Antibody Cross-Reactivity between Proteins of Chia Seed ( Salvia hispanica L.) and Other Food Allergens.

Chia seeds are becoming increasingly common in Europe because of their functional and nutritional properties. Despite this, few studies have focused on the allergic potential and antibody cross-reactivity among storage proteins in chia seed and other plants. The aim of this study was to identify chia seed's immunoglobulin G (IgG) and immunoglobulin E (IgE) binding proteins ( Salvia hispanica L.) and to investigate the antibody cross-reactivity among its storage proteins and those of other seeds. Extracted chia seed proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunodetection was performed with commercial antibodies against sesame seed, hazelnut, and peanut and sera from 33 patients with a hazelnut allergy and five with a sesame allergy. Cross-reactivity of certain antibodies with storage proteins of chia seed, sesame seed, and hazelnut was assessed using an enzyme-linked immunosorbent assay (ELISA) inhibition, blot inhibition, and surface plasmon resonance (SPR) spectroscopy. IgG binding proteins were identified at molecular weight (MW) 70, 49, 34, 23, and 20 kDa by applying commercial antibodies. Furthermore, the interaction of chia proteins with sera from sesame-allergic patients led to identify IgE binding proteins at MW 49, 45, 31, 20, and 12 kDa, while IgEs in sera from hazelnut-allergic patients reacted with proteins at MW 300, 140, 49, 45, 31, 20, and 6 kDa. The results of ELISA inhibition and blot inhibition indicated chia seed proteins are similar to sesame seed and hazelnut proteins in the primary structure. The antisesame antibodies' binding to sesame proteins was more strongly inhibited by the chia globulin fraction (GLO) than the antihazelnut antibodies' binding to hazelnut proteins. SPR results confirmed the presence of IgG binding proteins in GLO and the high similarity of epitopes on globulins of chia seed and sesame seed. Thus, chia seed consumption might lead to cross-sensitization in patients with a sesame allergy.

Aptamers: Universal capture units for lateral flow applications.

The present work demonstrates the implementation of aptamers as capture molecules for a wide range of target classes in lateral flow assay applications. The targets were chosen in order to cover a wide range of target classes (small sized - metabolite, medium sized - protein, and large sized - whole cell/spore). For each target class one target molecule was selected as representative and appropriate aptamers were used for lateral flow assay development. The work points out that the implementation of aptamers as capture molecules in a universal lateral flow test platform was successful independent form target size. Furthermore, the limit of detection for p-aminohippuric acid in urine (200 ppm), lysozyme in white wine (20 ppm), and Alicyclobacillus spores in buffered orange juice (>8 CFU/mL) were determined using aptamers as capture molecules. The whole approach is considered as a proof of concept, regarding the ability of aptamers as an alternative to antibodies (in conjunction with directive 2010/63/EU on the protection of animals used for scientific purposes) in lateral flow applications.

Aptamer-based trapping of phytosphingosine in urine samples.

Usually, small molecules like single metabolites used in clinical diagnostic can be quantified by instrumental approaches like LC-MS or bioanalytical techniques using antibodies or aptamers as selective receptors. The present work comprises the generation of aptamers with an affinity towards the medically relevant metabolite phytosphingosine via the previously reported just in time-Selection approach (Hünniger et al., 2014). The whole approach could be seen as a proof of concept to extend the existing just in time-Selection protocol for selection towards small molecules with dissociation constants in the low nanomolar range. Moreover it is conceivable that the shown methods could be quickly adapted to further scopes. Aptamers could be applied for clean-up or concentration processes prior to further analysis. As an example, we used the selected aptamers towards phytosphingosine bound to magnetic particles for affinity enrichment in both selection buffer and urine samples. As an outcome, enrichment factors of up to 9-fold (selection buffer)/4-fold (urine samples) were achieved by this approach.

HPTLC-aptastaining - Innovative protein detection system for high-performance thin-layer chromatography.

Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations.

Food Targeting: A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification of Alicyclobacillus spp. Spores after Aptamer-Based Enrichment.

Spore-forming Alicyclobacillus spp. are able to form metabolites that induce even in small amounts an antiseptical or medicinal off-flavor in fruit juices. Microbial contaminations could occur by endospores, which overcame the pasteurization process. The current detection method for Alicyclobacillus spp. can take up to 1 week because of microbiological enrichment. In a previous study, DNA aptamers were selected and characterized for an aptamer-driven rapid enrichment of Alicyclobacillus spp. spores from orange juice by magnetic separation. In the present work, a direct quantification assay for Alicyclobacillus spp. spores was developed to complete the two-step approach of enrichment and detection. After mechanical treatment of the spores, the isolated DNA was quantified in a real-time PCR-assay targeting 16S rDNA. The assay was evaluated by the performance requirements of the European Network of Genetically Modified Organisms Laboratories (ENGL). Hence, the presented method is applicable for direct spore detection from orange juice in connection with an enrichment step.

Food sensing: selection and characterization of DNA aptamers to Alicyclobacillus spores for trapping and detection from orange juice.

The quality of the beverage industry's products has to be constantly monitored to fulfill consumers' high expectations. The thermo-acidophilic Gram-positive Alicyclobacillus spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to Alicyclobacillus spp. have been developed, analyzed, and characterized in terms of affinity and specificity.

Just in time-selection: A rapid semiautomated SELEX of DNA aptamers using magnetic separation and BEAMing.

A semiautomated two-step method for in vitro selection of DNA aptamers using magnetic separation and solid-phase emulsion polymerase chain reaction has been developed. The application of a magnetic separator allows the simultaneous processing of up to 12 SELEXs (systematic evolution of ligands by exponential enrichment) with different targets or buffer conditions. Using a magnetic separator and covalent target immobilization on magnetic beads, the selection process was simplified and the substeps of aptamer/target incubation, washing, and elution of the aptamers were merged into one automated procedure called "FISHing". Without further processing the resulting FISHing eluates are suitable for BEAMing (beads, emulsion, amplification, and magnetics), which includes the amplification by emPCR (emulsion polymerase chain reaction) and strand separation by the implementation of covalently immobilized reverse primers on magnetic beads. The novel selection process has been proved and validated by selecting and characterization of aptamers to the wine fining agent lysozyme.