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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.

Immunity to Influenza is dependent on MHC II polymorphism: study with 2 HLA transgenic strains

Luckey, David; Weaver, Eric A.; Osborne, Douglas G.; Billadeau, Daniel D.; Taneja, Veena
Sci Rep.
Dec 2019
10.1038/s41598-019-55503-1
Major histocompatibility complex II (MHC II) molecules are involved in antigen presentation and the development of a functional adaptive immune response. Evolutionary selection for MHC molecules that effectively clear infectious agents provides an advantage to humans. However, certain class II molecules are associated with autoimmune diseases. In this study we infected autoimmune-susceptible DRB1*0401.AEo and non-susceptible *0402.AEo mice with H1N1 influenza and determined clearance and protective immunity to H3N2 virus. *0401 mice generated a robust TLR-triggered immune response and cleared H1N1 influenza virus infection. After vaccination and challenge with H1N1, *0401 mice, when challenged with H3N2, generated cross-protective immunity to heterosubtypic H3N2 influenza strain whereas *0402 mice cleared the H1N1 infection but did not generate cross-protective immunity against the H3N2 influenza strain. The intracellular trafficking route of MHCII revealed that *0401 molecules traffic through the late endosome/lysosomes while *0402 molecules traffic into early endosomes. This suggested that trafficking of MHCII could affect the functional output of the innate immune response and clearance of viral infections. Also, DRB1*0401 mice live longer than HLA-DRB1*0402 mice. The study provides a potential hypothesis for evolutionary selection of *0401 molecule, even though it is associated with autoreactivity, which may be dependent on the availability of peptide repertoire of self-antigens.

Analysis of humoral immune responses in chikungunya virus (CHIKV) infected patients and individuals vaccinated with a candidate CHIKV vaccine

Henss, Lisa; Yue, Constanze; von Rhein, Christine; Tschismarov, Roland; Lewis-Ximenez, Lia Laura; Dölle, Albert; Baylis, Sally A; Schnierle, Barbara S
Dec 2019
10.1093/infdis/jiz658
Abstract Background Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu like symptoms. The acute symptoms disappear after one week, but chronic arthralgia can persist for years. Here, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Methods Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors and antibody epitope mapping was performed with a peptide array. Results Greatest CHIKV neutralization activity was observed 60-92 days after onset of symptoms. The amount of CHIKV-specific antibodies, their binding avidity and cross-reactivity with other alphaviruses increased over time. CHIKV and o’nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2–6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV and the E2 ASR1 was the major antibody target. Conclusion These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy.

Peptides of neuron specific enolase as potential ASD biomarkers: From discovery to epitope mapping

Ramirez-Celis, Alexandra; Edmiston, Elizabeth; Schauer, Joseph; Vu, Tam; Van de Water, Judy
Brain, Behavior, and Immunity.
Dec 2019
10.1016/j.bbi.2019.12.002
Autism spectrum disorder (ASD) is an important health issue and affects 1 in 59 children in the US. Prior studies determined that maternal autoantibody related (MAR) autism is thought to be associated with ~23% of ASD cases. We previously identified seven MAR-specific autoantigens including CRMP1, CRMP2, GDA, LDHA, LDHB, STIP1, and YBX1. We subsequently described the epitope peptide sequences recognized by maternal autoantibodies for each of the seven ASD-specific autoantigens. The aim of the current study was to expand upon our previous work and identify additional antigens recognized by the ASD-specific maternal autoantibodies, as well as to map the unique ASD-specific epitopes using microarray technology. Fetal Rhesus macaque brain tissues were separated by molecular weight and a fraction containing bands between 37 and 45 kDa was analyzed using 2-D gel electrophoresis, followed by peptide mass mapping using MALDI-TOF MS and TOF/TOF tandem MS/MS. Using this methodology, Neuron specific enolase (NSE) was identified as a target autoantigen and selected for epitope mapping. The full NSE sequence was translated into 15-mer peptides with an overlap of 14 amino acids onto microarray slides and probed with maternal plasma from mothers with an ASD child and from mothers with a Typically Developing child (TD) (ASD = 27 and TD = 21). The resulting data were analyzed by T-test. We found 16 ASD-specific NSE-peptide sequences for which four sequences were statistically significant (p < 0.05) using both the t-test and SAM t-test: DVAASEFYRDGKYDL (p = 0.047; SAM score 1.49), IEDPFDQDDWAAWSK (p = 0.049; SAM score 1.49), ERLAKYNQLMRIEEE (p = 0.045; SAM score 1.57), and RLAKYNQLMRIEEEL (p = 0.017; SAM score 1.82). We further identified 5 sequences that were recognized by both ASD and TD antibodies suggesting a large immunodominant epitope (DYPVVSIEDPFDQDDWAAW). While maternal autoantibodies against the NSE protein are present both in mothers with ASD and mothers of TD children, there are several ASD-specific epitopes that can potentially be used as MAR ASD biomarkers. Further, studies including analysis of NSE as a target protein in combination with the previously identified MAR ASD autoantigens are currently underway.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries

Pashov, Anastas; Shivarov, Velizar; Hadzhieva, Maya; Kostov, Victor; Ferdinandov, Dilyan; Heintz, Karen-Marie; Pashova, Shina; Todorova, Milena; Vassilev, Tchavdar; Kieber-Emmons, Thomas; Meza-Zepeda, Leonardo A.; Hovig, Eivind
Front. Immunol..
Dec 2019
10.3389/fimmu.2019.02796
Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients’ sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

A Low‐Cost Laser‐Based Nano‐3D Polymer Printer for Rapid Surface Patterning and Chemical Synthesis of Peptide and Glycan Microarrays

Eickelmann, Stephan; Tsouka, Alexandra; Heidepriem, Jasmin; Paris, Grigori; Zhang, Junfang; Molinari, Valerio; Mende, Marco; Loeffler, Felix F.
Adv. Mater. Technol..
Nov 2019
10.1002/admt.201900503
A low-cost laser-based printing setup is presented, which allows for the spot-wise patterning of surfaces with defined polymer nanolayers. These nanolayer spots serve as a “solid solvent,” embedding different chemicals, chemical building blocks, materials, or precursors and can be stacked on top of each other. By melting the spot pattern, the polymer-embedded molecules are released for chemical reaction. This enables researchers to quickly pattern a surface with different molecules and materials, mixing them directly on the surface for high-throughput chemical synthesis to generate and screen diverse microarray libraries. In contrast to expensive ink-jet or contact printing, this approach does not require premixing of inks, which enables in situ combinatorial mixing. Easy access and versatility of this patterning approach are shown by generating microarrays of various biomolecules, such as glycans for the first time, to screen interactions of antibodies and lectins. In addition, a layer-by-layer solid-phase synthesis of peptides directly on the microarray is presented. Amino acid–containing nanolayers are repeatedly laser-transferred and reacted with the functionalized acceptor surface in defined patterns. This simple system enables a reproducible array production, down to spot-to-spot distances of 100 µm, and offers a flexible and cheap alternative to expensive spotting robot technology.

Ara h 7 isoforms share many linear epitopes: Are 3D epitopes crucial to elucidate divergent abilities?

Ehlers, Anna M.; Klinge, Marco; Suer, Waltraud; Weimann, Yvonne; Knulst, André C.; Besa, Frithjof; Le, Thuy‐My; Otten, Henny G.
Clin Exp Allergy.
Nov 2019
10.1111/cea.13496
Background The peanut allergens Ara h 2, h 6, and h 7 are potent allergens and can trigger severe reactions. Ara h 7 consists of three isoforms differing in their ability to induce basophil degranulation, whereas the ability of Ara h 7.0201 is comparable to Ara h 2 and 6 as shown in previous literature. Objective To identify linear epitopes of Ara h 7.0101, Ara h 7.0201 and Ara h 7.0301 recognized by IgE and IgG4 from patients sensitized to Ara h 7 and to investigate their potential to elucidate divergent abilities of the Ara h 7 isoforms in inducing basophil activation. Methods Linear epitopes recognized by IgE and IgG4 were mapped by peptide microarray analysis containing 15-mer peptides of Ara h 2.0201, 6, 7.0101, 7.0201 and 7.0301 and 39 peanut allergic patients sensitized to Ara h 7 (discovery). For validation, 20-mer peptides containing the minimal epitope and surrounding amino acids were incubated with 25 sensitized patients and 10 controls (validation). Results Three out of 14 linear epitopes were unique for each isoform (Ara h 7.0101: aa 97-109; Ara h 7.0201: aa 122-133; Ara h 7.0301: aa 65-74) but scarcely recognized by IgE. The main linear IgE epitope (aa 51-57) located in the long flexible loop of all Ara h 7 isoforms was bound by antibodies from 31% of the patients (discovery and validation cohort). Regarding IgG4, 55% of the patients recognized an epitope present on all isoforms (aa 55-65), whereas epitope aa 129-137, only present on Ara h 7.0101/0.0301, was recognized by 38% of the patients. Recognition was highly individual, although 20% of the patients recognized any linear epitope neither by IgE nor by IgG4 despite a low mean z-score of ≥ 1.7. Remarkably, only 50% of the patients recognized one or more epitopes by IgE. Conclusion & Clinical Relevance Ara h 7 isoforms share many linear epitopes being easily accessible for antibody binding. Unique epitopes, essential to elucidate divergent potencies, were scarcely recognized, suggesting a crucial involvement of conformational epitopes.

Characterization of a sandwich ELISA for quantification of total human soluble neuropilin‐1

Gadermaier, Elisabeth; Tesarz, Manfred; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
J Clin Lab Anal.
Sep 2019
10.1002/jcla.22944
Background Neuropilin-1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique. Methods We developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti-human NRP1 antibodies were mapped with microarray technology. We included a sample pre-treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants. Results The ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma (heparin, EDTA, and citrate). Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N-terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra-assay: 6%), dilution linearity (95%-115%), specificity (98%), and spike recovery (81%-109%) meet the international standards of acceptance. Conclusion Our novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand-bound total NRP1.

A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B

Fritzler, Marvin J.; Brown, Rachael D.; Zhang, Meifeng
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy.
Aug 2019
10.1089/mab.2019.0016
Kinesin-like protein KIF20B, originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits in vitro microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It has been characterized as a slow molecular motor that moves toward the plus-end of microtubules. Human autoantibodies directed against KIF20B have been described in up to 25% of patients with idiopathic ataxia and less commonly in other neuropathies and autoinflammatory conditions. One of the limitations of research into the structure and function of KIF20B has been a reliable monoclonal antibody that can be used in a variety of applications. To establish a reference standard for anti-KIF20B immunoassays and facilitate studies on the role of KIF20B in developmental cell biology, we developed an IgG1 monoclonal antibody, 10C7, which reacts with the cognate KIF20B protein in Western immunoblots and in addressable laser bead immunoassays. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, indirect immunofluorescence studies showed that reactivity was mainly localized to a proportion of interphase nuclei, but during metaphase, it was redistributed throughout the cytoplasm and perichromatin mass. Later in telophase/anaphase, KIF20B was localized to the stem body and midzone of the midbody. 10C7 also showed remarkable staining of a subset of cells in the cerebellum, ovary, and testis tissues. KIF20B was shown to have extensive coiled-coil domains. The monoclonal antibody, 10C7, will be of value to diagnostic laboratory scientists interested in having a reliable reference standard for anti-KIF20B immunoassays as well as cell, molecular, and developmental biology researchers.

Immunization of cats to induce neutralizing antibodies against Fel d 1, the major feline allergen in human subjects

Thoms, Franziska; Jennings, Gary T.; Maudrich, Melanie; Vogel, Monique; Haas, Stefanie; Zeltins, Andris; Hofmann-Lehmann, Regina; Riond, Barbara; Grossmann, Jonas; Hunziker, Peter; Fettelschoss-Gabriel, Antonia; Senti, Gabriela; Kündig, Thomas M.; Bachmann, Martin F.
Journal of Allergy and Clinical Immunology.
Jul 2019
10.1016/j.jaci.2019.01.050
Background Cat allergy in human subjects is usually caused by the major cat allergen Fel d 1 and is found in approximately 10% of the Western population. Currently, there is no efficient and safe therapy for cat allergy available. Allergic patients usually try to avoid cats or treat their allergy symptoms. Objective We developed a new strategy to treat Fel d 1–induced allergy in human subjects by immunizing cats against their own major allergen, Fel d 1. Methods A conjugate vaccine consisting of recombinant Fel d 1 and a virus-like particle derived from the cucumber mosaic virus containing the tetanus toxin–derived universal T-cell epitope tt830-843 (CuMVTT) was used to immunize cats. A first tolerability and immunogenicity study, including a boost injection, was conducted by using the Fel-CuMVTT vaccine alone or in combination with an adjuvant. Results The vaccine was well tolerated and had no overt toxic effect. All cats induced a strong and sustained specific IgG antibody response. The induced anti–Fel d 1 antibodies were of high affinity and exhibited a strong neutralization ability tested both in vitro and in vivo. A reduction in the endogenous allergen level and a reduced allergenicity of tear samples, were observed. Conclusion Vaccination of cats with Fel-CuMVTT induces neutralizing antibodies and might result in reduced symptoms of allergic cat owners. Both human subjects and animals could profit from this treatment because allergic cat owners would reduce their risk of developing chronic diseases, such as asthma, and become more tolerant of their cats, which therefore could stay in the households and not need to be relinquished to animal shelters.

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