Peter Oroszlan

Zeptosens' protein microarrays: A novel high performance microarray platform for low abundance protein analysis

Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two‐dimensional gel electrophoresis (2‐DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000, 97, 9390–9395) [1], most spots in 2‐DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high‐throughput applications. Key factors to reach this goal are: high read‐out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein‐protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.

Fiber-optic Atrazine immunosensor☆

Abstract Fiber-optic sensors based on the excitation of luminescent chromophores by the evanescent field associated with light guiding in an optical fiber can be used for highly sensitive and selective biochemical affinity assays. Due to the small penetration depth of the evanescent field into the medrium, the generation and detection of luminescence are restricted to the close proximity of the fiber core, i.e., fluorophores in solution beyond the evanescent field will not contribute to the emission signal. Evanescent wave sensors allow the binding of fluorophores to the sensor surface to be monitored in real-time mode. These advantages make this approach especially useful for the determination of substances in complex media, such as blood, river water or soil extracts. An evanescent-wave fiber-optic immunosensor for the detection of the herbicide Atrazine has been developed. In the competitive assay format chosen, fluorescein-labeled and nonlabeled Atrazine in solution compete for the binding sites of anti-Atrazine antibodies immobilized on the surface of the optical fiber. A signal reproducibility of better than 5% within the working range of the sensor (0.5–200 nM Atrazine concentration) is achieved. The sensor performance in complex media has been investigated using samples of surface water and soil extracts.

Sandwich immunoassay for the hapten angiotensin II a novel assay principle based on antibodies against immune complexes

Immunoassays for haptens such as short peptides or drugs are usually based on the principle of competition for a limited number of binding sites on antibody molecules. Owing to the small size of these antigens it has been thought that two specific antibodies cannot simultaneously bind a hapten. However, antisera containing so called anti-metatypic antibodies have been reported (Voss et al. (1988) Mol. Immunol. 25, 751-759) that bind to hapten-mAb complexes in a reaction where conformational changes on the primary antibody are important. Here, we report on monoclonal antibody pairs able to form ternary complexes with the octapeptide angiotensin II. The first mAb (mAb1) is conventional and binds angiotensin II with high affinity (Kd 10(-11) M). The secondary (anti-metatypic) mAbs (mAbs2s) recognize the immune complex consisting of angiotensin II bound to mAb1, but only poorly recognize mAb1 alone. An immunization technique involving tolerization with uncomplexed mAb1 was used to generate mAb2s. None of the mAbs2s were able to bind angiotensin II by themselves but all efficiently bound the complex of angiotensin II and mAb1. All mAb2s stabilized the angiotensin II-mAb1 complex and one mAb2 distinctly improved the specificity of the assay for angiotensin II. By either labelling mAb1 and immobilizing mAb2 (or vice versa) two-site immunometric assays with detection limits of 1 pg/ml angiotensin II have been established. The kinetics of the complex formation was investigated by fiber optic biospecific interaction analysis (FOBIA), a system allowing real time observation of binding events on the surface of a glass fiber. The association rate towards the liganded conformation of mAb1 was higher than towards the free mAb1. By contrast, the mAb2s dissociated at similar rates from complexed and uncomplexed mAb1.

Sustained release of injectable zinc-recombinant hirudin suspensions : Development and validation of in vitro release model

In humans, recombinant hirudin (rHir), an anticoagulant protein, has a relatively short half-life (about 1 h). Therefore, a rHir formulation with sustained biological activity was previously proposed to result from complexing zinc salts and rHir (Zn-rHir). The purpose of this paper is to introduce and validate an in vitro release model for subcutaneous Zn-rHir formulations. In glass vials the formulations were suspended in agarose gel (2%) and coated with an extra layer of protein-free agarose. The agarose layers were covered with receiver solution, either buffered solutions (HEPES or PBS, pH 7.4) or human serum. To validate the release model and to demonstrate its potential to discriminate between different formulations, several commercial insulin and Zn-insulin formulations were also tested. The release profiles were evaluated by statistical moment analysis (mean times). Only in HEPES buffer was good discrimination between the investigated insulin formulations observed. The mean times of in vitro release of the insulin formulations and the proposed duration of their biological activities were in correlation. Low discrimination was found in PBS. For rHir, clear discrimination between the investigated rHir formulations was achieved in HEPES buffer, whereas low discrimination was found in PBS or in serum. The developed release model may be a sensitive in vitro test to assure the quality of subcutaneous insulin and rHir formulations, and may also be applicable to assess other slow-release protein and low molecular weight drug injectables.

A model system for the development of an optical biosensor based on lipid membranes and membrane-bound receptors

Abstract The reproducible and easy immobilization of receptors on sensor surfaces is a prerequisite for the development of receptor-based fibre optic biosensors. Using a fused silica fiber as the transducer, binding processes of luminescently labeled ligands can be monitored by evanscent wave sensor (EWS) technology. The vesicle fusion technique was chosen for the immobilization of membrane-bound receptors in order to preserve their binding specificity and activity, by embedding them in an environment similar to a lipid bilayer. The results of initial studies of repetitive cycles of lipid layer deposition and removal, indicating good reproducibility of lipid layer formation on the fiber, are presented. Using the binding of fluorescently labeled streptavidin to a biotinylated lipid layer as a model system for receptor-ligand interaction, good sensitivity, combined with low non-specific binding were observed.

Immunoassays in monitoring biotechnological drugs.

For the evaluation and interpretation of pharmacokinetic data reliable quantitative determinations are a requirement that can only be met by well-characterized and fully validated analytical methods. To cope with these requirements a method is being established that is based on an integrated and automated fiber-optic biospecific interaction analysis system (FOBIA) for immunoassays. Performance characteristics of this system used in monitoring of recombinant hirudin (CGP 39 393) are presented. Recombinant hirudin is a highly potent and selective inhibitor of human thrombin. Owing to its size and charge, recombinant hirudin is mainly eliminated by glomerular filtration. But only a fraction of the hirudin dose seems to be reabsorbed at the proximal tubule by luminal endocytosis and hydrolyzed by lysosomal enzymes, leaving approximately 50% of the dose to be extracted in the urine. Thus, renal clearance of recombinant hirudin in the absence of renal insufficiency appears to depend primarily on the glomerular filtration rate. During a 3-month i.v. tolerability study in dogs, some of the dogs developed antibodies against recombinant hirudin. The hirudin-antibody complex accumulated in plasma and apparent hirudin plasma concentrations were therefore much higher than expected from single-dose kinetics. Hirudin captured by antibodies showed an extended half-life and the hirudin-antibody complex is still pharmacologically active, as demonstrated by the observed increase in thrombin time. In conclusion, only appropriate analytical methods allow adequate monitoring and pharmacokinetic characterization of biotechnology drugs in biological materials.

Automated optical sensing system for biochemical assays

In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

Biochemical affinity sensing systems based on luminescence generation in the evanescent field of optical waveguides

We have developed a (bio)chemical analysis system based on luminescence generation and detection in the evanescent field associated with light guiding in an optical fiber. Our intention was directed towards optimization of not only the sensor, including the sensor handling and the immobilization of biochemical recognition elements, but also of the assay chemistry, with special emphasis on methods used for sensor regeneration, of the fluidic system, and of the experimental control software. Goals of this optimization process were not only to achieve high sensitivity, reproducibility and the related precision of the results, but also maximum regenerability of the sensors and system flexibility for a variety of different applications. Four examples of different bioaffinity assays, established on our sensor system, are presented: a competitive immunoassay for atrazine, a sandwich immunoassay for hirudin, a DNA hybridization assay, and first studies for the development of sensors based on membrane- bound receptors. In the atrazine assay, the sensor could be regenerated more than 300 times. In the hybridization assay, a detection limit of 7.5 multiplied by 10-14 M complementary fluorescein-labeled DNA was achieved. The performance of our system is compared with an established enzyme-linked immunosorbent assay (ELISA) on the example of the hirudin assay. In the concluding section of this paper, advantages and disadvantages of our fiberoptic, luminescence-based system, compared with commercialized systems, based on detection of changes of the effective refractive index, are discussed.

Receptor Based Fiberoptic Biosensor

This chapter describes a fiberoptic biosensor based on biological receptors and evanescent wave sensor (EWS). Biological receptors are potentially useful as sensor recognition elements because of their high binding specificity and selectivity. Receptor based biosensors are interesting for the analysis of toxicological and pharmacological relevant substances, and to monitor and characterize on a real-time basis receptor–ligand interactions. The general design and the high sensitivity of the EWS allow replacing radioactive labels, commonly used for receptor-ligand binding studies, by fluorescent labels. The high spatial selectivity is provided by the optical sensing principle: the evanescent field associated with a light ray guided in an optical fiber penetrates only parts of a wavelength (some hundred nanometers) into the surrounding medium. The first model system is based on the immobilization of lipid-anchored biotin, as an analog for the receptor binding site and fluorescently labeled streptavidin as ligand. Vesicles composed of an uncharged lipid and of biotinylated lipids are spread on the hydrophobic fiber surface. Specific binding of streptavidin to the biotinylated lipid layer is measured. A high sensitivity with low nonspecific binding of streptavidin to the lipids is achieved.

Faseroptische Evaneszentfeld-Sensoren für biochemische Assays

A fiber optic sensor for biochemical affinity assays, based on evanescent wave excitation of luminescently labeled biomolecules, is presented. As a typical application, a competitive immunoassay for the detection of herbicides will be shown. In this assay format, both luminescently labeled and nonlabeled antigens compete for the binding to antibodies which have been immobilized, as biomolecular recognition elements, on the fibers. When light of the adequate excitation wavelength is launched into the fiber, luminescently labeled antigens, which combine with the immobilized affinity partners within the penetration depth of the evanescent field, are excited and can be detected. Outside of the evanescent field no luminescence is generated.