The sphingolipid receptor S1PR2 is a receptor for Nogo-a repressing synaptic plasticity.

Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G13, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A(-/-) mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity.

HLA antibody affinity determination: From HLA-specific monoclonal antibodies to donor HLA specific antibodies (DSA) in patient serum.

Organs transplanted across donor-specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA. To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative. There are currently a number of biophysical technologies available that allow the assessment of antibody binding strength. However, these methods require prior knowledge of antibody concentrations. Our objective within this study was to develop a novel approach that combines the determination of DSA-affinity as well as DSA-concentration for patient sample evaluation within one assay. We initially tested the reproducibility of previously reported affinities of human HLA-specific monoclonal antibodies and assessed the technology-specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio-layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow-induced dispersion analysis (FIDA). While the first three (solid-phase) technologies revealed comparable high binding-strengths, suggesting measurement of avidity, the latter (in-solution) approach revealed slightly lower binding-strengths, presumably indicating measurement of affinity. We believe that our newly developed in-solution FIDA-assay is particularly suitable to provide useful clinical information by not just measuring DSA-affinities in patient serum samples but simultaneously delivering a particular DSA-concentration. Here, we investigated DSA from 20 pre-transplant patients, all of whom showed negative CDC-crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI). DSA-concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449-fold difference). In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%. To conclude, this study strengthens the presumption that pre-transplant patient DSA consists of various concentrations and different net affinities. Validation of these results in a larger patient cohort with clinical outcomes will be essential in a further step to assess the clinical relevance of DSA-concentration and DSA-affinity.

A simple approach to improve the sensitivity of enzyme-linked immunosorbent assay: using the IMAPlate 5RC96 for result readout.

In this article, we describe a new enzyme-linked immunosorbent assay (ELISA) setup to improve the sensitivity of commercial or homemade ELISAs. In the new ELISA setup, an IMAPlate 5RC96, a disposable multi-utility lab device developed by NCL New Concept Lab is used as a self-uptaking microcuvette array to read out the result of the ELISA that is performed in the normal 96-well plate with reduced substrate solution and stop solution. A commercial interleukin-6 (IL-6) ELISA reagent kit was used for the evaluation. Compared with the conventional ELISA setup, the new ELISA setup could easily increase the absorbance values by up to more than 10-fold. Therefore, the sensitivity (change in absorbance/change in concentration [DeltaAbs/DeltaConc]) is increased accordingly. In addition, methods to extend the upper detection limit of plate readers for the IMAPlate 5RC96 are described. This new ELISA setup may be more notable for the approach employed than for the specific analyte. It should generally be applicable to any conventional ELISA and should serve as an example of a simple solution that increases the detection sensitivity and/or detection range of other assays as well. We expect the approach to have a substantial practical impact on analytical methods and to accelerate discovery, research, and application of analytes at low concentration in life sciences and diagnostics.

Establishment of a miniaturized enzyme-linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate.

A miniaturized enzyme-linked immunosorbent assay (ELISA) with a reaction volume of 5 microl for human transferrin quantification has successfully been developed using an intelligent multifunctional analytical plate (IMAPlate 5RC96), the first miniature analytical platform capable of manually performing parallel liquid transfer, reaction, and analysis. This is the first article to validate the platform for the ELISA application. The data obtained from the standards in this miniaturized ELISA can well be fitted by a one-site binding reaction mode, the coefficient of variation (CV) of the whole plate for an artificial sample (spiking a known concentration of human transferrin into the assay diluent) is 7.0%, and the mean recovery is between 94 and 114% (n=96), comparable to the values from conventional ELISA in a 96-well format plate. The IMAPlate 5RC96-based miniaturized ELISA not only can reduce sample and reagent consumption to 5% of the conventional ELISA but also can shorten the reaction time. Combined with the advantages brought by miniaturization, the easy-to-handle, parallel, and simultaneous liquid transfer features of the IMAPlate 5RC96 provide a completely new lab tool for manually performing high-throughput ELISA. Our results demonstrate that the IMAPlate 5RC96 is a convenient, robust, high-throughput lab device feasible for miniaturized ELISA in an ordinary laboratory.

Self-Powered Multiparameter Health Sensor.

Wearable health sensors are about to change our health system. While several technological improvements have been presented to enhance performance and energy-efficiency, battery runtime is still a critical concern for practical use of wearable biomedical sensor systems. The runtime limitation is directly related to the battery size, which is another concern regarding practicality and customer acceptance. We introduced ULPSEK-Ultra-Low-Power Sensor Evaluation Kit-for evaluation of biomedical sensors and monitoring applications (http://ulpsek.com). ULPSEK includes a multiparameter sensor measuring and processing electrocardiogram, respiration, motion, body temperature, and photoplethysmography. Instead of a battery, ULPSEK is powered using an efficient body heat harvester. The harvester produced 171 W on average, which was sufficient to power the sensor below 25 C ambient temperature. We present design issues regarding the power supply and the power distribution network of the ULPSEK sensor platform. Due to the security aspect of self-powered health sensors, we suggest a hybrid solution consisting of a battery charged by a harvester.

Synthetic single domain antibodies for the conformational trapping of membrane proteins.

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.