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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.

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.

Syndecan-1-targeted therapeutic antibody impairs macropinocytosis and elicits antitumor immunity in pancreatic cancer

Yang, Zecheng; Theardy, Madelaine S.; Chen, Shuaitong; Wei, Yongkun; Takeda, Mitsunobu; Zeng, Yue; Wang, Xiaofei; Yao, Jun; Li, Jennifer; Thirasastr, Prapassorn; Park, Jangho; Zheng, Yangxi; Vien, Long T.; Wani, Khalida M.; Wang, Huamin; Gao, Sisi; Heffernan, Tim; Kwong, Lawrence; Wistuba, Ignacio I.; Bover, Laura; Draetta, Giulio F.; Ying, Haoqiang; Yao, Wantong
Cell Reports Medicine.
Feb 2026
10.1016/j.xcrm.2026.102613
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies, with a 5-year survival rate of just 13%. While the development and early clinical use of small molecules targeting oncogenic KRAS mutations, key drivers of PDAC, have shown promise, resistance to these targeted therapies remains a significant challenge. We recently identified Syndecan-1 (SDC1), a highly expressed heparan sulfate proteoglycan, as a critical KRAS effector protein that promotes nutrient salvage and tumor growth. Here, we report the development of a human-specific monoclonal antibody (anti-SDC1 mAb) that inhibits PDAC cell proliferation in vitro and suppresses PDAC tumor growth in vivo. Mechanistically, the anti-SDC1 mAb blocks macropinocytosis and induces antibody-dependent cellular cytotoxicity (ADCC). In vivo, anti-SDC1 mAb synergizes with standard chemotherapy, KRAS∗ inhibitors, and immunotherapies, resulting in tumor regression and near-complete response. These findings highlight the anti-SDC1 mAb as a promising therapeutic strategy for PDAC and potentially other KRAS∗ and SDC1-driven tumors.

Generation and characterization of monoclonal antibodies that recognize human and murine supervillin protein isoforms

Smith, Tara C.; Saul, Richard G.; Barton, Elisabeth R.; Luna, Elizabeth J.
PLoS ONE.
Oct 2018
10.1371/journal.pone.0205910
Supervillin isoforms have been implicated in cell proliferation, actin filament-based motile processes, vesicle trafficking, and signal transduction. However, an understanding of the roles of these proteins in cancer metastasis and physiological processes has been limited by the difficulty of obtaining specific antibodies against these highly conserved membrane-associated proteins. To facilitate research into the biological functions of supervillin, monoclonal antibodies were generated against the bacterially expressed human supervillin N-terminus. Two chimeric monoclonal antibodies with rabbit Fc domains (clones 1E2/CPTC-SVIL-1; 4A8/CPTC-SVIL-2) and two mouse monoclonal antibodies (clones 5A8/CPTC-SVIL-3; 5G3/CPTC-SVIL-4) were characterized with respect to their binding sites, affinities, and for efficacy in immunoblotting, immunoprecipitation, immunofluorescence microscopy and immunohistochemical staining. Two antibodies (1E2, 5G3) recognize a sequence found only in primate supervillins, whereas the other two antibodies (4A8, 5A8) are specific for a more broadly conserved conformational epitope(s). All antibodies function in immunoblotting, immunoprecipitation and in immunofluorescence microscopy under the fixation conditions identified here. We also show that the 5A8 antibody works on immunohistological sections. These antibodies should provide useful tools for the study of mammalian supervillins.

Gdf-15 as a diagnostic marker to predict the clinical outcome of a treatment with immune checkpoint blockers

WISCHHUSEN, Jörg; Haake, Markus; DUMMER, Reinhard; MEHLING, Matthias
Aug 2018
The present invention relates to methods for predicting the probability of a treatment response of a human cancer patient to an immune checkpoint blocker treatment e.g. with anti PD-1, and to methods for predicting the probability of survival of a human cancer patient following an immune checkpoint blocker treatment, and to apparatuses and kits which can be used in these methods.

Combinatorial Synthesis of Macromolecular Arrays by Microchannel Cantilever Spotting (µCS)

Atwater, Jordyn; Mattes, Daniela S.; Streit, Bettina; von Bojničić-Kninski, Clemens; Loeffler, Felix F.; Breitling, Frank; Fuchs, Harald; Hirtz, Michael
Adv. Mater..
Aug 2018
10.1002/adma.201801632
Surface-bound microarrays of multiple oligo- and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high-throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning-probe-lithography-based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high-density arrays with feature densities of 62 500 features per cm2. To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA- and FLAG-tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost-effective microarrays.

Reductionist Approach in Peptide-Based Nanotechnology

Gazit, Ehud
Annu. Rev. Biochem..
Jun 2018
10.1146/annurev-biochem-062917-012541
The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like β-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

A Trifunctional Linker for Purified 3D Assembled Peptide Structure Arrays

Mattes, Daniela S.; Rentschler, Simone; Foertsch, Tobias C.; Münch, Stephan W.; Loeffler, Felix F.; Nesterov-Mueller, Alexander; Bräse, Stefan; Breitling, Frank
Small Methods.
Feb 2018
10.1002/smtd.201700205
Microarrays are an important tool in modern research that allow the rapid screening of many different interactions simultaneously. Peptide arrays, which bear different peptides arranged in separate spots, permit high-throughput screening to investigate linear and cyclic binding sites. To study conformational or discontinuous binding sites, protein arrays are the major choice. However, the tremendous costs for the generation of high-density protein arrays of high purity restrict progress in protein research. Therefore, peptide-based arrays, which can mimic assembled peptide structures, have an enormous potential. Here, a method is presented to create such structures in the array format as an alternative to protein arrays. A trifunctional linker is developed with an azide, a protected alkyne, and a carboxyl group, which can react with two or three different peptides. Due to the spatial proximity, the peptides interact and can form an assembled peptide structure. As a proof of concept, assembled peptide structures are demonstrated on beads and on a polymer surface and the approach can be validated via matrix-assisted laser desorption/ionization spectrometry. Furthermore, a multistep transfer of peptide arrays is shown, generating purified assembled peptide structure arrays in high density.

Characterization of a sandwich ELISA for the quantification of all human periostin isoforms

Gadermaier, Elisabeth; Tesarz, Manfred; Suciu, Andreea Ana-Maria; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
J Clin Lab Anal.
Feb 2018
10.1002/jcla.22252
Background Periostin (osteoblast-specific factor OSF-2) is a secreted protein occurring in seven known isoforms, and it is involved in a variety of biological processes in osteology, tissue repair, oncology, cardiovascular and respiratory systems or allergic manifestations. To analyze functional aspects of periostin, or the ability of periostin as potential biomarker in physiological and pathological conditions, there is the need for a precise, well-characterized assay that detects periostin in peripheral blood. Methods In this study the development of a sandwich ELISA using monoclonal and affinity-purified polyclonal anti-human periostin antibodies was described. Antibodies were characterized by mapping of linear epitopes with microarray technology, and by analyzing cross-reactive binding to human periostin isoforms with western blot. The assay was validated according to ICH/EMEA guidelines. Results The monoclonal coating antibody binds to a linear epitope conserved between the isoforms. The polyclonal detection antibody recognizes multiple conserved linear epitopes. Therefore, the periostin ELISA detects all known human periostin isoforms. The assay is optimized for human serum and plasma and covers a calibration range between 125 and 4000 pmol/L for isoform 1. Assay characteristics, such as precision (intra-assay: ≤3%, inter-assay: ≤6%), spike-recovery (83%-106%), dilution linearity (95%-126%), as well as sample stability meet the standards of acceptance. Periostin levels of apparently healthy individuals are 864±269 pmol/L (serum) and 817±170 pmol/L (plasma) respectively. Conclusion This ELISA is a reliable and accurate tool for determination of all currently known periostin isoforms in human healthy and diseased samples.

Sensing Immune Responses with Customized Peptide Microarrays

Schirwitz, Christopher; Loeffler, Felix F.; Felgenhauer, Thomas; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Biointerphases.
Aug 2012
10.1007/s13758-012-0047-5
The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding. A new technology to produce peptide microarrays with a laser printer provides access to affordable and highly complex peptide microarrays. Such a peptide microarray can contain up to 775 peptide spots per cm², whereby the position of each peptide spot and, thus, the amino acid sequence of the corresponding peptide, is exactly known. Compared to other techniques, such as the SPOT synthesis, more features per cm² at lower costs can be synthesized which paves the way for laser printed peptide microarrays to take on roles as efficient and affordable biomedical sensors. Here, we describe the laser printer-based synthesis of peptide microarrays and focus on an application involving the blood sera of tetanus immunized individuals, indicating the potential of peptide arrays to sense immune responses.

Physical Characterization of the “Immunosignaturing Effect”

Stafford, Phillip; Halperin, Rebecca; Legutki, Joseph Bart; Magee, Dewey Mitchell; Galgiani, John; Johnston, Stephen Albert
Mol Cell Proteomics.
Apr 2012
10.1074/mcp.M111.011593
Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer’s disease display a characteristic and reproducible immunosignature on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.

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.

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