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Biomedical subjects

Thomas O Joos

Publications and source records attributed to Thomas O Joos.

15 recordsLinked to original sources
PubMed2007

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Affinity Labels↗
PubMed2005

Multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins.

BACKGROUND: More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity-purified recombinant HPV proteins. METHODS: Twenty-seven HPV proteins were expressed as glutathione S-transferase fusion proteins and affinity-purified in one step by incubation of glutathione-displaying beads in bacterial lysate. Spectrally distinct bead sets, each carrying one particular antigen, were mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer. RESULTS: Glutathione S-transferase fusion proteins bound with high affinity to glutathione-displaying beads (Kd = 6.9 x 10(-9) mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies were detected at serum dilutions >1:1,000,000. Imprecision (median CV) was < or = 5.4%, and assay reproducibility was high (R2 = 0.97). Results on clinical samples showed high concordance with ELISA (kappa = 0.846), but multiplex serology exhibited increased detection of weak antibody responses. Antibodies to the E6 oncoproteins of the rare HPV types 52 and 58 were associated with cervical cancer (P < 0.001). CONCLUSION: Multiplex serology enables antibody analyses of large numbers of sera against up to 100 antigens in parallel and has the potential to replace ELISA technology.

Antibodies, Viral↗
PubMed2005

Protein microarrays for antibody profiling: specificity and affinity determination on a chip.

Protein microarray technology facilitates the detection and quantification of hundreds of binding reactions in one reaction from a minute amount of sample. Proof-of-concept studies have shown that the set-up of sensitive assay systems based on protein arrays is possible, however, the lack of specific capture reagents limits their use. Therefore, the generation and characterisation of capture molecules is one of the key topics for the development of protein array based systems. Recombinant antibody technologies, such as HuCAL (human combinatorial antibody library; MorphoSys, Munich, Germany), allow the fast generation of highly specific binders to nearly any given target molecule. Although antibody libraries comprise billions of members, it is not the selection process, but the detailed characterisation of the pre-selected monoclonal antibodies that presents the bottleneck for the production of high numbers of specific binders. In order to obtain detailed information on the properties of such antibodies, a microarray-based method has been developed. We show that it is possible to define the specificity of recombinant Fab fragments by protein and peptide microarrays and that antibodies can be classified by binding patterns. Since the assay uses a miniaturised system for the detection of antibody-antigen interactions, the observed binding occurs under ambient analyte conditions as defined by Ekins (J. Pharm. Biomed. Anal. 1989, 7, 155-168). This allows the determination of a relative affinity value for each binding event, and a ranking according to affinity is possible. The new microarray based approach has an extraordinary potential to speed up the screening process for the generation of recombinant antibodies with pre-defined selection criteria, since it is intrinsically a high-throughput technology.

Antibodies, Monoclonal↗
PubMed2005

Protein microarrays: catching the proteome.

After the completion of the human genome sequencing project, DNA microarrays and sophisticated bioinformatics platforms give scientists a global view of biological systems. In today's proteome era, efforts are undertaken to adapt microarray technology in order to analyse the expression of a large number of proteins simultaneously and screen entire genomes for proteins that interact with particular factors, catalyse particular reactions, act as substrates for protein-modifying enzymes and/or as targets of autoimmune responses. In this review, we will summarise the current stage of protein microarray technology. We will focus on the latest fields of application for the simultaneous determination of a variety of parameters from a minute amount of sample. Future challenges of this cutting-edge technology will be discussed.

Animals↗
PubMed2005

Protein microarrays: applications and future challenges.

Within the last decade protein microarray technology has been successfully applied for the simultaneous identification, quantification and functional analysis of proteins in basic and applied proteome research. These miniaturized and parallelized assay systems have the potential to replace state-of-the-art singleplex analysis systems. However, prior to their general application in robust, reliable, routine and high-throughput applications it is mandatory that they demonstrate robustness, sensitivity, automation and appropriate pricing. In this review, the current state of protein microarray technology will be summarized. Recent applications for the simultaneous determination of a variety of parameters using only minute amounts of sample will be described and future challenges of this cutting-edge technology will be discussed.

Animals↗
PubMed2004

Protein microarrays and multiplexed sandwich immunoassays: what beats the beads?

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently applied for the identification, quantitation and functional analysis of proteins. Protein microarray technology is of major interest for proteomic research in basic and applied biology as well as for diagnostic applications. Miniaturized and parallelized assay systems have reached adequate sensitivity and hence have the potential to replace singleplex analysis systems. However, robustness and automation needs to be demonstrated before this technology will finally prove suitable for high-throughput applications. Miniaturized and parallelized sandwich immunoassays are the most advanced assays formats among the different protein microarray applications. Multiplexed sandwich immunoassays can be used for the identification of biomarkers and the validation of potential target molecules. In this review an overview will be given on the current stage of protein microarray technology with a special focus on miniaturized multiplexed sandwich immunoassays.

Automation↗
PubMed2003

Protein microarrays: promising tools for proteomic research.

Miniaturized and parallelized ligand binding assays are of great interest in postgenomic research because microarray technology allows the simultaneous determination of a large number of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are used for the identification and quantification of proteins as well as for the study of protein interactions. Protein affinity assays have been implemented that allow the analysis of interactions between proteins with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA. Microarray technology is an emerging technology used in global analytical approaches and has a considerable impact on proteomic research.

Animals↗
PubMed2002

Protein microarray technology.

Microarray technology allows the simultaneous analysis of thousands of parameters within a single experiment. Microspots of capture molecules are immobilised in rows and columns onto a solid support and exposed to samples containing the corresponding binding molecules. Readout systems based on fluorescence, chemiluminescence, mass spectrometry, radioactivity or electrochemistry can be used to detect complex formation within each microspot. Such miniaturised and parallelised binding assays can be highly sensitive, and the extraordinary power of the method is exemplified by array-based gene expression analysis. In these systems, arrays containing immobilised DNA probes are exposed to complementary targets and the degree of hybridisation is measured. Recent developments in the field of protein microarrays show applications for enzyme-substrate, DNA-protein and different types of protein-protein interactions. This article discusses theoretical advantages and limitations of any miniaturised capture-molecule-ligand assay system and discusses how the use of protein microarrays will change diagnostic methods and genome and proteome research.

Biotechnology↗
PubMed2002

Protein microarray technology.

This review summarizes the major activities in the field of protein microarray technology. A short summary of the theoretical concepts of miniaturized ligand binding assays explains why such microspot assays represent the most sensitive approaches for capture-target assays. The main focus of this review is centered on the applications using miniaturized and parallelized protein binding assays which rely on the product formation between immobilized capture molecules and their corresponding target molecules which are present in the sample. These types of ligand binding assays are useful tools for protein identification, quantification and protein affinity studies. Protein identification and quantification assays have a great potential in the field of diagnostics and proteomics where many different protein markers which are present in complex samples have to be analyzed in parallel. Protein affinity assays can be used to analyze interactions between proteins such as antibodies, receptors or enzymes with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA. Different applications of protein microarray-based assays and their huge potential for diagnostic and proteomic approaches will be discussed.

Animals↗
PubMed2002

Protein microarray technology.

Microarray technology allows the simultaneous analysis of thousands of parameters within a single experiment. Microspots of capture molecules are immobilized in rows and columns onto a solid support and exposed to samples containing the corresponding binding molecules. Readout systems based on fluorescence, chemiluminescence, mass spectrometry, radioactivity or electrochemistry can be used to detect complex formation within each microspot. Such miniaturized and parallelized binding assays can be highly sensitive, and the extraordinary power of the method is exemplified by array-based gene expression analysis. In these systems, arrays containing immobilized DNA probes are exposed to complementary targets and the degree of hybridization is measured. Recent developments in the field of protein microarrays show applications for enzyme-substrate, DNA-protein and different types of protein-protein interactions. Here, we discuss theoretical advantages and limitations of any miniaturized capture-molecule-ligand assay system and discusses how the use of protein microarrays will change diagnostic methods and genome and proteome research.

Gene Expression↗
PubMed2002

Oocytes and embryos of Xenopus laevis express two different isoforms of germ cell nuclear factor (GCNF, NR6A1).

The germ cell nuclear factor (GCNF) is a nuclear orphan receptor and a putative regulator of the pluripotent state of cells. Although it was first described in mouse germ cells, GCNF is also expressed in mouse and Xenopus embryos. By means of 5'RACE we have identified a novel isoform of Xenopus laevis GCNF that is predominantly expressed in germ cells, whereas both the oocyte and embryonic forms are expressed during Xenopus embryogenesis. EST database search revealed that the homologues of both isoforms are also transcribed in Xenopus tropicalis.

Amino Acid Sequence↗
PubMed2002

Miniaturised multiplexed immunoassays.

Miniaturised immunoassays are of general interest for applications that require the simultaneous determination of different parameters from a minute sample of material. Apart from planar microarray-based systems, bead-based flow cytometric approaches are well suited for the multiplexed detection of target molecules, especially when the number of parameters that have to be determined in parallel is limited.

Animals↗
PubMed2002

Cell microarrays: an emerging technology for the characterization of antibodies.

The possibility to miniaturize and parallelize biological assays has a great impact on the development of biomedical technologies. Here, we describe a simple, miniaturized, and parallelized method employing entire cells from different cell lines displaying a protein of interest on their surface, which were immobilized on a microarray slide. Antibodies were added to these cellular microarrays, and their specific binding to the cell surface proteins was monitored using appropriate fluorescently labeled detection molecules. This new method is applicable for rapidly screening cell surface-specific antibodies with respect to selectivity and cross-reactivity.

Animals↗