Walter Stöcklein

Molecular Basis for the Binding Promiscuity of an Anti-p24 (HIV-1) Monoclonal Antibody

Multiple binding capabilities utilized by specific protein-to-protein interactions in molecular recognition events are being documented increasingly but remain poorly understood at the molecular level. We identified five unrelated peptides that compete with each other for binding to the paratope region of the monoclonal anti-p24 (HIV-1) antibody CB4-1 by using a synthetic positional scanning combinatorial library XXXX[B1,B2,B3,X1,X2,X3]XXXX (14 mers; 68,590 peptide mixtures in total) prepared by spot synthesis. Complete sets of substitution analogs of the five peptides revealed key interacting residues, information that led to the construction of binding supertopes derived from each peptide. These supertope sequences were identified in hundreds of heterologous proteins, and those proteins that could be obtained were shown to bind CB4-1. Implications of these findings for immune escape mechanisms and autoimmunity are discussed.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

BackgroundAs a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.ResultsThe selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.ConclusionThe MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

Effects of organic solvents of semicontinuous immunochemical detection of coumarin derivatives

The effects of water-miscible organic solvents have been investigated with respect to the stability of immobilized antibodies, binding of hapten and conjugation to immobilized antibodies, and marker enzyme stability and activity. Polyclonal antibodies against a coumarin derivative have been isolated from sheep. Laccase and peroxidase are used as hapten labels for competitive enzyme-linked immunosorbent assay (ELISA) and flow-injection immunoanalysis (FIIA). Examples for improvements of the sensitivity and elution efficiency by the application of solvent-water mixtures are presented.

Electrochemical Behavior and Nitric Oxide Interaction of Immobilized Cytochrome c′ fromRhodocyclus gelatinosus

Cytochrome c′ from Rhodocyclus gelatinosus was immobilized at a mercaptosuccinic acid modified gold electrode for the first time and the electrochemical behavior was characterized. The conditions of protein adsorption were investigated. Buffer and pH variations indicated a mainly nonionic nature of interaction. The heterogeneous electron transfer rate constant of the immobilized protein was determined (ks=30 s –1). The adsorption was found to be stable in protein-free solution for about one week. The immobilized cytochrome c′ was accessible for nitric oxide molecules in solution. The interaction resulted in an enhanced reduction current in the cyclic voltammogram. When polarized at –220 mV (vs. Ag/AgCl) the electrode current showed a linear dependence upon NO concentration. The signal was proven to be sensitive within the nanomolar range.

Signal Amplification in Immunoassays Using Labeling via Boronic Acid Binding to the Sugar Moiety of Immunoglobulin G: Proof of Concept for Glycated Hemoglobin

Abstract 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.

Development of a bifunctional sensor using haptenized acetylcholinesterase and application for the detection of cocaine and organophosphates

We developed a dual piezoelectric/amperometric sensor for the detection of two unrelated analytes in one experiment that uses propidium to anchor acetylcholinesterases (AChE) at the surface. This mass-sensitive sensor does not only allow the examination of the interaction between AChE and the modified surface but also the detection of in situ inhibition of the surface-bound AChE. Here we describe the application of the propidium-based sensor in combination with a modified AChE. For this reason the cocaine derivative benzoylecgonine (BZE) was coupled via a 10A long hydrophilic linker - 1,8-diamino-3,4-dioxaoctane - to carboxylic groups of the AChE after EDC/NHS activation. Thus the modified AChE (BZE-AChE) possesses an additional recognition element besides the inhibitor binding site. After the deposition of BZE-AChE on the sensor surface the binding of an anti-BZE-antibody to the BZE-AChE can be monitored. This makes it possible to determine two analytes - cocaine and organophosphate - in one experiment by measuring antibody binding and decrease in enzymatic activity, respectively. Furthermore it was also shown that other cocaine-binding enzymes, e.g., butyrylcholinesterase, can bind to the modified BZE-AChE. The competitive immunoassay allowed the detection of cocaine with a dynamic range from 10(-9) to 10(-7)M. The organophosphate chlorpyrifos-oxon could be detected in concentrations from 10(-6) down to 10(-8)M after 20 min of injection time (equals to 500 microL sample volume.

Direct electrocatalytic determination of dissolved peroxidases

Abstract A novel voltammetric approach is described for the quantification of dissolved redox enzymes. The measurement of horseradish peroxidase and microperoxidase is based on the electrocatalytic current generated when the enzyme reduces hydrogen peroxide to water and oxidises at low potential a mediator which is regenerated in a cathodic electrode reaction. Since the mediator regeneration is sufficiently fast, the overall reaction rate is determined by the enzyme activity. As a consequence stationary current-time curves are obtained in dependence on the activity of peroxidase in solution. For the chemical modification the electrode was modified with 1-(N,N-dimethylamine)-4-(4-morpholine)benzene, a mediator of low solubility in reduced form and with favourably low formal potential. At a working potential of − 50 mV vs. 0.1 M Ag AgCl cathodic steady-state currents were obtained after injection of peroxidase in the presence of 1 mmol/1 hydrogen peroxide. The detection limit for horseradish peroxidase and microperoxidase-11 were 10ng/ml (250 pmol/1) and 20 ng/ml (10 nmol/1), respectively.

Electrochemical determination of human hemoglobin by using ferrocene carboxylic acid modified carbon powder microelectrode

Abstract A mediator modified carbon powder microelectrode was developed and applied for the electrochemical determination of human hemoglobin in whole blood. The electrode body contained acetylene carbon black blended with ferrocene carboxylic acid. A quantitative oxidation of oxyhemoglobin to methemoglobin by the modified electrode was justified spectroscopically. The electrode responded to the addition of Hb within 10 s with the sensitivity of 64 nA g−1 dL in the linear range between 0.05 and 0.6 g dL−1. Thus, a sample solution of 1/50 allows for measurement of hemoglobin in human whole blood in the physiological important concentration range.

Direct electron transfer of adrenodoxin—a [2Fe–2S] protein—and its mutants at modified gold electrode

Abstract Adrenodoxin can adsorb on polyallylamine modified surfaces at low ionic strength, because of local acidic surface. After a gold electrode was modified with a multilayer of mercaptoundecanoic acid and polyallylamine the direct heterogeneous electron transfer between adrenodoxin and the electrode has been achieved. The apparent electron transfer rate constant keto was estimated to be ∼0.14 s−1 and the midpoint redox potential to be −0.46 V±10 mV. Peak current is proportional to scan rate up to 150 mV s−1 indicating that wt-Adx was adsorbed on the electrode surface. Two mutants were investigated which show at the same modified electrode a redox potential shift of about 30 mV in the negative direction. An increase in the heterogeneous electron transfer rate and efficiency of cytochrome c reduction were observed with both mutants as compared to wild type adrenodoxin.

Electrochemical Immunosensors on the Route to Proteomic Chips

Publisher Summary This chapter aims to demonstrate how highly parallel protein analysis can be achieved by combining immunoassays and electrochemical biochip technology. Analytical systems that are based on electrochemical sensors have the potential for decentralized applications. Most electrochemical immunosensors are developed for the determination of single analytes. However, in the context of proteomics, thousands of proteins have to be analyzed in parallel. This could be reached by using a large number of different recognition spots on a chip surface. Methods originally developed for DNA chips could be the basis for the immobilization of antibodies. The chapter presents possible ways to overcome two major problems in the developing electrochemical protein chips: (1) the cross talk between the recognition spots during indication must be avoided, and (2) the microelectrodes on the chip Electrochemical Immunosensors on the Route to Proteomic Chips should be read out simultaneously.