Peptide-protein microarrays and surface plasmon resonance detection: biosensors for versatile biomolecular interaction analysis.

International audienceBiosensors in microarray format provide promising tools for high-throughput analyses of complex samples. Although they are able to detect, quantify and characterize a multitude of compounds, most of the available devices are specialized in the analysis of one type of interaction, limiting their application to a define area. The aim of our work was to develop and characterize versatile protein (or peptide) microarrays suitable for the simultaneous analysis of a large panel of biological interactions. Our system involved a simple procedure to immobilized proteins or peptides, based on pyrrole electropolymerization, and ligand binding was detected by imaging the surface plasmon resonance. We demonstrated its suitability in three different contexts, i.e. humoral response characterization, ion binding analysis and cell detection. This work evidences the potentiality of this approach which allows multiparametric, high-throughput and label-free analysis of biological samples suitable for the detection of compounds as various as proteins, ions or cells and the characterization of their interaction with peptides or proteins

A new role for complement C3: regulation of antigen processing through an inhibitory activity.

International audienceIncreasing evidence underlines the involvement of complement component C3 in the establishment of acquired immunity which appears to play a complex role and to act at different levels. As antigen proteolysis by antigen presenting cells is a key event in the control of antigen presentation efficiency, and consequently in the quality of the immune response, we investigated whether C3 could modulate this step. Our results demonstrate for the first time that C3 can interfere with antigen proteolysis: (i) proteolysis of tetanus toxin (TT) by the lysosomal fraction from a human monocytic cell line (U937) is impaired in the presence of C3, (ii) this effect is C3-specific and involves the C3c fragment of the protein, (iii) C3c is effective even after disulfide disruption, but none of its three constitutive peptides is individually accountable for this inhibitory effect and (iv) the target-protease(s) exhibit(s) a serine-protease activity. The physiological relevance of our results is demonstrated by experiments showing a subcellular colocalisation of TT and C3 after their uptake by U937 and the reduction of TT proteolysis once internalised together with C3. These results highlight a novel role for C3 that broadens its capacity to modulate acquired immune response

Des puces à protéines/peptides pour des applications en recherche fondamentale et clinique

Les biopuces sont des systèmes miniaturisés qui permettent des analyses en parallèle et à haut débit. Ainsi, les puces à protéines/peptides sont très utiles dans l'étude des interactions entre diverses molécules biologiques. Nous avons réalisé des puces à protéines et peptides en se basant sur la technologie des polymères conducteurs : la méthode consiste à immobiliser les molécules sondes (protéines ou peptides) sur des supports par électrocopolymérisation à partir d'un mélange de pyrrole et de pyrrole lié à la molécule sonde. Le support consiste en un prisme recouvert d'un film d'or permettant l'analyse des interactions protéine-protéine ou antigène-anticorps par imagerie de la résonance plasmonique de surface. Cette méthode optique, compatible avec des mesures en parallèle, permet une analyse directe, en temps réel et donne accès aux paramètres cinétiques des interactions. Ces puces, de réalisation facile, présentent une très bonne spécificité et une bonne sensibilité. Elles peuvent être régénérées un grand nombre de fois et sont compatibles avec l'analyse d'échantillons biologiques complexes tels que du sérum non dilué. Ces caractéristiques permettent un champ d'application très large, en recherche fondamentale ou clinique mais aussi en diagnostic.GRENOBLE1-BU Sciences (384212103) / SudocCACHAN-ENS (940162301) / SudocSudocFranceF

Real time and label-free analysis of cellular activity on chip

Publisher: IEEE, 345 E 47TH ST, New York, NY 10017 USA Accession Number ISI:000279891700386International audienceBiochips for cellular applications are of considerable interest to both fundamental research and diagnostic fields. In this study, we demonstrate the use of Surface Plasmon Resonance imaging (SPRi) for the analysis of blood cell activity on biochip. This method previously used to detect antigen-antibody interactions was adapted for the detection of antibodies secreted from B-cell hybridoma using specific antigens grafted on the chip by electropolymerization. For the first time, living cells were maintained in culture on the biochip and the kinetic of their secretion was monitored by SPFti. Unlike traditional methods such as ELISA or ELISPOT, this novel technique permits a real-time and label-free analysis. As a result, antibodies secreted were detected only few minutes after the loading of cells on the chip. Thus, this sensor provides a promising tool for cells phenotype analysis and could be useful in analyzing other secreting cells, like T-cells, widely involved in the inflammatory response and several pathologies

Real-time detection of lymphocytes binding on an antibody chip using SPR imaging.

International audienceWe demonstrate the use of SPR imaging for the detection of site-specific binding of either B or T lymphocyte populations on an electrochemically-grafted antibody array

On chip real time monitoring of B-cells hybridoma secretion of immunoglobulin.

International audienceThe secretions of molecules by cells are of tremendous interest for both fundamental insights studies and medical purposes. In this study, we propose a new biochip-based approach for the instantaneous monitoring of protein secretions, using antibody production by B lymphocytes cultured in vitro. This was possible thanks to the Surface Plasmon Resonance imaging (SPRi) of a protein biochip where antigen proteins (Hen Egg Lysozyme, HEL) were micro-arrayed along with series of control proteins. B cell hybridomas were cultured on the chip and the secretion of immunoglobulins (antibody) specific to HEL was monitored in real-time and detected within only few minutes rather than after a 30-60 min incubation with standard ELISA experiments. This fast and sensitive detection was possible thanks to the sedimentation of the cells on the biochip sensitive surface, where local antibody concentrations are much higher before dilution in the bulk medium. An other interesting feature of this approach for the secretion monitoring was the independence of the SPR response--after normalization--regarding to the density of the surface-immobilized probes. Such biosensor might thus pave the way to new tools capable of both qualitative and semi-quantitative analysis of proteins secreted by other immune cells

C3b complexation diversifies naturally processed T cell epitopes.

International audienceIn addition to its well-established role in innate immunity, the complement component C3 is of critical importance in modulating the humoral response. In this study, we examined the effect of C3b linkage to tetanus toxin (TeNT) in the production of antigenic peptides inside human APC. We purified HLA-DR associated peptides isolated either from TeNT or TeNT-C3b pulsed cells. This study revealed that TeNT-C3b derived antigenic peptides are different and more numerous than TeNT derived peptides. This increased production of antigenic peptides correlated with a C3b-induced TeNT modification of proteolysis. These findings argue in favour of a new role for C3b in the modulation of T cell epitope during antigen processing and presentation

Design and application of a microarray for fluorescence and and Surface Plasmon Resonance imaging analysis of peptide-antibody interactions.

International audienceIn the past few years, protein and peptide microarrays have shown a great potential in fundamental research, medical diagnostics and drug discovery. We have developed a new method based on covalent immobilization of peptides on a microarray gold surface for detecting antibodies by either fluorescence or surface plasmon resonance imaging (SPRi). The fluorescence-based revelation approach allows for an indirect analysis of protein-antibody binding via labeled molecules, whereas the SPRi-based assay is a real-time and label-free detection system suitable for the direct analysis of biological interactions. We illustrate our peptide chip's efficiency in specifically binding serum antibodies and compare the fluorescence and SPRi approaches to detect peptide-antibody interactions. Moreover, we used a monoclonal antibody against the hepatitis C virus nucleocapsid protein to provide evidence that our system is a powerful tool to identify which epitope is recognized by a given antibody. This novel technology therefore provides a promising tool for both antibody screening and epitope mapping

Clinically related protein-peptide interactions monitored in real time on novel peptide chips by surface plasmon resonance imaging.

International audienceBACKGROUND: Developing rapid, high-throughput assays for detecting and characterizing protein-protein interactions is a great challenge in the postgenomic era. We have developed a new method that allows parallel analysis of multiple analytes in biological fluids and is suitable for biological and medical studies. METHODS: This technology for studying peptide-antibody interactions is based on polypyrrole-peptide chips and surface plasmon resonance imaging (SPRi). We generated a chip bearing a large panel of peptide probes by successive electro-directed copolymerizations of pyrrole-peptide conjugates on a gold surface. RESULTS: We provide evidence that (a) the signal produced by antibody binding is highly specific; (b) the detected signal specifically reflects the antibody concentration of the tested solution in a dose-dependent manner; (c) this technique is appropriate for analyzing complex media such as undiluted sera, a novelty with respect to previous techniques; and (d) correlation between classic ELISA results and the SPRi signal is good (P = 0.008). We also validated this system in a medical model by detecting anti-hepatitis C antibodies in patient-derived sera. CONCLUSION: Because of its characteristics (easy preparation of the peptide chip; high-throughput, label-free, real-time detection; high specificity; and low background), this technology is suitable for screening biological samples and for large-scale studies