DNA G-Quadruplex as a Reporter System for Sensor Development

The versatile DNA G-quadruplex structure has emerged as an interesting alternative reporter system applied in different biosensor platforms. In comparison to the conventional reporter systems like enzymatic or fluorescent, DNA G-quadruplex has some distinct advantages, as it is thermostable, easy to produce, low cost and most importantly able to be amplified. Such remarkable advantages have led many researchers to exploit DNA G-quadruplex as the reporter system in colorimetric, fluorescence and luminescence sensors. There has also been integration of DNA G-quadruplex with electrochemical methods and quantum dot for sensing applications. Therefore, this chapter highlights some recent examples of different biosensor platforms that use DNA G-quadruplex as a reporter system with different detection methods

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

Recombinant human antibody technology has been the cornerstone of the uprising of biologics in the pharmaceutical industry. The introduction of various display technologies like phage, yeast, bacterial, ribosomal, mRNA, DNA display and mammalian cell surface display has allowed improved antibody generation programs. The ability to generate recombinant antibodies from available human antibody libraries by using in vitro display methods pave the way to select recombinant human antibodies against almost every antigen. The libraries are a close representation of the B-cell response elicited by the natural immune system. The introduction of various methods to fine tune the antibody affinities has made recombinant antibody technology highly sought after. The ability to engineer specific characteristics of each antibody by design is possible utilizing advanced in vitro strategies. This chapter will focus on the technologies commonly applied in antibody display technologies to engineer improved affinities

Phage Display‐Derived Antibodies: Application of Recombinant Antibodies for Diagnostics

Antibodies are produced by the human body in response towards infections as a means of protection. The in vivo production of antibodies by B-cells involves a series of intricate gene editing processes resulting in a highly diverse pool of antibodies. However, this diversity can be replicated in vitro using phage display. Phage display offers the potential to present the antibody phenotype together with the cloned genotype of the specific antibody in a single-phage particle. Antibodies are highly sought after for diagnostic applications owing to its specificity and affinity towards a target antigen. The advent of recombinant antibody (rAb) technology allows for a faster and more cost-effective solution for antibody generation. It also provides diagnostic developers with the possibility to customize the antibodies. Antibodies have been utilized successfully in various diagnostic platforms ranging from standard immunoassays to lateral-flow assays, nanoparticles, microfluidics, DNA‐integrated assays and others. The limitless application of antibodies in the field of diagnostics has made it a critical component in any diagnostic development platform. This chapter focuses on the processes involved in antibody discovery including the various forms of antibody libraries for phage display and panning processes. We also highlight some diagnostic platforms that apply recombinant antibodies

Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen

Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications

Therapeutic Phage Display-Derived Single-Domain Antibodies for Pandemic Preparedness

Driven by necessity, the COVID-19 pandemic caused by SARS-CoV-2 has accelerated the development and implementation of new vaccine platforms and other viral therapeutics. Among these is the therapeutic use of antibodies including single-domain antibodies, in particular the camelid variable heavy-chain fragment (VHH). Such therapies can provide a critical interim intervention when vaccines have not yet been developed for an emerging virus. It is evident that an increasing number of different viruses are emerging and causing epidemics and pandemics with increasing frequency. It is therefore imperative that we capitalize on the experience and knowledge gained from combatting COVID-19 to be better prepared for the next pandemic

Dengue serotyping with a label-free DNA sensor

Dengue virus (DENV) is one of the most important mosquito-borne viruses in tropical and subtropical regions. Development of severe forms of dengue viral infection such as dengue fever (DF) and dengue hemorrhagic fever (DHF) has claimed many lives. The standard methods for detecting dengue virus are time consuming, laborious, and require skilful personnel. In this study, we propose a method whereby DENV RNA extracted from dengue infected mosquitoes was converted into DNA for probe hybridization to generate silver nanocluster strands that could be visualised under UV light. Label-free silver nanocluster based DNA sensors are able to provide strong fluorescence upon DNA hybridization. Highly specific DNA sequence detection is possible by taking advantage of the specificity of DNA hybridization kinetics. The proposed system is capable of detecting all four dengue DNA serotypes (DENV1-4) without any cross-reactivity. A single tube assay format showed better hybridisation efficiency with higher fluorescence intensity generated and a lower detection limit compared to a cocktail probe assay format. The method was able to detect as low as 100 nM of amplified double stranded dengue DNA targets using both single and cocktail probe assays. This provides an interesting alternative approach for multiplex DNA sensing utilizing DNA silver nanoclusters as a reporter system. © 2018 The Royal Society of Chemistry

ゾル−ゲル転移を示す生体適合ポリマー材料の開発と応用 (2)

(1) Title: Bulk pH Responsive DNA Quadruplex Hydrogels Prepared by Liquid-Phase Large Scale DNA SynthesisJournal: ACS Macro Letters(2) Title: Communication—DNA Quadruplex Hydrogel Beads Showing Peroxidase ActivityJournal: Journal of The Electrochemical SocietyDOI: http://dx.doi.org/10.1149/2.0441909je

Einfluss unterschiedlicher Parameter auf die Erstellung von Phagen-Display- Bibliotheken hinsichtlich einer verbesserten Erzeugung menschlicher Antikörper

The main rationale for the titanic flexibility of the immune system to engage various types of entities deemed foreign by the body is down to the huge diversity of the antibody repertoire. With the prospective potential of antibodies in therapy and diagnostics, effective and efficient strategies to increase in vitro generation of human antibodies have been investigated. The quality of antibody libraries is influenced by several factors including antibody formats, display levels, sequence diversity, expression level, tendency to multimerize, compatibility with in vitro screening, affinity maturation and ease of format conversion to other antibody formats or display and selection systems. The aim of the thesis was to investigate different parameters that contribute to the generation of human antibodies by phage display. Here, the influence of different antibody scaffolds on display efficiency and antibody panning were analysed using different Fab Formats. IgG Fab performed better than IgD Fab with a 8-fold presentation efficiency. Further, the interchange of V-regions between individual formats and the influence on binding characteristics was evaluated. From the format conversion experiments, in between three formats - scFv, IgD Fab and IgG Fab - IgG Fab was the best format as it showed the lowest decrease in binding characteristics when converted to the either scFv or IgD Fab. Once the best interchangeable format for antibody library design was determined, the amplification procedure to generate an IgG Fab library from human donor materials was assessed. First, a set of V-gene primers was designed and evaluated. The primer set was analyzed against V-gene sequences from the VBASE2 database. The primer set analyzed showed a significant coverage of known V-gene sequences. 98% coverage of functional and putatively functional genes were covered. Other parameters effecting the amplification of V-genes from donor materials were assessed, namely, cDNA generation, polymerase amplification efficiency as well as the influence of additives for improving PCR amplification yields. Form the outcome of these studies, a standard operating procedure could be described allowing efficient amplification of antibody gene fragments for library generation.Die gigantische Flexibilität des Immunsystems, die es in die Lage versetzt, verschiedenste Typen von Entitäten angreifen zu können, die als fremd erkannt werden, liegt in der enormen Diversität des Antikörper-Repertoires begründet. In Hinblick auf das prospektive Potenzial von Antikörpern in Therapie und Diagnostik wurden effektivere und effizientere Strategien untersucht, die zur Verbesserung der in vitro-Erzeugung menschlicher Antikörper führen sollen. Die Qualität von Antikörper-Bibliotheken wird von verschiedenen Faktoren beeinflusst, darunter Antikörperformate, “display level”, Sequenzdiversität, Expressionsstärke, Tendenz zur Multimerbildung, der Kompatibilität mit in vitro Screening-Verfahren, der Affinitätsreifung und der Leichtigkeit, mit der Formatkonversionen in andere Antikörperformate oder Display- und Selektionssysteme möglich sind. Das Ziel der vorliegenden Arbeit war, verschiedene Parameter zu untersuchen, die Einfluss auf die Erzeugung menschlicher Antikörper durch Phagen-Display haben. Um den Einfluss verschiedener Antikörpergerüste auf die Effizienz des Displays und die Antikörpersuche zu untersuchen, wurde zwei unterschiedliche Fab-Formate untersucht. Im Kontext der beiden untersuchten Fab-Formate, zeigte IgG Fab eine bessere Leistung als IgD Fab mit einer 8-fach erhöhten Effizienz der Präsentation. Des weiteren wurde der Austausch von V-Regionen zwischen individuellen Formaten und deren Einfluss auf die Bindungscharakteristik untersucht. Unter den drei Formaten – scFv, IgD Fab und IgG Fab – erwies sich IgG Fab als das geeignetste Format, da es den geringsten Verlust der Bindungseingeschaft aufwies, wenn es anschließend entweder nach scFv oder IgD Fab konvertiert wurde. Nachdem das beste Austauschformat für das Antikörper- Bibliotheksdesign ermittelt war, wurde die Amplifikationsprozedur, die zur Erzeugung einer IgG Fab-Bibliothek aus menschlichem Spendermaterial verwendet wurde, untersucht. Zunächst wurde ein Satz an V-Gen-Primern entworfen und bewertet. Der Primersatz wurde gegen V-Gen-Sequenzen aus der VBASE2-Datenbank abgeglichen. Insgesamt konnten 98% aller funktionellen und putativ funktionellen V-Genen mit dem Primersatz abgedeckt werden. Anschließend wurden weitere Parameter untersucht, die einen Einfluss auf die Amplifikation menschlicher V-Gene aus Spendermaterial haben, insbesondere die cDNA Synthese, die Effizienz unterschiedlicher DNA-Polymerasen und Additive hinsichtlich der PCR-Ausbeute. Aus den Resultaten dieser Vergleiche konnte ein optimiertes Standard-Protokoll entwickelt werden, das die effiziente Amplifikation von Antikörpergenfragmenten zur Erzeugung von Bibliotheken erlaubt

Studying the role of periplasmic chaperones in Fab presentation on phage surface.

In phage display technology, antibodies presented on phage particles are often a smaller variant of the antibody molecule. The Fab format consists of a pair of heavy chain and light chain that is linked together via a disulphide bridge. The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formatio

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some ‘fine tuning’ may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications