Development of Immunodiagnostic Strip Tests for Mycotoxin Screening of Food and Feed

Development of Immunodiagnostic Strip Tests for Mycotoxin Screening of Food and Feed

B. Cvak*, A. Molinelli*, R. Krska ** *Romer Labs Division Holding GmbHTechnopark 1, 3430 Tulln, Austria

**Center for Analytical Chemistry,Department for Agrobiotechnology (IFA-Tulln),University of Natural Resources and Life Sciences, ViennaKonrad-Lorenz-Straße 20, A-3430 Tulln, Austria

Mycotoxins are toxic natural secondary metabolites produced by several species of fungi on agricultural commoditiesin the field or during storage. The Type A trichothecenes HT-2 and T-2 toxin as well as Zearalenone are produced bythe genus Fusarium. Ingestion of Type A trichothecenes may cause skin irritation, emesis, diarrhea or multiplehaemorrhage and they are also potent inhibitors of protein and DNA synthesis and known to causeimmunsuppression. Zearalenone and its derivatives possess highly anabolic and oestrogenic properties. Ochratoxin A isa widespread food contaminating mycotoxin. Besides cytotoxic properties it is classified as a possible humancarcinogen. Aflatoxin B1 is produced by fungi of the genus Aspergillus and known to be a human carcinogen. Due tothis various toxic effects of mycotoxins on humans and animals it is important to develop rapid methods for screeningon-site besides standard analytical methods. Lateral flow devices (LFDs), also known as strip tests, are immunoassay-based methods which allow qualitative or semi-quantitative determination of mycotoxins within a few minutes. Thedevelopment of this rapid immunochemical test system for the screening of the mycotoxins Zearalenone (ZON), sum ofT-2/HT-2 toxin, Aflatoxin B1, B2, G1 and G2 (Afla), and Ochratoxin A (OTA) is the main focus of the presented work.

INTRODUCTIONINTRODUCTION

CONCLUSION AND OUTLOOKCONCLUSION AND OUTLOOK

The authors thank Romer Labs, FEMtechand bmvit for financial support.

ACKNOWLEDGEMENTSACKNOWLEDGEMENTSCorrespondence should be addressed to:Barbara Cvak, M.S.barbara.cvak@romerlabs.com

BACKGROUNDBACKGROUND

RESULTS AND DISCUSSIONRESULTS AND DISCUSSION

This paper describes the development of LFDs for the mycotoxins zearalenone andT-2/HT-2 toxin, Aflatoxins, and Ochratoxin A. These LFDs are intended to be usedfor the qualitative as well as semi-quantitative determination of these mycotoxins.No sample preparation other than 3 min extraction and 3 min test time is requiredprior to subsequent relative reflectance reading of the test lines.

EXPERIMENTALEXPERIMENTAL

The presented rapid tests are based on acompetitive assay format. Specific mycotoxin-monoclonal as well as polyclonal antibodieswere labeled with colloidal gold as signalreagent. A test zone leads to the formation ofa colored line which allows measuring thetoxin concentration and a control lineconfirms the correct test performance. Due tothe competition between free andimmobilized targets, the intensity of the testlines is indirectly proportional to the amountof target mycotoxin in the sample. The colorintensity of the lines is measured with aphotometric reader (XReader, Romer LabsSingapore Pte. Ltd., Singapore).

Strip productionStrips were produced using different overlapping membranes. The reagents for test and controllines were immobilized onto a nitrocellulose membrane by using a contact tip dispenser(BioDot, Irvine, CA, USA). The membrane was immobilized on a plastic backing card and a wickmembrane was used for absorbing excess sample liquid.

Labeling Antibodies with colloidal goldColloidal gold was synthesized in a particle size ofØ 40 nm as signal reagent for the test system. Specific antibodiesagainst ZON, T-2/HT-2 toxin, Afla and OTA were employed and witheach of them antibody/colloidal gold (Ab/AuC) conjugates produced.

Sample preparationMilled maize samples were extracted with extraction buffer (XBuffer,Romer Labs Division Holding, Tulln, Austria) for 3 min and thesample was left 10 min for extract sedimentation. Blank sampleextract was spiked with the four mycotoxins for calibration.

Test performance50 µL of sample extract was added to a microtiter welltogether with the same amount of Ab/AuC conjugate mix. Astrip was inserted into the well, and the mix was allowed tomigrate onto the strip membrane for 3 min.

Preliminary results with spiked maize extracts indicate linear working ranges. As largedifferences are usually observed in maize between spiked and naturally contaminatedsamples further strip test optimization and calibration will be performed using naturallycontaminated maize samples characterized by reference methods and, if available,certified reference materials.

R. Krska, A. Molinelli. 2009. Rapid test strips for analysis of mycotoxins in food and feed. Anal. Bioanal. Chem. 393, 67-71

REFERENCESREFERENCES

T-2/HT-2 toxin and Zearalenone – “Multi Mycotoxin Strip Tests”A picture of prototype double strips with calibrations for ZON and the sum ofT-2/HT-2 toxin in a range of 0-200 µg/kg are shown in Figure 1. By using twodifferent ready-to-use gold conjugate mixes due to the two test lines, a relativedark background can be seen. Therefore it was difficult for the photometric

Aflatoxin B1

Ochratoxin A

0 20 40 60 80 100 10 120 160 200

© D. Pum

Colloidal gold solution and TEM image

Ready-to-use gold conjugate mix in a 2 mL tube and LFDs in microtiter wells

Figure1: Picture of Prototype Double-Strip Tests for T-2/HT-2 Toxin and ZON

Figure2: Calibration Curve of Double-Strip Tests for T-2/HT-2 Toxin and ZON

reader to measure theintensities of the testlines (TL) for the highertoxin concentrations.For this reason the HT-2 toxin calibrationcurve (red one) shownin Figure 2 rangesfrom 0-80 µg/kg only.For the second test line(ZON test line) theintensities were higher,obtaining a calibrationcurve in the range of0-160 µg/kg.

0 0.1 0.2 0.4 2 3 4 6 8 10

Figure 4: Picture of the Aflatoxin B1 Calibration Curve in a Range of 0-10 µg/kg

y = 307,45e-0,333x

R² = 0,961

0,0

50,0

100,0

150,0

200,0

250,0

300,0

350,0

400,0

0 2 4 6 8 10 12

XReader absorbance

µg Afla B1/kg maize

Figure 3: Calibration Curve for Aflatoxin B1 (0-10 µg/kg)

A matrix-matched calibration with spiked maize sampleswas performed. Results are shown in Figure 3, where itcan be seen that the intensity of the test line is indirectlyproportional to the mycotoxin concentration. Figure 4shows the decreasing signal intensities on the developedLFDs for the calibration range depicted in Figure 3.

Control lineTL ZON

TL T-2/HT-2

y = -9,0114x + 378,8R² = 0,9856

y = -6,5867x + 208,23R² = 0,9449

0

50

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350

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450

0 2 4 6 8 10 12

XReader absorbance

OTA in solution [ppb]

BSA test line ConA test line

Two toxin-protein conjugates OTA-BSA and OTA-ConA wereimmobilized onto nitrocellulose membranes as test lines.Figure 5 shows calibration with spiked buffer to check theseconjugates in strip test format. The curves are nearlysimilar, except that the intensities with OTA-BSA as test linewere higher.

Figure 5: OTA Calibration [0-10 ppb OTA in solution]

Control lineTest Line

Colloidal gold labeled with anti-mycotoxin antibodies

Mycotoxin 1 coupled to carrier protein

Free Mycotoxin 1

Mycotoxin 2 coupled to carrier protein

Free Mycotoxin 2

Anti-species antibody for control line

LEGEND

On the left hand side an originalpicture of a developed strip testwith 2 test lines (TL) and 1 controlline (CL) can be seen.The cartoon on the right hand sideshows the test principle. Each partof the cartoon is described in detailin the legend.

CL

TL 1

TL 2

y = -1,3575x + 266,6R² = 0,8495

y = -1,0263x + 94,754R² = 0,781

0

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300

350

0 20 40 60 80 100 120 140 160 180

XReader absorbance

µg toxin/kg maize

ZON_Calibration HT-2_Calibration