Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time

On the viscometric examination of cement pastes

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General In Vitro Method to Analyze the Interactions of Synthetic Polymers with Human Antibody Repertoires

Recent reports on the hitherto underestimated antigenicity of poly­(ethylene glycol) (PEG), which is widely used for pharmaceutical applications, highlight the need for efficient testing of polymer antigenicity and for a better understanding of its molecular origins. With this goal in mind, we have used the phage-display technique to screen large, recombinant antibody repertoires of human origin in vitro for antibodies that bind poly­(vinylpyrrolidone) (PVP). PVP is a neutral synthetic polymer of industrial and clinical interest that is also a well-known model antigen in animal studies, thus allowing the comparison of in vitro and in vivo responses. We have identified 44 distinct antibodies that bind specifically to PVP. Competitive binding assays show that the PVP-antibody binding constant is proportional to the polymerization degree of PVP and that specific binding is detected down to the vinylpyrrolidone (VP) monomer level. Statistical analysis of anti-PVP antibody sequences identifies an amino-acid motif that is shared by many phage-display-selected anti-PVP antibodies that are similar to a previously described natural anti-PVP antibody. This suggests a role for this motif in specific antibody/PVP interactions. Interestingly, sequence analysis also suggests that only a single antibody chain containing this shared motif is responsible for antibody binding to PVP, as confirmed upon systematic deletion of either antibody chain for 90% of selected anti-PVP antibodies. Overall, a large number of antibodies in the human repertoires we have screened bind specifically to PVP through a small number of shared amino acid motifs, and preliminary comparison points to significant correlations between the sequences of phage-display-selected anti-PVP antibodies and their natural counterparts isolated from immunized mice in previous studies. This study pioneers the use of antibody phage-display to explore the antigenicity of biotechnologically relevant polymers. It also paves the way for a fast, cost-effective, and systematic in vitro analysis, thus reducing the need for animal immunization experiments. Moreover, identifying the encoding DNA sequence of polymer-binding antibodies via phage-display enables future applications of a molecular biology approach to protein–polymer conjugation, based on protein–antibody fusion

Engineering recombinant antibodies for polymer biofunctionalization

The attachment of recognition elements such as antibody fragments to polymeric substrates can be used to mediate cell- or protein-specific interactions. In this work, single-chain Fv (scFv) antibody fragments were isolated against two cell types of interest and expressed in an Escherichia coli expression platform. The scFvs were engineered at their C-terminus to incorporate a cysteine-containing linker, for reaction with maleimide-linked polymers, or a heptasaccharide glycan for complexation with surface amine moieties. Antigen binding of the modified scFvs was unchanged, and expression yields of the glyco-engineered scFvs were similar to the unmodified molecules, while cys-tagged scFv yields varied between scFv variants. Targeted immobilization of the scFvs via either modification resulted in three-to five-fold higher binding of ligands over adsorbed molecules. The study demonstrates a simple and efficient antibody engineering and modification approach for effective targeted immobilization on polymeric substrates. Copyright (C) 2015 John Wiley & Sons, Ltd.This work was supported by Irish Research Council (IRC) grantPD/2010/1689 (MJH), Science Foundation Ireland Grant 07/SRC/B1163 (CC, AS), Enterprise Ireland Science and TechnologyAgency Grant PC/2007/021 (SR) and European Union “EPICstent”Project Grant FP7-PEOPLE-2012-IAPP-324514 (SA)

Selection of Arginine-Rich Anti-Gold Antibodies Engineered for Plasmonic Colloid Self-Assembly

Antibodies are affinity proteins with a wide spectrum of applications in analytical and therapeutic biology. Proteins showing specific recognition for a chosen molecular target can be isolated and their encoding sequence identified in vitro from a large and diverse library by phage display selection. In this work, we show that this standard biochemical technique rapidly yields a collection of antibody protein binders for an inorganic target of major technological importance: crystalline metallic gold surfaces. Twenty-one distinct anti-gold antibody proteins emerged from a large random library of antibodies, and they were sequenced. The systematic statistical analysis of all the protein sequences reveals a strong occurrence of arginine in anti-gold antibodies, which corroborates recent molecular dynamics predictions on the crucial role of arginine in protein/gold interactions. Once tethered to small gold nanoparticles using histidine tag chemistry, the selected antibodies could drive the self-assembly of the colloids onto the surface of single crystalline gold platelets as a first step toward programmable protein-driven construction of complex plasmonic architectures. Electrodynamic simulations based on the Green Dyadic Method suggest that the antibody-driven assembly demonstrated here could be exploited to significantly modify the plasmonic modal properties of the gold platelets. Our work shows that molecular biology tools can be used to design the interaction between fully folded proteins and inorganic surfaces with potential applications in the bottom-up construction of plasmonic hybrid nanomaterials

General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires

International audienceRecent reports on the hitherto underestimated antigenicity of poly(ethylene glycol) (PEG), which is widely used for pharmaceutical applications, highlight the need for efficient testing of polymer antigenicity and for a better understanding of its molecular origins. With this goal in mind, we have used the phage-display technique to screen large, recombinant antibody repertoires of human origin in vitro for antibodies that bind poly(vinylpyrrolidone) (PVP). PVP is a neutral synthetic polymer of industrial and clinical interest that is also a well-known model antigen in animal studies, thus allowing the comparison of in vitro and in vivo responses. We have identified 44 distinct antibodies that bind specifically to PVP. Competitive binding assays show that the PVP-antibody binding constant is proportional to the polymerization degree of PVP and that specific binding is detected down to the vinylpyrrolidone (VP) monomer level. Statistical analysis of anti-PVP antibody sequences identifies an amino-acid motif that is shared by many phage-display-selected anti-PVP antibodies that are similar to a previously described natural anti-PVP antibody. This suggests a role for this motif in specific antibody/PVP interactions. Interestingly, sequence analysis also suggests that only a single antibody chain containing this shared motif is responsible for antibody binding to PVP, as confirmed upon systematic deletion of either antibody chain for 90% of selected anti-PVP antibodies. Overall, a large number of antibodies in the human repertoires we have screened bind specifically to PVP through a small number of shared amino acid motifs, and preliminary comparison points to significant correlations between the sequences of phage-display-selected anti-PVP antibodies and their natural counterparts isolated from immunized mice in previous studies. This study pioneers the use of antibody phage-display to explore the antigenicity of biotechnologically relevant polymers. It also paves the way for a fast, cost-effective, and systematic in vitro analysis, thus reducing the need for animal immunization experiments. Moreover, identifying the encoding DNA sequence of polymer-binding antibodies via phage-display enables future applications of a molecular biology approach to protein-polymer conjugation, based on protein-antibody fusion