Abstract The development of an enzyme-immunoassay (ELISA)for the detection of the Fusarium mycotoxin zearalenone (ZON)is described. In contrast to the common antibody isolation frommammal serum, chicken were immunized in order to isolatespecific antibodies from the egg yolk. Five weeks after the startof the immunization a titer of 1:76 000 resulted from three in-jections without any adverse effects for the animals. Utilizingthe indirect competitive assay format ZON could be detected ina concentration range between 10 and 200 µg/L.

Introduction

Zearalenone (ZON) [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-β-resorcyclic acid lactone] is one of the most important Fusar-ium mycotoxins which shows frequent occurrence in Europeancorn and wheat and is characterized as to be estrogenic as canbe shown in animal experiments [1]. A number of chemicalmethods has been developed for the determination of ZON indifferent cereals, especially by use of HPLC [2–4] or TLC [5–7]. In addition, also immunoassays based on polyclonal ormonoclonal antibodies (AB) are available for quantitative de-tection of several mycotoxins [8–10]. In contrast to the pre-sent invasive techniques, the immunization of chicken fol-lowed by the isolation of the specific antibodies from the eggyolk shows several advantages, e.g. resistance against acid andheat [11] and repeated freezing and thawing [12] combinedwith high AB yield [13] and less influence on animal welfare[14], and represents therefore an interesting alternative. In thiswork the first approach for the realization of an indirect com-petitive ELISA (enzyme-linked immunosorbent assay) for thedetection of ZON in cereals by use of yolk antibodies is de-scribed.

Experimental

Immunization. 250 µg of the immunogen (ZON conjugated tobovine serum albumin) were dissolved in 250 µL phosphate-buffered saline (PBS) and mixed with the same volume Freund’scomplete adjuvans. The emulsion was injected into the breastmuscle of the chicken. This procedure was repeated twice aftertwo and four weeks using Freund’s incomplete adjuvans. Thespecific AB were isolated from the egg yolk by the polyethyl-ene glycol precipitation method according to Polson [15].

Immunoassay. Microtiter plates (MTP) were coated with 150 µL/well of antigen solution (ZON-conalbumin conjugate 0.5 µg/mL in 0.05 M carbonate buffer pH 9.6) overnight at 4 °C. Afterwashing three times with 0.05 M PBS containing 0.02% Tween20 the MTP were blocked with a solution of 0.1% gelatin and0.1% Tween 20 in 0.1 M PBS at 37 °C for 1 h. For the final em-ployment of the ELISA, yolk AB were diluted 1:50000 with0.1 M PBS containing 0.1% Tween 20 and 0.1% BSA (assaybuffer). For preincubation 200 µL/well of this solution weredispensed on a blocked MTP followed by an addition of 100 µLZON in acetonitrile/water 30:70 at different concentration lev-els. This mixture was incubated at room temperature for 1 h.150 µL of each well were transferred to the above mentionedantigen coated MTP and incubated again for one hour at roomtemperature. After washing 150 µL/well of peroxidase labeledrabbit anti-chicken-IgG diluted at 1:1500 in assay buffer wereadded to the MTP. The plates were incubated at room temper-ature for 2 h and washed again. Finally 200 µL of tetramethyl-benzidine solution (0.2 M citrate buffer pH 4.0 containing 0.2 µL/mL of 30% H2O2) were added. After 15 min incubation at roomtemperature the enzyme reaction was stopped by addition of 50 µL/well of 1 M sulfuric acid. The absorbance at 450 nm ofeach well was immediately determined on an MTP reader(TECAN SLT instruments, Grödig, Austria).

Results and discussion

After the injections of the immunogen, no adverse effects forthe animals like inflammation of the skin were observable. Fiveweeks after the first immunization a maximum AB titer of1:76000 could be obtained, which is a very quick immune re-sponse compared to the immunization of mammals. The opti-mization of the coating antigen concentration and AB dilutionby checkerboard titration was followed by an optimization ofthe dilution of the peroxidase labeled second antibody. As canbe seen in Fig. 1 the ELISA permits ZON determination be-tween 10 and 200 µg/L which is less sensitive than comparableELISAs using serum or monoclonal AB [10]. However, takinginto consideration the rather high threshold level of ZON incorn (usually > 60 µg/kg) this type of assay should be appro-priate for monitoring the concentration of ZON in cereals. Ad-ditionally, current immunization experiments with larger timeintervals between the immunogen injections should lead to amore sensitive assay. This work shows the potential of en-zyme-immunoassays using yolk antibodies. It should be alsoemphasized that the isolation of specific AB from egg yolk rep-resents an important way of the responsible realization of ani-mal experiments. Further optimization studies for the detectionof ZON in wheat and maize extracts are currently carried out in

Fresenius J Anal Chem (1998) 362 :176–177 – © Springer-Verlag 1998

SHORT COMMUNICATION

Dedicated to the memory of Professor Dr. Robert Kellner

H. Pichler · R. Krska (Y) · M. GrasserbauerCenter for Analytical Chemistry, Institute for Agrobiotechnology (IFA-Tulln), Konrad Lorenz Strasse 20, A-3430 Tulln, Austria

A. SzékácsPlant Protection Institute, Hungarian Academy of Sciences,POB 102, H-1525 Budapest, Hungary

H. Pichler · R. Krska · A. Székács · M. Grasserbauer

An enzyme-immunoassay for the detection of the mycotoxin zearalenone by use of yolk antibodies

Received: 3 January 1998 / Revised: 16 March 1998 / Accepted: 20 March 1998

our laboratory combining specificity and precision determina-tion together with comparison to HPLC. A more sensitive di-rect competitive immmunoassay is also presently developed.

Acknowledgments This study was financed by the Jubiläums-fond der Oesterreichischen Nationalbank (No. 6571) and addi-tionally supported by the Hungarian-Austrian Bilateral Collab-oration Program (ÖAD and National Technical DevelopmentCouncil, OMFB A-24/96) and from EC INCO-COPERNICUSGrant ERBIC15CT960802. The authors are especially gratefulto Dr. Marcela Hermann and her team for their excellent ani-mal husbandry at the Institute for Molecular Genetics of theUniversity of Vienna.

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Fig. 1 Inhibition of antibodybinding by zearalenone atdifferent concentration levels