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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

Detection of specific IgE against linear epitopes from Gal d 1 has additional value in diagnosing hen’s egg allergy in adults

Ehlers, Anna M.; Otten, Henny G.; Wierzba, Eva; Flügge, Ulrike; Le, Thuy-My; Knulst, André C.; Suer, Waltraud
Background Although hen’s egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffers from a persistent or newly-onset hen’s egg allergy, making accurate diagnosis in adults necessary. However, sensitisation to hen’s egg extracts, components and linear epitopes are solely studied in children. Methods Hen’s egg allergic (n=16) and tolerant (n=20) adults were selected by sensitisation towards recombinant components rGal d 1 and/or 3. Sensitisation profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterisation of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3. Results Overall, sIgE titres against hen’s egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitisation to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3. Conclusion and Clinical Relevance Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen’s egg allergic adults with objective symptoms.

Rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays

Heiss, Kirsten; Heidepriem, Jasmin; Fischer, Nico; Weber, Laura K; Dahlke, Christine; Jaenisch, Thomas; Loeffler, Felix F
J. Proteome Res..
Sep 2020
Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-2, responsible for yet more than 500 000 deaths globally. Outbreaks caused by viruses such as SARS-CoV-2, HIV, Ebola, influenza, and Zika have increased over the last decade, underlining the urgent need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides. Using these highly diverse libraries, covering tens of thousands of peptides, allow for the in-depth analysis of antibody signatures in a multiplexed, high-throughput fashion. In this review, we highlight synthesis platforms that facilitate fast and highly flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines, to quickly encounter pandemic threats.

On‐Chip Neo‐Glycopeptide Synthesis for Multivalent Glycan Presentation

Mende, Marco; Tsouka, Alexandra; Heidepriem, Jasmin; Paris, Grigori; Mattes, Daniela S.; Eickelmann, Stephan; Bordoni, Vittorio; Wawrzinek, Robert; Fuchsberger, Felix F.; Seeberger, Peter H.; Rademacher, Christoph; Delbianco, Martina; Mallagaray, Alvaro; Loeffler, Felix F
Chem. Eur. J..
Apr 2020
Single glycan–protein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laser-based array synthesis technology allows for flexible and rapid on-surface synthesis of different peptides. By combining this technique with click chemistry, neo-glycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well-defined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacing-, density-, and ligand-dependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin

Garcia-Bonete, Maria-Jose; Jensen, Maja; Recktenwald, Christian V.; Rocha, Sandra; Stadler, Volker; Bokarewa, Maria; Katona, Gergely
Sci Rep.
Dec 2017
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays

Weber, Laura K.; Isse, Awale; Rentschler, Simone; Kneusel, Richard E.; Palermo, Andrea; Hubbuch, Jürgen; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F.
Eng. Life Sci..
Oct 2017
Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

Peptide array functionalization via the Ugi four-component reaction

Ridder, B.; Mattes, D. S.; Nesterov-Mueller, A.; Breitling, F.; Meier, M. A. R.
Chem. Commun..
May 2017
The Ugi four-component reaction was investigated as a tool for the functionalization of peptide arrays via post-synthetic side-chain modification, mimicking post-translational processes. Additionally, as a proof of concept for the synthesis of peptidomimetics on arrays, the integration of an Ugi unit into a growing peptide chain was demonstrated.

Autoantikehad täppismeditsiinis

Jaks, Viljar; Uibo, Raivo
Immuuntolerantsi häirumine, mille üheks väljundiks on antikehade teke organismile omaste biomolekulide vastu, on oluline patogeneetiline mehhanism mitmete laialdaselt levinud haiguste puhul ja seetõttu on autoantikehade määramine kujunenud oluliseks diagnostiliseks vahendiks. Artiklis on käsitletud autoantikehade esinemise olulisust haiguste tekke ja kulu prognoosimisel. Kuigi sellekohane info on veel üsna napp, on selge, et organismi immuunstaatuse muutus eelneb aastaid haiguse ilmnemisele ning autoimmuunset komponenti sisaldava haiguse kulg ja prognoos on seotud patsiendil esinevate kindlate autoantikehadega. Sellest tulenevalt võime loota, et organismi immuunstaatuse uurimine, eriti aga autoantikehade spektri iseloomustamine, on tulevikus geneetilise info analüüsimise kõrval üks täppismeditsiini olulisemaid tööriistu.

Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants

Havenith, Heide; Kern, Karolin; Rautenberger, Paul; Spiegel, Holger; Szardenings, Michael; Ueberham, Elke; Lehmann, Jörg; Buntru, Matthias; Vogel, Simon; Treudler, Regina; Fischer, Rainer; Schillberg, Stefan
Biotechnol. J..
Feb 2017
Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agents.

Antibody repertoire profiling with mimotope arrays

Pashova, Shina; Schneider, Christoph; von Gunten, Stephan; Pashov, Anastas
Human Vaccines & Immunotherapeutics.
Jan 2017
Large-scale profiling and monitoring of antibody repertoires is possible through next generation sequencing (NGS), phage display libraries and microarrays. These methods can be combined in a pipeline, which ultimately maps the antibody reactivities onto defined arrays of structures – peptides or carbohydrates. The arrays can help analyze the individual specificities or can be used as complex patterns. In any case, the targets recognized should formally be considered mimotopes unless they are proven to be epitopes driving the antibody synthesis. Here, the advantages and disadvantages of the major profiling techniques as well as their current and future application in disease prediction and vaccination are discussed.

Purification and Characterization of Anacardium occidentale (Cashew) Allergens Ana o 1, Ana o 2, and Ana o 3

Reitsma, Marit; Bastiaan-Net, Shanna; Sforza, Stefano; van der Valk, Johanna P. M.; van Gerth van Wijk, Roy; Savelkoul, Huub F. J.; de Jong, Nicolette W.; Wichers, Harry J.
J. Agric. Food Chem..
Feb 2016
In this study a fast and simple purification procedure for the three known allergens from cashew (7S globulin Ana o 1, 11S globulin Ana o 2, and 2S albumin Ana o 3) is described. The purified allergens are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, glycoprotein stain, and protein identification. The purified proteins still bind IgE, and this IgE binding varied between different pools of patient serum. Ana o 1 was found to be a glycoprotein. Ana o 3 has been studied more in detail to identify both the small and large subunits, both displaying microheterogeneity, and epitope mapping of Ana o 3 has been performed.

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies: Peptide Arrays for Antibody Selectivity Studies

Vernet, Thierry; Choulier, Laurence; Nominé, Yves; Bellard, Laure; Baltzinger, Mireille; Travé, Gilles; Altschuh, Danièle
J. Mol. Recognit..
Oct 2015
Antibody selectivity represents a major issue in the development of efficient immuno-therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high-throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross-reactive capacity of an antibody fragment using the PEPperCHIP® technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore® technology; GE-Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N-terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence 6TAMFQDPQER15) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross-reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity.

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