Real-Time Immunosensor for Small-Molecule Monitoring in Industrial Food Processes

Industrial food processes are monitored to ensure that food is being produced with good quality, yield, and productivity. For developing innovative real-time monitoring and control strategies, real-time sensors are needed that can continuously report chemical and biochemical data of the manufacturing process. Here, we describe a generalizable methodology to develop affinity-based biosensors for the continuous monitoring of small molecules in industrial food processes. Phage-display antibody fragments were developed for the measurement of small molecules, as exemplified with the measurement of glycoalkaloids (GAs) in potato fruit juice. The recombinant antibodies were selected for use in a competition-based biosensor with single-molecule resolution, called biosensing by particle motion, using assay architectures with free particles as well as tethered particles. The resulting sensor measures GAs in the micromolar range, is reversible, has a measurement response time below 5 min, and enables continuous monitoring of GAs in protein-rich solutions for more than 20 h with concentration measurement errors below 15%. The demonstrated biosensor gives the perspective to enable a variety of monitoring and control strategies based on continuous measurement of small molecules in industrial food processes.</p

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target_s orthologues, focusing mainly on post-translational modifications. Findings: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection

Heterodimerization regulates RNase MRP/RNase P association, localization, and expression of Rpp20 and Rpp25

Rpp20 and Rpp25 are subunits of the human RNase MRP and RNase P endoribonucleases belonging to the Alba superfamily of nucleic acid binding proteins. These proteins, which bind very strongly to each other, transiently associate with RNase MRP. Here, we show that the Rpp20-Rpp25 heterodimer is resistant to both high concentrations of salt and a nonionic detergent. The interaction of Rpp20 and Rpp25 with the P3 domain of the RNase MRP RNA appeared to be strongly enhanced by their heterodimerization. Coimmunoprecipitation experiments demonstrated that only a single copy of each of these proteins is associated with the RNase MRP and RNase P particles in HEp-2 cells. Both proteins accumulate in the nucleoli, which in case of Rpp20 is strongly dependent on its interaction with Rpp25. Finally, the results of overexpression and knock-down experiments indicate that their expression levels are codependent. Taken together, these data indicate that the Rpp20-Rpp25 heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, which suggests that their interaction is also crucial for their role in RNase MRP/P function