Combining culture and microbead-based immunoassay for the early and generic detection of bacteria in platelet concentrates.

BACKGROUND: Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. STUDY DESIGN AND METHODS: A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. RESULTS: Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. CONCLUSION: In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

Parallel kinetic analysis and affinity determination of hundreds of monoclonal antibodies using the ProteOn XPR36.

The production of antibodies for diagnostic and therapeutic applications is a major focus for biotechnology and pharmaceutical companies, and it requires the development of fast, high-throughput methodologies for screening and selecting appropriate candidate antibodies for development. These candidates must have very high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR 36 protein interaction array system for the rapid screening and selection of high-affinity antibodies--"one-shot" kinetics. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies simultaneously in one experiment. We have used this new methodology to screen hundreds of monoclonal supernatants containing antibodies against two proteins of potential clinical interest: human interleukin 12 (IL-12) and a human hemoglobin (Hb) variant. In fact, approximately 250 supernatants raised against each antigen were screened in approximately 17 h, providing several high-affinity candidate monoclonal antibodies for each of these antigens.

Tissue-type plasminogen activator controls neuronal death by raising surface dynamics of extrasynaptic NMDA receptors.

N-methyl-d-aspartate receptors (NMDARs) are ion channels whose synaptic versus extrasynaptic localization critically influences their functions. This distribution of NMDARs is highly dependent on their lateral diffusion at the cell membrane. Each obligatory subunit of NMDARs (GluN1 and GluN2) contains two extracellular clamshell-like domains with an agonist-binding domain and a distal N-terminal domain (NTD). To date, the roles and dynamics of the NTD of the GluN1 subunit in NMDAR allosteric signaling remain poorly understood. Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator (tPA), well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic NMDARs. This process explains the previously reported ability of tPA to promote NMDAR-mediated calcium influx. In parallel, we developed a monoclonal antibody capable of specifically blocking the interaction of tPA with the NTD of the GluN1 subunit of NMDAR. Using this original approach, we demonstrate that the tPA binds the NTD of the GluN1 subunit at a lysine in position 178. Accordingly, when applied to mouse neurons, our selected antibody (named Glunomab) leads to a selective reduction of the tPA-mediated surface dynamics of extrasynaptic NMDARs, subsequent signaling and neurotoxicity, both in vitro and in vivo. Altogether, we demonstrate that the tPA is a ligand of the NTD of the obligatory GluN1 subunit of NMDAR acting as a modulator of their dynamic distribution at the neuronal surface and subsequent signaling.

Local carotid atherosclerotic plaque proteins for the identification of circulating biomarkers in coronary patients.

OBJECTIVE: To identify circulating biomarkers that originate from atherosclerotic vulnerable plaques and that could predict future cardiovascular events. METHODS: After a protein enrichment step (combinatorial peptide ligand library approach), we performed a two-dimensional electrophoresis comparative analysis on human carotid plaque protein extracts (fibrotic and hemorrhagic atherosclerotic plaques). In silico analysis of the biological processes was applied on proteomic data. Luminex xMAP assays were used to quantify inflammatory components in carotid plaques. The systemic quantification of proteins originating from vulnerable plaques in blood samples from patients with stable and unstable coronary disease was evaluated. RESULTS: A total of 118 proteins are differentially expressed in fibrotic and hemorrhagic plaques, and allowed the identification of three biological processes related to atherosclerosis (platelet degranulation, vascular autophagy and negative regulation of fibrinolysis). The multiplex assays revealed an increasing expression of VEGF, IL-6, IL-8, IP-10 and RANTES in hemorrhagic as compared to fibrotic plaques (p<0.05). Measurement of protein expressions in plasmas from patients with stable and unstable coronary disease identified a combination of biomarkers, including proteins of the smooth muscle cell integrity (Calponin-1), oxidative stress (DJ-1) and inflammation (IL-8), that allows the accurate classification of patients at risk (p=0.0006). CONCLUSION: Using tissue protein enrichment technology, we validated proteins that are differentially expressed in hemorrhagic plaques as potential circulating biomarkers of coronary patients. Combinations of such circulating biomarkers could be used to stratify coronary patients.

N-truncated Aß peptides in complex fluids unraveled by new specific immunoassays.

Previous studies have highlighted the potential physiopathological and diagnostic role of N- and C-terminally truncated amyloid-ß (Aß) peptides in Alzheimer's disease. However, our knowledge about their production remains incomplete, in part due to the lack of very specific and sensitive tools for their detection. We thus developed specific monoclonal antibodies that target either Aß11-x or Aß17-x species, which result from the combined cleavages by ß/γ- or α/γ-secretases, respectively. The presence of Aß peptides truncated at residue 11 and 17 peptides was qualitatively and quantitatively assessed, using surface enhanced laser desorption ionization-time of flight mass spectrometry and xMAP (Multi-Analyte Profiling) immunoassays, in the supernatant of HEK293 cells that overexpress wild type or mutant Aß protein precursor or in which α- and ß-secretase activities had been modulated. Our results show a differential secretion of Aß11-40 and Aß17-40 species by these HEK293 cell lines. Finally, Aß11-40 concentration in human cerebrospinal fluid (measured with the new xMAP immunoassays) from a first pilot study was higher in cerebrospinal fluid samples from patients with Alzheimer's disease than in samples from patients with other types of dementia.

Production and characterization of mouse monoclonal antibodies reactive to Chikungunya envelope E2 glycoprotein.

Chikungunya fever is an arbovirosis of major impact in public health in Asia and Africa. Chikungunya (CHIK) virus is member of the genus Alphavirus and belongs to the Semliki Forest (SF) antigenic complex. We describe for the first time a panel of monoclonal antibodies (MAbs) reactive to CHIK envelope E2 glycoprotein. For the screening of E2-specific MAbs, we expressed a recombinant soluble CHIK E2 protein in Drosophila S2 cells. Analyzed by immunological methods, MAbs 3C3, 3E4, and 8A4 were selected on the basis of their reactivity. Their epitopes are located to the outer surface of CHIK virion. These MAbs have no cross reactivity with related members of SF antigenic complex with the notable exception of Igbo-Ora virus. Anti-CHIK E2 MAbs 3C3, 3E4, and 8A4 should be helpful for studying the biology of CHIK virus and pathogenesis of disease. The combination of 8A4 and 3E4 is suitable for developing a specific antigen-capture ELISA.

High-affinity uranyl-specific antibodies suitable for cellular imaging.

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO2(2+)-DCP-BSA (DCP, 1,10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO2(2+)-DCP-casein, we selected two highly specific mAbs against uranyl-DCP ( K D 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO2(2+), Fe (3+), Zn2+, Cu2+, and Ca2+) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO2(2+) equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immunolabeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its paratopes can accommodate a wide variety of chelating groups.