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Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.
Regardless of its ban in agricultural use, the triazine derivative terbutryn is still used as a biocide against biofouling in building materials, such as façade colors and roof tiles. From there, it is washed off and may contaminate soil and surface waters, where it shows ecotoxicological behavior. For understanding of these leaching processes, analytical methods that can detect terbutryn in traces are necessary. In contrast to separation methods (Gas (GC) or liquid chromatography (LC)) coupled to mass spectrometry (MS), enzyme immunoassays (EIAs) have proven to be adequate tools for environmental monitoring. Compared to GC-or LC-MS, they are cheap, require only limited time and experimental expertise and enable high throughput. For quantification of terbutryn, EIAs have been established, with a limit of detection (LOD) of 50 ng L-1. For leaching experiments however, a lower LOD would be advantageous.
For detection of terbutryn we developed a direct competitive ELISA based on a monoclonal antibody: Terbutryn and an enzyme-terbutryn conjugate (“tracer”) compete for the binding sites of the immobilized antibody. The bound tracer converts a colourless substrate into a blue-coloured product, which can be detected via spectrophotometry. For quantification of terbutryn, a sigmoidal calibration curve is used. For establishing of the EIA, a new tracer based on a terbutryn surrogate (“hapten”) was synthesized. After optimization of the experimental conditions, the immunoassay exhibited a more than ten-fold increased sensitivity (LOD: 3 ng L-1), high stability against sample compounds (Ca2+, NaCl) and lower cross-reactivities against structurally related compounds, than reported before. Analysis of spiked samples was possible with high precision (inter-assay CV: < 10 %) and accuracy (recoveries from spiked samples: 89 % ± 10%). We therefore propose it as quick and reliable method for leaching studies of terbutryn out of building materials.
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.
Preactivation crosslinking – An efficient method for the oriented immobilization of antibodies
(2019)
Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.