Human IgE mAbs identify major antigens of parasitic worm infection.

Background: Much of our understanding of the targets of IgE comes from studies of allergy, though little is known about the natural immunogenic targets seen after parasitic worm infections.

Objective: We used human monoclonal antibodies (mAbs) for an unbiased and comprehensive characterization of the immunodominant antigens targeted by IgE in conditions like allergy or helminth infection that are associated with elevated levels of IgE.

Methods: Using human hybridoma technology to immortalize IgE encoding B-cells from peripheral blood of subjects with filarial infections and elevated IgE, we generated naturally occurring human IgE mAbs.

Estimating Detection Limits of Potentiometric DNA Sensors Using Surface Plasmon Resonance Analyses.

As the signals of potentiometric-based DNA ion-selective field effect transistor (ISFET) sensors differ largely from report to report, a systematic revisit to this method is needed. Herein, the hybridization of the target and the probe DNA on the sensor surface and its dependence on the surface probe DNA coverage and the ionic strength were systematically investigated by surface plasmon resonance (SPR). The maximum potentiometric DNA hybridization signal that could be registered by an ISFET sensor was estimated based on the SPR measurements, without considering buffering effects from any side interaction on the sensing electrode.

Structural Changes of Mercaptohexanol Self-Assembled Monolayers on Gold and Their Influence on Impedimetric Aptamer Sensors.

Despite a large number of publications describing biosensors based on electrochemical impedance spectroscopy (EIS), little attention has been paid to the stability and reproducibility issues of the sensor interfaces. In this work, the stability and reproducibility of faradaic EIS analyses on the aptamer/mercaptohexanol (MCH) self-assembled monolayer (SAM)-functionalized gold surfaces in ferri- and ferrocyanide solution were systematically evaluated prior to and after the aptamer-probe DNA hybridization. It is shown that the EIS data exhibited significant drift, and this significantly affected the reproducibility of the EIS signal of the hybridization.

Systematic Approach to the Development of Microfabricated Biosensors: Relationship between Gold Surface Pretreatment and Thiolated Molecule Binding.

Despite the increasing popularity of microfabricated biosensors due to advances in technologic and surface functionalization strategies, their successful implementation is partially inhibited by the lack of consistency in their analytical characteristics. One of the main causes for the discrepancies is the absence of a systematic and comprehensive approach to surface functionalization. In this article microfabricated gold electrodes aimed at biosensor development have been systematically characterized in terms of surface pretreatment, thiolated molecule binding, and reproducibility by means of X-ray photoelectron scattering (XPS) and cyclic voltammetry (CV).

Considerations in producing preferentially reduced half-antibody fragments.

Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated.

Site-directed antibody immobilization techniques for immunosensors.

Immunosensor sensitivity, regenerability, and stability directly depend on the type of antibodies used for the immunosensor design, quantity of immobilized molecules, remaining activity upon immobilization, and proper orientation on the sensing interface. Although sensor surfaces prepared with antibodies immobilized in a random manner yield satisfactory results, site-directed immobilization of the sensing molecules significantly improves the immunosensor sensitivity, especially when planar supports are employed. This review focuses on the three most conventional site-directed antibody immobilization techniques used in immunosensor design.

Determination of antibodies against human growth hormone using a direct immunoassay format and different electrochemical methods.

A direct immunoassay format with human growth hormone (hGH) immobilized on a self assembled monolayer modified surface plasmon resonance (SPR) chip was chosen to detect specific antibodies (anti-hGH) using different electrochemical techniques. Atomic force microscopy imaging and SPR were used as control methods for the evaluation and confirmation of the antigen-antibody complex formation. The applicability and sensitivity of candidate electrochemical techniques to develop an accurate and sensitive electrochemical immunosensor were investigated.

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry.

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics.