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The Short Course “Immunoanalytical Methods for Environmental, Food and Clinical Analysis” offers training in antibody-based analytical techniques. The course is suitable also for beginners. The Short Course comprises Lecture Units teaching the concepts, equipment, reagents and explains the protocols to be used in the practical part. Simple “Hands On” units are offered employing portable instrumentation. – The Short Course spans from antigen production, generation of antibodies, assay development and optimization, data evaluation to the different formats that have been developed. Examples are provided from the areas of environmental analysis (pharmaceuticals, hormones and anthropogenic markers in water and wastewater), food analysis (caffeine in beverages, mycotoxins) and clinical diagnostics (infarction biomarkers). The attendants will be enabled to select an appropriate method and to judge its applicability for their given analytical problem.
Immunoassays are in the majority of formats heterogeneous assays with one of the commodities – antibody or antigen – immobilized on a solid substrate or sometimes to nanoparticles or beads. All these formats require a washing step in order to separate bound from non-bound species before generating the measurement signal.
Measuring fluorescence polarization is a method to distinguish between a fluorescent macromolecule and a low-molecular weight fluorophore. After irradiation of the sample with polarized light, a macromolecule like e.g. an antibody, will emit still highly polarized light (high polarization, resp. small difference in degree of polarization to the background). In a fluorescence polarization immunoassay, the fluorescence of an analyte surrogate (a fluorescence “tracer”) is followed. Depolarization is high and so the measured polarization is low. When antibody is added, the tracer is bound, depolarization decreases and the in polarization is high. Increasing amounts of analyte more and more impede the tracer being bound by the tracer and thus the decreases, the typical sigmoidal relationship is obtained (Fig. 1). We dispose of 5 polarization platforms reading in plate mode, strip mode and cuvette mode. One is able to register polarization changes with time and so allows for kinetic measurements another one is hand-held. We have been comparing FPIA formats before [1-4] and now a comprehensive view on sensitivities, the importance of fast vs. slow binding kinetics as well as the choice of format can be presented.
Background. Aflatoxin B1 (AFB1) is a toxic low-molecular-weight secondary fungal metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus not only at pre-harvest time but also at post-harvest stages including storage. AFB1 was classified as a Group I carcinogen by the World Health Organization for Research on Cancer in 1993. AFB1 is able to naturally contaminate medicinal plants and therefore causing serious health issues for humans consuming the related medicine. This study aimed to develop an efficient fluorescence polarization immunoassay (FPIA) and for the first time a rapid (5-10 min), low-cost, and simple membrane-based flow-through immunoassay (MBA) for determination of AFB1 in medical herbs based on a monoclonal antibody.
Methods. Two different techniques for AFB1detection in medical herbs (Herba Orígani vulgáris, Folia Urticae, Fructus Rubi idaei) were developed and compared, namely an easy-to-use semi-quantitative flow-through membrane-based enzyme immunoassay (MBA), and a homogeneous method which needs no separation or washing steps (assay time 10 min), a quantitative fluorescence polarization immunoassay (FPIA).
Results. A cut-off level of the developed MBA can vary wide from 0.8 ppb to 1 ppb. The FPIA method showed a linear working range of 8.6 ppb to 64 ppb, IC50 of FPIA is 24 ppb. The results were in good correlation with the ELISA results (IC50 = 0.1 ppb). LC–MS/MS was used to confirm the results, too.
Conclusion. MBA can be used for a quick on-site AFB1 detection in medical herbs (Herba Orígani vulgáris (Oríganum vulgáre), Folia Urticae (Urtíca dióica), Fructus Rubi idaei (Rúbus idáeus)), the developed test does not require special equipment and is not time-consuming (5-10min), includes several simple steps and can be performed directly on-site. The major advantages of the developed FPIA are its simplicity and suitability for a rapid screening of a large number of samples.