Enzyme immunoassay for tenuazonic acid in apple and tomato products.

The Alternaria mycotoxin tenuazonic acid was derivatized with succinic anhydride and conjugated to keyhole limpet hemocyanin (KLH) and to horseradish peroxidase (HRP), respectively. The KLH conjugate was used to produce polyclonal antibodies in rabbits. A competitive direct enzyme immunoassay (EIA) for tenuazonic acid was established, which was moderately sensitive for tenuazonic acid [50% inhibition concentration (IC(50)): 320 ± 130 ng/mL] but strongly reacted with tenuazonic acid acetate (IC(50): 23.3 ± 7.5 ng/mL). Therefore, an optimized EIA protocol was established, which employed acetylation of standard and sample extract solutions. The mean standard curve detection limit (IC(30)) for tenuazonic acid acetate was 5.4 ± 2.0 ng/mL, enabling detection limits for tenuazonic acid in apple and tomato products of 25-50 ng/g (150 ng/g in tomato paste). Recoveries in a concentration range of 50-2000 ng/g were 60-130% in apple juice and tomato juice and 40-150% in other tomato products. Tenuazonic acid was detected in apple juice and tomato products from German retail shops at levels of 50-200 ng/g. In conclusion, this novel EIA for tenuazonic acid could be useful within a screening program for Alternaria mycotoxins in food.

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies.

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC20) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 µg/kg, alternariol at 1-13 µg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.

Production and characterization of antibodies against fumigaclavine A.

Polyclonal antibodies against fumigaclavine A (FuA) were obtained from rabbits after immunization with a FuA-keyhole limpet hemocyanine conjugate prepared by formaldehyde condensation. Using these antibodies and a FuA-bovine serum albumine conjugate, a competitive indirect enzyme immunoassay (EIA) was established. The antiserum obtained from one rabbit enabled highly sensitive detection of FuA, with an IC50 level and detection limit of the standard curve of 3.3 ng/ml and approx. 1 ng/ml, respectively. The EIA was very specific for FuA, with 1.3% cross-reactivity with FuB. Several other lysergic acid derivatives (ergonovine, ergotamine, alpha-ergocryptine) were tested but did not cross-react in the FuA EIA. This is the first description of antibodies against FuA and the first development of an EIA for FuA.