A miniaturized sandwich immunoassay platform for the detection of protein-protein interactions

<p>Abstract</p> <p>Background</p> <p>Analysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this traditional large scale resin-based coIP is too laborious and time consuming. To overcome this problem, we developed a miniaturized sandwich immunoassay platform (MSIP) by combining antibody array technology and coIP methods.</p> <p>Results</p> <p>Based on anti-FLAG antibody spotted aldehyde slides, MSIP enables simple, rapid and large scale detection of PPIs by fluorescent labeling anti-myc antibody. By analyzing well-known interacting and non-interacting protein pairs, MSIP was demonstrated to be highly accurate and reproducible. Compared to traditional resin-based coIP, MSIP results in higher sensitivity and enhanced throughput, with the additional benefit of digital read-outs. In addition, MSIP was shown to be a highly useful validation platform to confirm PPI candidates that have been identified from yeast two hybrid systems.</p> <p>Conclusions</p> <p>In conclusion, MSIP is proved to be a simple, cost-saving and highly efficient technique for the comprehensive study of PPIs.</p

Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families

Induction of antigen-specific tolerance through hematopoietic stem cell-mediated gene therapy: the future for therapy of autoimmune disease?

Based on the principle that immune ablation followed by HSC-mediated recovery purges disease-causing leukocytes to interrupt autoimmune disease progression, hematopoietic stem cell transplantation (HSCT) has been increasingly used as a treatment for severe autoimmune diseases. Despite clinically-relevant outcomes, HSCT is associated with serious iatrogenic risks and is suitable only for the most serious and intractable diseases. A further limitation of autologous HSCT is that relapse rates can be high, suggesting disease-causing leukocytes are incompletely purged or the environmental and genetic determinants that drive disease remain active. Incorporation of antigen-specific tolerance approaches that synergise with autologous HSCT could reduce or prevent relapse. Further, by reducing the requirement for highly toxic immune-ablation and instead relying on antigen-specific tolerance, the clinical utility of HSCT could be significantly diversified. Substantial progress has been made exploring HSCT-mediated induction of antigen-specific tolerance in animal models but studies have focussed on primarily on prevention of autoimmune diseases. However, as diagnosis of autoimmune disease is often not made until autoimmune disease is well developed and populations of autoantigen-specific pathogenic effector and memory T cells have become well established, immunotherapies must be developed to address effector and memory T-cell responses which have traditionally been considered the key impediment to immunotherapy. Here, focusing on T-cell mediated autoimmune diseases we review progress made in antigen-specific immunotherapy using HSCT-mediated approaches, induction of tolerance in effector and memory T cells and the challenges for progression and clinical application of antigen-specific ‘tolerogenic’ HSCT therapy

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Silver Decahedral Nanoparticles-Enhanced Fluorescence Resonance Energy Transfer Sensor for Specific Cell Imaging

We report on a silver decahedral nanoparticles (Ag₁₀NPs)-based FRET (fluorescence resonance energy transfer) sensor for target cell imaging. Fluorophores-functionalized aptamers (Sgc8-FITC) were bound with Ag₁₀NPs via the SH group on the aptamer to form Ag₁₀-Sgc8-FITC. Then, quencher-carrying strands (BHQ-1) were hybridized with Sgc8-FITC to form a Ag₁₀NPs-based FRET sensor (Ag₁₀-Sgc8-F/Q). The sensor interacted with membrane protein tyrosine kinase-7 (PTK-7) on the CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) cell surface to attain fluorescence imaging of CCRF-CEM cells. The addition of CCRF-CEM cells resulted in many sensors binding with cells membrane and the displacement of BHQ-1, thus disrupting the FRET effect and the enhanced fluorescence intensity of FITC. It was found that Ag₁₀NPs largely enhanced the fluorescence intensity of FITC. The results also showed that the Ag₁₀NPs-based FRET sensor (Ag₁₀-Sgc8-F/Q) was not only superior to the bare FRET sensor (Sgc8-F/Q) and sensor Ag-Sgc8-F/Q but also highly sensitive and specific for CCRF-CEM cells imaging

Simultaneous detection of α-Lactoalbumin, β-Lactoglobulin and Lactoferrin in milk by Visualized Microarray

Abstract Background α-Lactalbumin (a-LA), β-lactoglobulin (β-LG) and lactoferrin (LF) are of high nutritional value which have made ingredients of choice in the formulation of modern foods and beverages. There remains an urgent need to develop novel biosensing methods for quantification featuring reduced cost, improved sensitivity, selectivity and more rapid response, especially for simultaneous detection of multiple whey proteins. Results A novel visualized microarray method was developed for the determination of a-LA, β-LG and LF in milk samples without the need for complex or time-consuming pre-treatment steps. The measurement principle was based on the competitive immunological reaction and silver enhancement technique. In this case, a visible array dots as the detectable signals were further amplified and developed by the silver enhancement reagents. The microarray could be assayed by the microarray scanner. The detection limits (S/N = 3) were estimated to be 40 ng/mL (α-LA), 50 ng/mL (β-LG), 30 ng/mL (LF) (n = 6). Conclusions The method could be used to simultaneously analyze the whey protein contents of various raw milk samples and ultra-high temperature treated (UHT) milk samples including skimmed milk and high calcium milk. The analytical results were in good agreement with that of the high performance liquid chromatography. The presented visualized microarray has showed its advantages such as high-throughput, specificity, sensitivity and cost-effective for analysis of various milk samples