Zoltán Konthur

Bundesanstalt für Materialforschung und -prüfung | BAM · Department of Analytical Chemistry (Dpt.1)

Objectives Porphyromonas gingivalis ( P.g .) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g . CH2007 (RA CH2007 -PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (C...

In this review, we summarize the current knowledge concerning the eukaryotic protozoan parasite Leishmania tarentolae, with a main focus on its potential for biotechnological applications. We will also discuss the genus, subgenus, and species-level classification of this parasite, its life cycle and geographical distribution, and similarities and d...

Background Porphyromonas gingivalis (P g.) is involved in triggering self-reactive immune responses when cirtullinating bacterial or human proteins. However, first evidence to link anti-ribosomal T and B cells responses to rheumatoid arthritis (RA) has been published but the mechanism and its influence on therapy is not clear (1). Infection based a...

Background: The continuing increase of anti-citrullinated protein autoanti-bodies (ACPA) titers together with epitope spreading close to onset of disease, suggests that antibody responses to different citrullinated anti-gens may be critical in rheumatoid arthritis (RA) pathogenesis. Interestingly , monoclonal antibodies demonstrate reactivity to mu...

Career situation of first and presenting author Young investigator. Introduction Porphyromonas gingivalis (P g.) is involved in triggering self-reactive immune responses when citrullinating bacterial or human proteins. However, first evidence to link anti-ribosomal T and B cells responses to rheumatoid arthritis (RA) has been published but the mec...

Panning is a common process used for antibody selection from phage antibody libraries. There are several methods developed for a similar purpose, namely streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips, magnetic beads, polystyrene immunotubes, and microtiter plate. The advantage of using a magnetic particle pro...

Directed evolution is a proven approach to fine tune or modify biomolecules for various applications ranging from research to industry. The process of evolution requires methods that are capable of not only generating genetic diversity but also to distinguish the variants of desired characteristics. One method that is synonymous with directed evolu...

Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We pe...

The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning e...

Objectives Rituximab (RTX) has shown clinical efficacy but up to 40% of RTX treated rheumatoid arthritis (RA) patients are poor responders (Ann-Rheum-Dis. 2005 Feb;64(2):246–52) and the commonly used RA biomarkers (RF/ACPA) are poor predictors for therapy response. In this study the autoantibody repertoire analysed on protein macorarrays from RA pa...

Background Rituximab (RTX) has shown clinical efficacy but up to 40% of RTX treated rheumatoid arthritis (RA) patients are poor responders (Ann-Rheum-Dis. 2005 Feb;64 (2):246–52) and the commonly used RA biomarkers (RF/ACPA) are poor predictors for therapy response. In this study the autoantibody repertoire analysed on protein macorarrays from RA p...

Background Rheumatoid arthritis triggers the formation of prion-like stress granules. To investigate which members of the heterogeneous nuclear ribonucleoprotein (hnRNP)-family, components of functionally important subcellular particles are targeted by autoantibodies from RA and other systemic rheumatic diseases. Methods Using a protein macroarray...

Background Rituximab (RTX) has shown clinical efficacy but up to 40% of RTX treated rheumatoid arthritis (RA) patients are poor responders (ref:1) and the commonly used RA biomarkers (RF/ACPA) are poor predictors for therapy response. Others reported associations between BAFF/BLyS levels, Fcg RIII and IL-6 genotype, interferon type I signatures and...

Background Rheumatoid arthritis is believed to be induced by environmental, genetic and immunological factors. Monoclonal Anti-citrullinated protein autoantibodies ACPA target different proteins and their role in RA is still largely unknown Objectives As a prerequisite to identify genetic factors involved in rheumatoid arthritis, we analyzed the s...

Background So far no mechanism for non response to biologicals targeting TNFα has been described despite one third of rheumatoid arthritis patients treated are non-responders. Objectives We investigated the differences in seroreactivity of patients responding and not responding to TNF therapies prior and after therapy and identified diagnostically...

Objective Monoclonal Anti-citrullinated protein autoantibodies ACPA target different proteins and their role in RA is still largely unknown. We analysed the susceptibility of PWD Musmusculusmusculus subspecies, separated from Musmusculusdomesticus about one million years ago to collagen antibody-induced arthritis (CAIA) and the role of ACPA antibod...

Background So far no mechanism for non response to biologicals targeting TNFα has been described, despite one third of rheumatoid arthritis patients treated are Non-Responder. A link to the gut microbiota and its ability to drive an autoimmune disease (Immunity. 2010, 32(6):815–27) can be made via segmented filamentous bacteria (SFB) that cooperate...

Prader-Willi Syndrome (PWS) is a neurogenetic disorder caused by the deletion of imprinted genes on the paternally inherited human chromosome 15q11-q13. This locus harbours a long non-protein-coding RNA (U-UBE3A-ATS) that contains six intron-encoded snoRNAs, including the SNORD116 and SNORD115 repetitive clusters. The 3'-region of U-UBE3A-ATS is tr...

Guanine quadruplex (G-quadruplex) motifs in the 5' untranslated region (5'-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophy...

A variety of animal models suggest that TLR signalling is important in the pathogenesis of RA and the generation of specific autoantibodies. This study was conducted with sera from patients with rheumatoid arthritis, as well as with arthritis animal models to identify identical autoantigens dependent on TLR 7 and 9 in human and animal models for di...

Leishmania tarentolae is a non-human-pathogenic Leishmania species of growing interest in biotechnology, as it is well-suited for the expression of human recombinant proteins. For many applications it is desirable to express recombinant proteins with a tag allowing easy purification and detection. Hence, we adopted a scheme expressing recombinant p...

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we...

Proteins identified as significantly altered in expression after 1 h or 48 h incubation with sAPPalpha. Proteins altered in both treatment conditions (1 h and 48 h) are highlighted in grey. (PDF)

Human serum has the potential for mesenchymal progenitor cell recruitment in repair of articular cartilage lesions. It is unclear which factor(s) in serum mediate this migratory effect. Our goal was to identify cell recruiting factors in human serum fractions obtained by ion exchange chromatography. The recruiting activity of serum fractions on hum...

Background and Objectives A variety of animal models suggest that TLR signalling is important in the pathogenesis of RA and the generation of specific autoantibodies. This study was conducted with sera from patients with rheumatoid arthritis, as well as with arthritis animal models to identify identical autoantigens dependent on TLR7 and 9 in human...

Soluble amyloid precursor protein alpha (sAPPalpha) is a cleavage product of the amyloid precursor protein (APP), the etiologic agent in Alzheimer disease (AD). Reduced expression of sAPPalpha was previously found in the brains of Alzheimer disease patients and it was suggested, that sAPPalpha might counteract neurotoxic effects of Abeta, another A...

Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-st...

The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombin...

Clustering of donors according to coincident appearance of most frequent VDJ rearrangements in IgM and IgG subisotypes. (PDF)

Supporting experimental data. (PDF)

VDJ rearrangements 100-fold over the median frequency of all VDJ recombination patterns within the cohort. (PDF)

Variability as a function of unique VDJ recombination patterns in each isotype in proportion to all isotypes within donors. (PDF)

Statistical analysis of relative amount of obtained sequences per isotype over the total number of sequences from the elderly group. (PDF)

VDJ recombination pattern distributions of 14 donors incorporating heavy chain isotype information. (PDF)

Clustering of donors according to coincident appearance of most frequent VDJ rearrangements in IgM with all CSR-dependent isotypes. (PDF)

Clustering of donors according to coincident appearance of most frequent VDJ rearrangements in IgG with subisotypes of IgG and with IgM. (PDF)

Regular expression filter used to integrate IMGT/High V-Quest alleles into genes. (PDF)

Clustering of donors according to coincident appearance of most frequent VDJ rearrangements in IgM with IgA1 and IgA2. (PDF)

Statistical analysis of relative amount of obtained sequences per isotype over the total number of sequences from all 14 donors. (PDF)

Statistical analysis of relative amount of obtained sequences per isotype over the total number of sequences from the young adult group. (PDF)

Unique VDJ recombination per isotype in proportion to all isotypes in all donors. (PDF)

Unique VDJ recombination per isotype in proportion to all isotypes in elderly donors. (PDF)

Analysis of changes in the VDJ rearrangement pattern distribution by entropy in the elderly. (PDF)

Unique VDJ recombination per isotype in proportion to all isotypes in young donors. (PDF)

Analysis of changes in the VDJ rearrangement pattern distribution by entropy over all donors. (PDF)

Analysis of changes in the VDJ rearrangement pattern distribution by entropy in the young adults. (PDF)

Secretory signal peptides (SPs) are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1...

SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large...

list of clones, sequenced by Sanger method. (DOC)

Top100 of Illumina-sequenced library pool (Bank40). (XLS)

Efficiency of Illumina sequencing and barcoding. Insert refers to reads of expected length of approximately 40 bases. (DOC)

PCR yields from selection-round amplification. (DOC)

list of oligonucleotides used in this study. (DOC)

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA...

Conventional analysis of molecular interactions by surface plasmon resonance is achieved by the observation of optical density changes due to analyte binding to the ligand on the surface. Low molecular weight interaction partners are normally not detected. However, if a macromolecule such as DNA can extend beyond the evanescent field and analyte in...

Many experiments require a fast and cost-effective method to monitor nucleic acid sequence diversity. Here we describe a method called diversity visualization by endonuclease (DiVE) that allows rapid visualization of sequence diversity of polymerase chain reaction (PCR) products based on DNA hybridization kinetics coupled with the activity of a sin...

Compartmentalization of polymerase chain reaction (PCR) reduces artifacts, especially when complex libraries are amplified. It allows clonal amplification of templates from complex mixtures in a bias-free manner. Here we describe a rapid, straightforward, and easy protocol for PCR in a water-in-oil emulsion (ePCR) including sample recovery by DNA p...

Background One third of rheumatoid arthritis (RA) patients treated with biologicals targeting tumour necrosis factor α (TNFα) are therapy non-responders. The authors investigated the differences in seroreactivity of patients responding and not responding to TNF therapies prior and after therapy to deduce diagnostically applicable autoantigenicity p...

Objective This study was conducted with sera from patients with rheumatoid arthritis, systemic lupus erythematosus as well as with arthritis and lupus-like disease animal models to identify identical autoantigens in human and animal models for disease modifying use. Methods Using protein filter technology (28000 human protein filter) the autoantige...

Gene transcription is controlled by transcriptional regulators acting with specific co-regulators to allow gene activation and repression. Here, we report the identification of the KRAB-containing zinc-finger transcriptional regulator, ZBRK1, as an interaction partner of the SCA2 gene product ataxin-2. Furthermore, we discovered that an elevated ZB...

We wish to alert your readers to MIAPAR, the minimum information about a protein affinity reagent. This is a proposal developed within the community as an important first step in formalizing standards in reporting the production and properties of protein binding reagents, such as antibodies, developed and sold for the identification and detection o...

For studying human antibody variable (V)-gene usage in any group of individuals or for the generation of recombinant human antibody libraries for phage display, quality and yield of the amplified V-gene repertoire is of utmost importance. Key parameters affecting the amplification of full antibody repertoires are V-gene specific primer design, comp...

Automation in combination with high throughput screening methods has revolutionised molecular biology in the last two decades. Today, many combinatorial libraries as well as several systems for automation are available. Depending on scope, budget and time, a different combination of library and experimental handling might be most effective. In this...

Cervical cancer is the second most common cancer among women worldwide.Every year approximately 500.000 new cases are diagnosed and 270.000 women die from this malignancy. The tumor is caused by a persistent infection with high-risk human papillomaviruses (HPV). The recently developed vaccination prevents infections with high-risk HPV 16 and HPV 18...

In recent years the improvements in high-throughput gene expression analysis have led to the discovery of numerous non-protein-coding RNA (npcRNA) molecules. They form an abundant class of untranslated RNAs that have shown to play a crucial role in different biochemical pathways in the cell. Reverse transcription quantitative real-time polymerase c...

Objective This study was conducted with sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and arthritis and lupus-like disease animal models to identify innate immune system-dependent and -independent autoantigens.

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cov...

Phage display of combinatorial antibody libraries is a very efficient method for selecting recombinant antibodies against a wide range of molecules. It has been applied very successfully for the generation of therapeutic antibodies for more than a decade. To increase robustness and reproducibility of the selection procedure, we developed a semi-aut...

The generation of recombinant antibodies by phage display in high-throughput demands fast downstream technologies and streamlined processes for the identification and initial characterisation of individual binders. Next to standard immunological methods such as enzyme-linked immunosorbent assays (ELISA) and Western-blot, protein microarrays offer a...

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cov...

Targeted RNases (TRs) are immunoenzymes with ribonucleases as cytotoxic effector domains, which are less immunogenic as plant or bacterial toxin components of classical immunotoxins. In this study, we show the generation and production of the first entirely human TR (huTR) directed against CD30+ lymphomas. The scFv-Fc-RNase construct was produced i...

IntroductionGeneral Considerations for the Automation of Antibody GenerationDevelopment of an Antibody Generation PipelineConclusion

Die Entwicklung der DNA-Sequenzierungstechnologien wurde durch die Aufgabe, das ge- samte menschliche Genom zu entschlüsseln, vorangetrieben. Fünf Jahre nach Ende des Humangenomprojektes ist das Verständnis der Funktion der durch die rund 23.000 Gene kodierten Proteine des menschlichen Genoms jedoch immer noch rudimentär. Einer der limitierenden Fa...

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-Eu...

Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still req...