Development of sensitive immundiagnostics for determination of toxic residues (mycotoxins, drugs) in biological fluids and animal feeds

Abstract

Recent high publicity of the greatest food – scandals in some European countries (e.g. BSE, dioxin, mycotoxicosis, heavy metal contaminants, illegal hormone treatment or drug residues in meat) underline the importance of food and feed safety and has lead for the building up of a more rigorous food control system. This system focused mainly on the detection of harmful components, among them the control of toxic components e.g. mycotoxins or veterinary drugs and antibiotics are on the fist place. In my dissertation the work was focused on the detection and screening of some of these toxic components. As head of the Diagnostic laboratory of Agricultural Biotechnology Center (Gödöllő) I conducted and coordinated these scientific activities. A direct, competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody has been developed for quantitative determination of ochratoxin A (OA) in different cereals. A dichloromethane/citric acid mixture was used for extraction of OA in cereals. This cleanup procedure proved to be as effective for ochratoxin A extraction as protocols using strong acids. The mean within-assay and interassay coefficients of variation for the standard curve was <10 %. The range of this test is 1-10 ng/g, with a detection limit of 0.5 ng/g OA. The toxin recovery from cereals contaminated with 0.5-100 ng/g OA varied between 90 and 130%. This test was commercialised in 1996 with branded name of TOXIKLON and has been used routinely for screening of samples in control laboratories. The test was modified and used for detection of OA content in human sera. The measuring range of this test (without sample dilution) was 0.2-2.0 ng/ml, and the detection limit was 0.2 ng/ml. The OA concentration of 355 sera samples varied from <0.2 to 10 ng/ml. OA but 75% of the samples contained 0.2-1.0 ng/ml. In some cases (6.8%), more than 1.0 ng/ml OA was measured, which is probably a result of elevated intake of OA, which may even exceed the “virtually safe dose”. Our data indicated that, like in many other countries, OA is present in food or feed products available in Hungary, and in order to save the health of consumers, their regular control is desirable. Another experiment was carried out to clear whether or not OA can be detected in seminal plasma after feeding of breeding boars with high amount of OA. The test was validated for semen and pig sera as well. Our findings suggested that OA might have an effect on the quality of semen. Another experiment was set up with broiler chickens after being fed with OA. The distribution of OA in different tissues (liver, kidney, thigh and breast muscles) was investigated. The test was validated for these organs as well. We could detect the highest amount of OA in liver and kidney, while the OA content in breast muscle was very low. A direct, monoclonal antibody based, competitive ELISA for the quantitative determination of fumonisin B1 (FB1) in cereals has also been developed. The measuring range of this test is 10-500 ng/g, with a detection limit of 7.6 ng/g FB1. The toxin recovery from cereals contaminated with 50-200 ng/g of FB1 varied between 61 and 84%. For practical application a test-kit, branded name of TOXIKLON has been developed. Our commercialised kit proved to be suitable for the rapid screening of food and feed samples for the presence of FBs. For screening of drug residues (gentamicin and sulphonamides) in biological fluids, direct competitive ELISA tests have been developed. The gentamicin-ELISA test was validated for cattle milk and pig sera as well. The milk could be measured directly, the measuring range was 0.1-10 ng/ml, with detection limit of 0.03 ng/ml GE. The matrix effect caused by sera could be avoided with the dilution of sera (1:10) with 0.1%ANS/PBS/Tween 20 buffer, the measuring range of this test was 1.5-100 ng/ml, with detection limit of 1.2 ng/ml GE. For detection of sulphadiazine in milk and sera, a sensitive monoclonal antibody based ELISA test has been developed as well. The measuring range and the detection limit of this test meets well with the requirement of the EU (100 µg/kg). Our laboratory has obtained an ISO 9001:(2001) certification for “production of specific monoclonal antibodies, development and manufacture of “ELISA” diagnostic tests for measurement of mycotoxins and antibiotics”.