig=AFQjCNEWrUI3l9wh1Zae0iNBkGKW0DtpYA&ust=1365773108280158

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BEADYPLEXUnmet needs for a screening test

Adding Multi-Matrix dimension

1. Multiple families ID in a single test (vs ELISA)

2. Sensitive for all/most residues (vs microbiological methods)

3. High Throughput (+100 samples/day)

4. Cost-effective and time-saving

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BEADYPLEX - “All-In-One” screeningFlow Cytometric Immunoassays – Assay principle

Immunoanalysis: detection of interaction events(receptor & antibodies).

Flow Cytometry: multi-parametric characterizationof microparticles (beads) by lazer.

1. Different sizes2. Different Internal fluorescence intensities

Multiplexing via microparticles characterized by:

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FCIA: simultaneous detection of residues of antibiotics from 10 families in one sample

in one analysis

10 different ELISA kits combined in one test

BEADYPLEX - “All-In-One” screeningFlow Cytometric Immunoassays – Assay principle

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BEADYPLEX - “All-In-One” screeningFlow Cytometric Immunoassays – Assay principle

FLOW CYTOMETRIC COMPETITIVE IMMUNOASSAY

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BEADYPLEX - “All-In-One” screeningFlow Cytometric Immunoassays – Assay principle

BL RL

Classification of particles

Determination of assay signal intensity

Size Internalfluorescence

Flow cytometer (NovoCyte 2000, ACEA Biosciences)

Automatic Reading 3 4 Automatic Reporting

BEADYPLEXWorkflow for Antibiotic residues

1Sample preparation 2 Immunoassay

BEADYPLEXAnalytical performance – Validation

Following CRLs Guidelines for the validation of Screening methods for residues of

veterinary medicines (20/01/2010) based on Commission Decision 2002/657/EC

Specificity/selectivity

Cut-Off values

Detection capabilities

False positive rates

Robustness

Applicability

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BEADYPLEXAnalytical performance

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Detection Limits (CCβ)

✓CCβ determined for all detected molecules

✓Most of CCβ ≤ MRL

CCβ (β error ≤ 5%) → concentration for each antibiotic recognized by the FCIA

for which at least 95% of the samples fortified at this level are identified as “screened positive”

according to the established assay cut-off values

BEADYPLEXAnalytical performance

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Profile of antibiotic detection (non exhaustive list)

Detection Limits (CCβ)

BEADYPLEXAnalytical performance

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False positive rate

Bead n Blank samplesn Screen positive

False positive rate (%)

AMINOGLYCOSIDES_A 117 0 0

AMINOGLYCOSIDES_B 117 0 0

BETA-LACTAMS 107 0 0

LINCOSAMIDES 123 0 0

COLISTIN 122 0 0

MACROLIDES_A 113 0 0

MACROLIDES_B 113 0 0

SULFONAMIDES 107 0 0

PHENICOLS 118 0 0

TETRACYCLINES 117 0 0

(F)QUINOLONES 110 0 0

PLEUROMUTILINES 122 2 1,6

False positive rate < to those

usually atributed to other

screening methods for

antibiotics

Parameter Condition 1 (control) Condition 2 Condition 3

SAMPLEEXTRACTION

EXTRACTION VOLUME 1 ML 0,8 ML 1,2 ML

AGITATION EXTRACTION TIME 10 MINUTES 5 MINUTES 30 MINUTES

AGITATION SPEED 6,5 AGITELEC 4,5 AGITELEC 2,5 AGITELEC

SAMPLE EXTRACTS STORAGE EXTRACTION + ANALYSIS 4°C ON -20°C ON

EXTRACTION METHOD AGITATION + CENTRIFUGATION STOMACHER -

ASSAYPROCEDURE

NUMBER OF PLATE WASHING STEPS 3 1 2

TEST INCUBATION TEMPERATURE RT 4°C 37°C

INCUBATION TIME COMPETITIVE STEP 30 MINUTES 15 MINUTES 60 MINUTES

INCUBATION TIME REPORTERS STEP 15 MINUTES 5 MINUTES 30 MINUTES

PLATE READING DELAY TEST + READING 1H DELAY ON DELAY

OTHER

REAGENTS (BEADS AND PRIMARYBINDERS)

BATCH 1 BATCH 2 BATCH 3

OPERATOR A B C

CYTOMETERS NOVOCYTE ACEA ACCURI C6 BD -

REAGENTS STABILITY SAME DAY 3 MONTHS 4°C 6 MONTHS 4°C

BEADYPLEXAnalytical performance

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Robustness

✓No impact of any of the

evaluated parameters on

assay performance

✓Extraction and analysis

need to be performed

the same day

✓Very robust method

BEADYPLEXAnalytical performance

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Applicability

✓Porcine muscle

✓Bovine muscle

✓Poultry muscle

✓Fish (salmon and colin)

✓Milk

Applicable to

Honey

Eggs

FLEXIBILITY

✓Extraction procedure

adaptation for different

matrices

✓Cut-off modification for

modulation of applicability

or sensitivity

BEADYPLEXAnalytical performance - Conclusions

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BEADYPLEXAnalytical performance - Conclusions

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BEADYPLEXDevelopments

HormonesSedatives

Anti-inflammatory

drugs

Mycotoxines

Food allergens

Foodbornepathogenic

bacteria

Antibioticsresidues

Allergens

FLOW CYTOMETRIC SANDWICH IMMUNOASSAY

BEADYPLEXFlow Cytometric Immunoassays – Assay principle

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page09

BL RL

BEADYPLEXFlow Cytometric Immunoassays – Assay principle

Classification of particles

Determination of assay signal intensity

B

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BEADYPLEXLOD/LOQ

Range

BLG 0,625 – 40 ppm

Casein 0,625 – 40 ppm

Egg 1,25 – 80 ppm

Peanut 1,25 – 80 ppm

Soy 1,25 – 80 ppm

Hormones Sedatives Anti-inflammatory drugs

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BEADYPLEXHormones Sedatives Anti-inflammatory drugs

Corticosteroids

Growthpromoters

Screening multi-class and multi-residue of veterinary drugs in meat/milk or urine by FCIA

Post-mortem test

Ante-mortem test

Thank you for your attention

FCIA team

Alessandro Gallo

Alexandre Monteforte

Cédric Pety de Thozée

Celia Suárez Pantaleón