Refine
Year of publication
Document Type
- Article (36)
- Conference Proceeding (1)
- Postprint (1)
Is part of the Bibliography
- yes (38)
Keywords
- A viruses (1)
- Anisotropy (1)
- Cell-free (1)
- Energy-transfer probe (1)
- Exciplex (1)
- Fluorescence (1)
- In vitro translation (1)
- Insect lysate (1)
- Single chain antibody (scFv) (1)
- Surface plasmon resonance (1)
Institute
Anthracyclines like daunorubicin (DRN) and doxorubicin (DOX) play an undisputed key role in cancer treatment, but their chronic administration can cause severe side effects. For precise anthracycline analytical systems, aptamers are preferable recognition elements. Here, we describe the detailed characterisation of a single-stranded DNA aptamer DRN-10 and its truncated versions for DOX and DRN detection. Binding affinities were determined from surface plasmon resonance (SPR) and microscale thermophoresis (MST) and combined with conformational data from circular dichroism (CD). Both aptamers displayed similar nanomolar binding affinities to DRN and DOX, even though their rate constants differed as shown by SPR recordings. SPR kinetic data unravelled a two-state reaction model including a 1 : 1 binding and a subsequent conformational change of the binding complex. This model was supported by CD spectra. In addition, the dissociation constants determined with MST were always lower than that from SPR, and especially for the truncated aptamer they differed by two orders of magnitude. This most probably reflects the methodological difference, namely labelling for MST vs. immobilisation for SPR. From CD recordings, we suggested a specific G-quadruplex as structural basis for anthracycline binding. We concluded that the aptamer DRN-10 is a promising recognition element for anthracycline detection systems and further selected aptamers can be also characterised with the combined methodological approach presented here.
Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/MuteSwan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.
Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis
(2015)
Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.
Para-maleimidophenyl (p-MP) modified gold surfaces have been prepared by one-step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N-terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p-MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read-out for a broad variety of biomolecular interactions on the same chip. Copyright (c) 2014 John Wiley & Sons, Ltd.
Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.
The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltarnmetric measurement of the coupled ferroceneboronic acid. Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied. In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an EO' of +200 mV versus Ag/AgC1 (1 M KC1). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times.
A novel electrochemical immunoassay based on the multiple affinity labeling of the indicator antibody with an electro-active tag is presented. The concept is illustrated for the determination of the glycated hemoglobin HbA1c in hemoglobin samples. Hemoglobin is adsorbed to the surfactant-modified surface of a piezoelectric quartz crystal. Whereas the quartz crystal nanobalance is used to validate the total Hb binding, the HbA1c on the sensor surface is recognized by an antibody and quantified electrochemically after the sugar moieties of the antibody have been labeled in-situ with ferroceneboronic acid. The sensitivity of this sensor is about threefold higher than the sensitivity of a hemoglobin sensor, where the ferroceneboronic acid is bound directly to HbA1c.
A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.