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

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Characterization of antibodies against the replication protein (Rep) encoded by bovine meat and milk factors (BMMFs)

Frehtman, Veronika; Shukla, Gunjan; Gentz, Michael; Müller, Marcus; Duduyemi, Oladimeji Paul; Grewe, Imke; Ernst, Claudia; Tessmer, Claudia; Didier, Andrea; Hofmann, Ilse; Bund, Timo; Leuchs, Barbara
Appl Microbiol Biotechnol.
Apr 2026
10.1007/s00253-026-13809-x
Abstract Bovine Meat and Milk Factors (BMMFs) are DNA elements with similarity to bacterial plasmids, are frequently identified in bovine meat and milk and were proposed to contribute to cancer development. All known BMMFs encode a conserved replication protein (Rep), allowing for histologic BMMF detection in clinical specimens based on Rep-directed mouse monoclonal antibodies (mAbs), which, however, have only been partially characterized so far. Here, 20 anti-BMMF Rep antibodies were assessed for biophysical properties, reactivity, specificity and binding sensitivity to five distinct BMMF Reps and other prokaryotic/eukaryotic target antigens using an enzyme-linked immunosorbent assay (ELISA)-based anti-BMMF Rep antibody binding assay. We demonstrated sensitive and specific antibody reaction with their respective Rep targets, according to the antibody immunization. Consensus antibodies raised against defined peptides of conserved Rep amino acid stretches interacted with most of the Rep antigens. Antibodies produced based on immunization with the Rep encoded on the BMMF isolate H1MSB.1, including rabbit and human chimeric variants, reacted only with the cognate H1MSB.1 Rep, with only two outliers targeting additional Reps. Completely new antibodies raised against the Rep of another isolate (C1HB.4) specifically detected the cognate C1HB.4 Rep antigen – not interacting with other Reps. New antibodies generated by triple Rep immunization (H1MSB.2/C1MI.3M.1/C1MI.9M.1 Rep) reacted to either all three or two immunization antigens without interacting with any other Reps. None of the antibodies cross-reacted against Reps of bacteria occurring during milk production or lysates of mammalian hosts. Competitive inhibition confirmed antigen-specificity across the antibody panel, which additionally did not show aberrancies concerning purity or antibody size for the majority of the tested Abs. These findings authenticate a highly specific panel of anti-BMMF Rep antibodies, which can serve as tools for BMMF detection in cancer and chronic diseases.**Key Points** • Anti-BMMF Rep antibodies are important to judge BMMFs’ role as cancer risk factors. • Selective binding of anti-BMMF Rep antibodies to BMMF Rep antigens. • No cross-reactivity of anti-BMMF Rep antibodies with bacterial and mammalian outgroup specimens.

Identification of a conformational epitope on the E antigen implicated in anti-E alloimmunization

Matsuura, Hideaki; Yamada, Ayuna; Doi, Hiroki; Fujii, Sumie; Miura, Yasuo
Blood Adv.
Mar 2026
10.1182/bloodadvances.2025018046

Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

Kim, Byoungmo; Lee, Ha Kyeong; Azam, Zulfikar; Choi, Jeong Uk; Wahab, Riajul; Lee, Na Kyeong; Ko, Yoon Gun; Choi, So‐Young; Lee, Se‐Ra; Shim, Wan Seob; Kim, Taeeung; Kim, In‐San; Alam, Farzana; Kim, Sang Yoon; Kim, Seong Who; Byun, Youngro; Al‐Hilal, Taslim A
Advanced Science.
Mar 2026
10.1002/advs.202512975
ABSTRACT Tip endothelial cells (TipEC), the leading edge of angiogenic sprouts, are essential for pathological neo‐vascularization but remain difficult to target due to the lack of specific druggable markers. Here, we identify Doppel as a selective and druggable regulator of endothelial tip cell function. Doppel expression enhances TipEC selection, directional migration, and regulates tip‐stalk cell dynamics by spatially controlling VEGFR2/Dll4/Src pathway. Genetic ablation of PRND (Doppel) reduces tip cell formation without affecting the stalk cells (StalkECs) number in tumors, indicating its selective role in TipECs. Importantly, depletion of TipECs using the first‐in‐class monoclonal antibodies against a highly conserved WQF‐motif of Doppel robustly decreased the growth of tumors by selectively downregulating VEGFR2+ TipECs but not StalkECs. These findings position Doppel as a tumor TipEC‐specific, druggable target that may offer a new avenue to enhance and refine anti‐angiogenic therapies in cancer treatment.

Systematic analysis of the RGS2 degron reveals characteristics of substrate recognition by the F-box protein FBXO44

McNabb, Harrison J.; Cho, Eugene; Pitman, Mary; Rushton, Phillip S.; Mobley, David; Sjögren, Benita
Journal of Biological Chemistry.
Nov 2025
10.1016/j.jbc.2025.110757
Regulator of G protein signaling 2 (RGS2) negatively modulates signaling downstream of G protein–coupled receptors by accelerating GTP hydrolysis at Gα subunits of heterotrimeric G proteins. Decreased RGS2 levels are implicated in numerous diseases, including cardiovascular disease and asthma. Thus, identifying selective means of enhancing RGS2 protein levels would be a viable therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin–proteasomal pathway, and we previously identified F-box only protein 44 (FBXO44) as the substrate recognition component of the E3 ligase responsible for facilitating RGS2 degradation. As such, the RGS2–FBXO44 interaction is a potential target for pharmacological intervention. Detailed information on the FBXO44 recognition site (degron) in RGS2 will aid in structure-based small-molecule inhibitor design, as well as in identifying additional FBXO44 targets, which would help predict possible side effects of targeting this interaction. Thus, the goal of this study was to dissect the molecular properties for FBXO44 binding of the RGS2 degron. We used a peptide array utilizing systematic residue substitution, combined with AlphaFold modeling and molecular dynamics simulations, to identify several amino acid changes that altered binding both positively and negatively. Finally, we experimentally confirmed our results in cells through coimmunoprecipitation and proteasomal inhibition, using full-length RGS2. Altogether, these results provide structural insights into RGS2–FBXO44 binding, which will aid in structure-guided drug discovery efforts. It also provides a framework for building a consensus recognition motif for FBXO44, which could aid in identifying more substrates for this understudied F-box protein.

Preclinical characterization of an active immunotherapy targeting calcitonin gene-related peptide

Boyd, Justin D.; Wang, Shixia; Lin, Hsiao-Wen; Hsieh, Yueh-Ting; Sun, Yu Shuang; Thibodeaux, Brett A.; Lu, Hanxin; Sahni, Jaya; Wiggins, Jonathan; Longo, Matthew S.; Brooks, Jeanne K.; Vroom, Madeline M.; Chang, Yi-Pin; Liu, Zhi; Ding, Shuang; Dodart, Jean-Cosme
Commun Med.
Apr 2025
10.1038/s43856-025-00870-2
Abstract **Background** The success of passive immunotherapies targeting Calcitonin gene-related peptide (CGRP) for managing migraine has prompted our efforts towards developing an active immunotherapy that induces the production of endogenous antibodies against CGRP. Achieving efficacious antibody titers via immunization could provide a more convenient and cost-effective treatment alternative to anti-CGRP monoclonal antibody (mAb) therapies. However, immunization against endogenous CGRP faces multiple challenges such as breaking immune tolerance, inducing sufficient antibody titers, and avoiding immune response-associated toxicity. **Methods** Synthetic peptide immunogens formulated in adjuvants were delivered intramuscularly. Serum samples were collected post immunization and used to measure antibody titers as well as for the isolation of antibodies specific to CGRP. Antibodies were characterized for their binding affinities and specificities. The capsaicin-induced increase in dermal blood flow model was used in rats for the assessment of the pharmacodynamic effect of immunization. **Results** Here we demonstrate that a peptide-based active immunotherapy designed to induce antibodies against CGRP promotes robust antibody titers across preclinical species. Characterization of the immune response strongly suggests that this peptide immunogen primarily stimulates a humoral response and only induced CGRP-specific antibodies. Antibodies produced by immunization are primarily IgG1 and demonstrate binding and activity potencies similar to marketed monoclonal antibodies against CGRP. Finally, immunization demonstrates in vivo efficacy in a rat pharmacodynamic model. **Conclusion** Our results strongly suggest that a peptide-based active immunotherapy against CGRP could provide an affordable and convenient therapeutic for the prevention of migraine.

A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples

He, Xiaohua; Patfield, Stephanie; Hnasko, Robert; Rasooly, Reuven; Mandrell, Robert E.
PLoS ONE.
Oct 2013
10.1371/journal.pone.0076368
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

Optimised ‘on demand’ protein arraying from DNA by cell free expression with the ‘DNA to Protein Array’ (DAPA) technology

Schmidt, Ronny; Cook, Elizabeth A.; Kastelic, Damjana; Taussig, Michael J.; Stoevesandt, Oda
Journal of Proteomics.
Aug 2013
10.1016/j.jprot.2013.02.002
We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. Biological significance: DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes.

Provocation of an Autoimmune Response to Cardiac Voltage-Gated Sodium Channel NaV1.5 Induces Cardiac Conduction Defects in Rats

Korkmaz, Sevil; Zitron, Edgar; Bangert, Anna; Seyler, Claudia; Li, Shiliang; Hegedüs, Peter; Scherer, Daniel; Li, Jin; Fink, Thomas; Schweizer, Patrick A.; Giannitsis, Evangelos; Karck, Matthias; Szabó, Gábor; Katus, Hugo A.; Kaya, Ziya
Journal of the American College of Cardiology.
Jul 2013
10.1016/j.jacc.2013.04.041
Objectives: This study sought to test the hypothesis that inducing an autoimmune response against the cardiac sodium channel (NaV1.5) induces arrhythmias. Background: Sporadic evidence supports the concept that autoantibodies may cause cardiac arrhythmias but substantial experimental investigations using in vivo models have been lacking to date. The NaV1.5 is essential for cardiac impulse propagation and its dysfunction has been linked to conduction disease. Methods: Rats were immunized with a peptide sequence derived from the third extracellular loop of the first domain of NaV1.5. After 28 days, we evaluated in vivo both the electrical and mechanical parameters of cardiac function. Histopathology, myocardial gene and protein expression were assessed. Whole-cell patch-clamp was used to measure sodium current (INa) density in isolated cardiomyocytes. Results: NaV1.5-immunized rats had high titers of autoantibodies against NaV1.5. On ECG recording, NaV1.5-immunized animals showed significantly prolonged PR-intervals. During Holter ECG-monitoring we observed repeated prolonged episodes of third-degree atrioventricular and sinoatrial block in every NaV1.5-immunized animal, but not in controls. Immunization had no effect on cardiac function. In comparison to controls, myocardial NaV1.5 mRNA and protein levels were decreased in immunized rats. INa density was reduced in cardiomyocytes incubated with sera from NaV1.5-immunized rats and from patients with idiopathic atrioventricular block (AVB) in comparison to sera from respective controls. In patients with idiopathic AVB, we observed autoantibodies against NaV1.5 that were absent in sera from healthy controls. Conclusions: Provocation of an autoimmune response against NaV1.5 induces conductance defects probably caused by a reduced expression level and an inhibition of NaV1.5 by autoantibodies, resulting in decreased INa.

Complex Formation between NheB and NheC Is Necessary to Induce Cytotoxic Activity by the Three-Component Bacillus cereus Nhe Enterotoxin

Heilkenbrinker, Uta; Dietrich, Richard; Didier, Andrea; Zhu, Kui; Lindbäck, Toril; Granum, Per Einar; Märtlbauer, Erwin
PLoS ONE.
Apr 2013
10.1371/journal.pone.0063104
The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml−1, but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.

Purification of High-Complexity Peptide Microarrays by Spatially Resolved Array Transfer to Gold-Coated Membranes

Schirwitz, Christopher; Loeffler, Felix F.; Felgenhauer, Thomas; Stadler, Volker; Nesterov-Mueller, Alexander; Dahint, Reiner; Breitling, Frank; Bischoff, F. Ralf
Adv. Mater..
Mar 2013
10.1002/adma.201203853
A method for the one-step purification of high-complexity peptide microarrays is presented. The entire peptide library is transferred from the synthesis support to a gold coated polyvinylidenfluoride (PVDF) membrane, whereby only full-length peptides covalently couple to the receptor membrane via an N-terminally added cysteine. Highly resolved peptide transfer and purification of up to 10 000 features per cm2 is demonstrated.

Combinatorial Peptide Synthesis on a Microchip

Schirwitz, Christopher; Block, Ines; König, Kai; Nesterov, Alexander; Fernandez, Simon; Felgenhauer, Thomas; Leibe, Klaus; Torralba, Gloria; Hausmann, Michael; Lindenstruth, Volker; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Current Protocols in Protein Science.
Aug 2009
10.1002/0471140864.ps1802s57
Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom-built microchips can be equipped with an amino-modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer-coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high-complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm2 can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background-free immunostaining on the chip-based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high-density peptide arrays.

Particle-Based Synthesis of Peptide Arrays

Breitling, Frank; Felgenhauer, Thomas; Nesterov, Alexander; Lindenstruth, Volker; Stadler, Volker; Bischoff, F. Ralf
ChemBioChem.
Mar 2009
10.1002/cbic.200800735
Lithographic methods allow for the combinatorial synthesis of >50,000 oligonucleotides per cm(2), and this has revolutionized the field of genomics. High-density peptide arrays promise to advance the field of proteomics in a similar way, but currently lag behind. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. Combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A color laser printer or a chip addresses the different charged particles consecutively to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids to the support in one single coupling reaction. The method should allow for the translation of entire genomes into sets of overlapping peptides to be used in proteome research.

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