No abstract

c Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 g/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus, with detection limits of 2 to 60 ng/ml.

Food and crop contaminations with mycotoxins are a severe health risk for consumers and cause high economic losses worldwide. Currently, different chromatographic- and immuno-based methods are used to detect mycotoxins within different sample matrices. There is a need for novel, highly sensitive detection technologies that avoid time-consuming procedures and expensive laboratory equipment but still provide sufficient sensitivity to achieve the mandated detection limit for mycotoxin content. Here we describe a novel, highly sensitive, and portable aflatoxin B1 detection approach using competitive magnetic immunodetection (cMID). As a reference method, a competitive ELISA optimized by checkerboard titration was established. For the novel cMID procedure, immunofiltration columns, coated with aflatoxin B1-BSA conjugate were used for competitive enrichment of biotinylated aflatoxin B1-specific antibodies. Subsequently, magnetic particles functionalized with streptavidin can be applied to magnetically label retained antibodies. By means of frequency mixing technology, particles were detected and quantified corresponding to the aflatoxin content in the sample. After the optimization of assay conditions, we successfully demonstrated the new competitive magnetic detection approach with a comparable detection limit of 1.1 ng aflatoxin B1 per mL sample to the cELISA reference method. Our results indicate that the cMID is a promising method reducing the risks of processing contaminated commodities.

Cholera is a life-threatening disease caused by the cholera toxin (CT) as produced by some Vibrio cholerae serogroups. In this research we present a method which directly detects the toxin’s B subunit (CTB) in drinking water. For this purpose we performed a magnetic sandwich immunoassay inside a 3D immunofiltration column. We used two different commercially available antibodies to capture CTB and for binding to superparamagnetic beads. ELISA experiments were performed to select the antibody combination. The beads act as labels for the magnetic frequency mixing detection technique. We show that the limit of detection depends on the type of magnetic beads. A nonlinear Hill curve was fitted to the calibration measurements by means of a custom-written python software. We achieved a sensitive and rapid detection of CTB within a broad concentration range from 0.2 ng/ml to more than 700 ng/ml.

10SARS-CoV-2 causes ongoing infections worldwide, and identifying people with immunity is 11 becoming increasingly important. Available point-of-care diagnostic systems as lateral flow assays 12 have high potential for fast and easy on-site antibody testing but are lacking specificity, sensitivity 13 or possibility for quantitative measurements. Here, a new point-of-care approach for SARS-CoV-2 14 specific antibody detection in human serum based on magnetic immuno-detection is described and 15 compared to standard ELISA. For magnetic immuno-detection, immunofiltration columns were 16 coated with a SARS-CoV-2 spike protein peptide. SARS-CoV-2 peptide reactive antibodies, spiked 17 at different concentrations into PBS and human serum, were rinsed through immunofiltration 18 columns. Specific antibodies were retained within the IFC and labelled with an isotype specific 19 biotinylated antibody. Streptavidin-functionalized magnetic nanoparticles were applied to label the 20 secondary antibodies. Enriched magnetic nanoparticles were then detected by means of frequency 21 magnetic mixing detection technology, using a portable magnetic read-out device. Measuring 22 signals corresponded to the amount of SARS-CoV-2 specific antibodies in the sample. Our 23 preliminary magnetic immuno-detection setup resulted in a higher sensitivity and broader 24 2 detection range and was four times faster than ELISA. Further optimizations could reduce assay 25 times to that of a typical lateral flow assay, enabling a fast and easy approach, well suited for point-26 of-care measurements without expensive lab equipment.