There is a growing need for diagnostic technologies which can provide accurate disease detection using sensitive, reliable, and inexpensive methods. A lot of research has been directed towards developing efficient point-of-care devices that enable the parallel detection of multiple analytes, in small-volume samples, with high sensitivity and in a short time. Flow cytometry, used for bead-based immunoassays are expensive and bulky. Using a microfluidic based optical detection system makes the whole process convenient and cheap. This motivates us to explore this platform for multiplexed testing by incorporating microbead-based assays. Hence, we aim to develop a microfluidics based optical detection system, which can measure multiple analytes at the same time for diseases like dengue. Dengue detection using NS1-4 (non-structural protein), which inflates on the first day of virus with the IgG antibody, can give significant information for the treatment. Simultaneous detection of dengue antigen and antibody is crucial for prevention and diagnosis of dengue infection. The outcome of this study generates the first-generation prototype of a universal fluorescence detection device capable of both classifying the microspheres and measuring the amount of specific biomarker.
Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approved cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined antibody engineering and functional genomics screens to identify putative mechanisms of resistance or synthetic lethality with antibody-mediated anti-proliferative effects. We developed a synthetic antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization, and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 antibody therapeutics.
Chromatic dispersion is utilized to separate fluorescent signals in different fluorescent dyes. The fluorescence intensity of allophycocyanin is used as the barcode fluorophore for specific biomarkers, enabling a four-plex biomarker detection.
Les exosomes sont des microvésicules dérivées des endosomes, contenant différents constituants cellulaire et sécrétées de façon active par de nombreux types cellulaires. Nous illustrons ici, à partir d’un exemple récent de la littérature, comment des exosomes sécrétés par les fibroblastes du stroma tumoral activés, et capturés par les cellules cancéreuses, sont capables d’activer dans celles-ci une voie de signalisation modifiant leur motilité et promouvant leur capacité métastatique.
Antigen-binding fragments (Fabs) with synthetic antigen-binding sites were isolated from phage-displayed libraries with restricted complementarity-determining region (CDR) diversity. Libraries were constructed such that solvent-accessible CDR positions were randomized with a degenerate codon that encoded for only four amino acids (tyrosine, alanine, aspartate, and serine). Nonetheless, high-affinity Fabs ( K d = 2–10 nM) were isolated against human vascular endothelial growth factor (hVEGF), and the crystal structures were determined for two distinct Fab-hVEGF complexes. The structures revealed that antigen recognition was mediated primarily by tyrosine side chains, which accounted for 71% of the Fab surface area that became buried upon binding to hVEGF. In contrast, aspartate residues within the CDRs were almost entirely excluded from the binding interface. Alanine and serine residues did not make many direct contacts with antigen, but they allowed for space and conformational flexibility and thus played an auxiliary role in facilitating productive contacts between tyrosine and antigen. Tyrosine side chains were capable of mediating most of the contacts necessary for high-affinity antigen recognition, and, thus, it seems likely that the overabundance of tyrosine in natural antigen-binding sites is a consequence of the side chain being particularly well suited for making productive contacts with antigen. The findings shed light on the basic principles governing the evolution of natural immune repertoires and should also aid the development of improved synthetic antibody libraries.
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