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

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Peptide Epitopes of NC16A BP180 in the Diagnostics of Bullous Pemphigoid

Lytton, Simon D.; Wagger, Christine; Meyersburg, Damian; Mussnig, Birgit; Lang, Roland; Maglie, Roberto; Anzengruber, Florian; Antiga, Emiliano; Hall, Russell P.; Bauer, Johann W.
JID Innovations.
Nov 2025
10.1016/j.xjidi.2025.100415

Anti-TRPV2 Autoantibody Linked to Sudden Infant Death Syndrome

Maguy, Ange; Tessier, Agnès; Mahendran, Yuvaraj; Denis, Manon; Lauzier, Benjamin; Charpentier, Flavien; Li, Jin
Jul 2025
10.1161/circulationaha.125.073748
As a leading cause of infant death, sudden infant death syndrome (SIDS) remains a perplexing diagnosis with no clear underlying biological substrate.1 In the past decade, studies have emerged demonstrating that circulating autoantibodies targeting cardiac antigens can underlie life-threatening arrhythmias.2 Because autoimmunity as a cause of SIDS has not yet been explored, we screened infant serum samples for the presence of autoantibodies targeting cardiac ion channels and examined how immunoglobulins may play a driving role in the pathogenesis of SIDS. Comparing cases of SIDS and accidental suffocation and strangulation in bed with healthy controls, we established the autoantibody profile of 47 serum samples using peptide microarray (Figure [A]), as previously described.2 Strikingly, only 1 single autoantibody targeting the transient receptor potential vanilloid 2 (TRPV2) channel (PTGPNATESVQPMEGQEDEG) was significantly associated with SIDS (P=0.028 versus controls, the default correction in limma). Collectively, we detected anti-TRPV2 autoantibodies in 84.6% of infants with SIDS compared with 50.0% in cases of accidental suffocation and strangulation in bed and 25.0% in controls.

Mapping autoantibody targets of full-length C-reactive protein in systemic lupus erythematosus: importance for neutrophil function and classical complement activation

Karlsson, Jesper; Wirestam, Lina; Duàn, Hanna; Ahmad, Suhana; Appelgren, Daniel; Enocsson, Helena; Wetterö, Jonas; Sjöwall, Christopher
Front. Immunol..
May 2025
10.3389/fimmu.2025.1578372
C-reactive protein (CRP) is an important pattern recognition molecule of innate immunity. Autoantibodies targeting CRP are common in patients with systemic lupus erythematosus (SLE) and the levels correlate with disease activity. The purpose of this study was to investigate binding sites of IgG autoantibodies on the full linear sequence of CRP and identify potential associations with clinical variables in well-characterized SLE patients; a secondary aim was to investigate the effect of an epitope-based synthesized peptide motif on neutrophil functions. The levels of anti-CRP and SLE-associated antibodies were assessed, and a microarray-based linear epitope mapping was performed to detect binding sites on the full CRP monomer. We observed that anti-CRP antibodies bind to a variety of linear epitopes with a higher prevalence in SLE compared to healthy blood donors. Eleven unique epitopes were identified, of which five were found exclusively in SLE. Furthermore, we show that patients with anticardiolipin IgG and/or anti-β2GPI IgG antibodies have a higher number of positive CRP epitopes, and some CRP autoantibody-specificities associate with antiphospholipid antibodies, disease activity, and classical complement activation. In addition, one identified motif was selected, synthesized, and used for studying neutrophil function. This peptide showed modulatory capacity on neutrophil oxidative burst and chemotaxis, but not on neutrophil extracellular trap formation. Our results implicate a wide variation of anti-CRP autoantibody binding motifs of the linear structure of CRP in SLE patients. Some epitopes have the potential to modify innate host responses of relevance to the pathogenesis of SLE.

Paediatric autoimmune uveitis is associated with intraocular antibodies against Epstein–Barr virus Nuclear Antigen 1 (EBNA-1)

Hendrikse, Jytte; Bont, Louis J.; Schellekens, Peter A.W.J.F.; De Groot-Mijnes, Jolanda D.F.; De Boer, Joke H.; Kuiper, Jonas J.W.
eBioMedicine.
Mar 2025
10.1016/j.ebiom.2025.105681
**Background** Non-infectious uveitis is an immune-mediated disease characterized by vision-threatening inflammation within the eye. Increasing evidence indicates that microbial agents promote non-infectious uveitis, but the natural history of immune responses to pathogens in patients remains unexplored. We determined intraocular antibodies against pathogens in paediatric uveitis. **Methods** We used peptide microarrays containing 3760 linear B-cell epitopes from 196 human pathogens to profile IgG levels in eye fluid biopsies and paired serum samples from 18 Dutch paediatric patients and 6 age-matched controls. We compared intensities of single epitopes and clusters based on overlapping amino acid sequence of peptides. Next-generation sequencing data was obtained to determine the HLA-DRB1∗15:01 genotype. **Findings** Intraocular antibody profiles largely matched serum profiles and were characterized by high IgG against the conserved PALTAVET-motif of enterovirus family members, as well as broad epitope reactivity against Epstein–Barr virus (EBV). The aqueous humour of patients showed elevated levels of antibodies against peptides containing the RRPFFHPV-motif of Epstein–Barr Virus Nuclear Antigen 1 [EBNA-1]. Antibody levels against the RRPFFHPV-motif of EBNA1 were significantly higher in individuals that carry the HLA-DRB1∗15:01 risk allele of paediatric uveitis. **Interpretation** Intraocular antibodies against an immunogenic epitope of EBV showed an association with paediatric uveitis, particularly HLA-DRB1∗15:01 positive uveitis, indicating a potential link between EBV-specific immune responses and autoimmune uveitis. **Funding** Funding for this research was received from Fischer Stichting (UZ2022-3), ODAS (2021-02), LSBS and ANVVB.

The antibody repertoire of autoimmune sensory neuronopathies targets pathways of the innate and adaptative immune system. An autoantigenomic approach.

Moritz, Christian P.; Tholance, Yannick; Boutahar, Nadia; Borowczyk, Coralie; Berger, Anne-Emmanuelle; Paul, Stéphane; Antoine, Jean-Christophe; Camdessanché, Jean-Philippe
Journal of Translational Autoimmunity.
Jan 2025
10.1016/j.jtauto.2025.100277
Sensory neuronopathies (SNN) encompasses diverse etiologies, with autoimmunity playing a major role through both cellular and humoral responses. To investigate the humoral autoantibody repertoire in autoimmune SNN, we conducted a retrospective cohort study using large Human Proteome-wide protein microarrays (HuProt 3.1, HuProt 4.0, ProtoArrays). We specifically focused on immune system pathways within the repertoire of targeted antigens (the autoantigenome). We included 131 participants: 44 patients with non-paraneoplastic autoimmune SNN (12 with anti-FGFR3 and/or anti-AGO antibodies), 8 with paraneoplastic SNN and 79 controls. Results were validated in an independent cohort of 16 SNN patients. Overrepresentation of immune-system-related proteins was assessed via the Reactome database, and serum levels of IFN-γ and IL-6 were measured using the Bio-Plex Pro™ Reagent Kit. Autoimmune SNN sera interact with more immune system proteins than healthy controls (ProtoArrays: 271/863 vs. 14/863, HuProt: 112/1694 vs. 39/1694, both p<0.0001). Overrepresentation was observed in all immune sub-pathways, including innate, adaptive immune responses, and cytokine signaling. Anti-FGFR3-positive SNN patients were more reactive with immune system proteins than negative ones. The independent SNN cohort validated the finding of overrepresentatively targeted immune system pathways. Validation with dot blot and ELISA confirmed reactivity to TRIM21 and IL-6, and identified anti-IFN-γ-positive SNN patients. IFN-γ levels correlated weakly with levels of anti-IFN-γ antibodies (Pearson’s r = 0.22, p=0.03). We conclude that the antibody repertoire of autoimmune SNN targets pathways of the innate and adaptative immune system, potentially reflecting key disease-related immune pathways and highlighting the systemic role of immune dysregulation in SNN.

High-throughput identification of immunoreactive peptides and corresponding proteins from Anaplasma platys and Ehrlichia canis using peptide microarray chips

Llanes, Alejandro; Madesh, Swetha; Brangulis, Kalvis; Rajeev, Sreekumari
Front Cell Infect Microbiol.
Jan 2025
10.3389/fcimb.2025.1671309
INTRODUCTION: Anaplasma platys and Ehrlichia canis are rickettsial pathogens infecting dogs, with a worldwide distribution. Both species are obligate intracellular pathogens and colonize bone marrow-derived cells, with coinfections frequently reported in dogs. Although E. canis immunodominant proteins have been thoroughly characterized, very few high-throughput studies have been conducted to identify immunogenic proteins from Anaplasma spp. In this study, we used a methodology based on peptide microarray chips to identify immunoreactive peptides, either shared or species-specific, in the complete theoretical proteomes of both pathogens. METHODS: B-cell epitopes were predicted in the corresponding proteins from both species and ranked for synthesis on the peptide microarrays. These microarrays were screened with serum samples from antibody-positive dogs, as well as negative control sera from unexposed dogs. Additionally, we assessed the feasibility of integrating evidence gathered at the level of individual peptides to identify potentially immunogenic proteins contributing to the patterns of immunoreactivity observed on microarrays. RESULTS: Screening of peptide microarrays resulted in complex antibody reactivity patterns against thousands of peptides. After discarding peptides with cross-reactivity to negative control sera, we identified over 1,200 immunoreactive peptides, including ~80 peptides shared between the two species with almost identical sequences. Despite screening linear peptides, we were able to identify proteins previously reported as immunodominant in E. canis, some of which contain predominantly conformational epitopes. DISCUSSION: Our results suggest that a high-throughput strategy based on peptide microarrays is an effective approach for the rapid identification of immunoreactive peptides and the underlying immunogenic proteins. This study provides a foundation for developing novel diagnostic tools and vaccine candidates against A. platys and E. canis, including potential combined or multivalent formulations targeting both pathogens.

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.

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.

Predicting HBsAg clearance in genotype A chronic hepatitis B using HBsAg epitope profiling: A biomarker for functional cure

Walsh, Renae; Hammond, Rachel; Yuen, Lilly; Deerain, Joshua; O’Donnell, Tanya; Leary, Thomas; Cloherty, Gavin; Gaggar, Anuj; Kitrinos, Kathryn; Subramanian, Mani; Wong, Darren; Locarnini, Stephen
Liver Int.
Nov 2019
10.1111/liv.14207
Background and Aim Functional cure is the major goal of chronic hepatitis B (CHB) therapy though few biomarkers predict this outcome. HBsAg epitope occupancy can be influenced by therapeutic and immune pressure. The aim of this study was to map the HBsAg epitope profiles during long-term nucleos(t)ide analogue therapy in patients with genotype A CHB, in the context of HBsAg loss (SL)/seroconversion. Methods We evaluated 25 genotype A CHB patients in the GS-US-174-0103 trial of HBeAg-positive CHB patients treated with tenofovir or adefovir for 4 years, 14 who achieved SL whilst 11 had no change. We epitope mapped the major domains of HBsAg to identify those patients with HBsAg clearance profile (CP) (loss of binding at both loops 1 and 2 epitopes of the ‘a’ determinant) vs non-clearance profile (no change in epitope recognition, or loss of epitope binding at one loop only), correlating this to on-treatment HBsAg responses. Complexed anti-HBs was also measured. Results Analysis of the HBsAg epitope profiles of the 25 patients at baseline identified no predictive correlation with SL. In contrast, analysis at week 48 and end of study (week 192) or prior to SL identified significant predictive associations between development of HBsAg CPs and outcome of functional cure. The detection of a CP also correlated with the development of an alanine aminotransferase flare and detection of anti-HBs complexed with HBsAg. Conclusion The detection of HBsAg CPs by epitope mapping represents a novel viral biomarker, reflecting an emerging anti-HBs selection pressure prior to functional cure.

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.

Immunization of mice with chimeric antigens displaying selected epitopes confers protection against intestinal colonization and renal damage caused by Shiga toxin-producing Escherichia coli

Montero, David A.; Del Canto, Felipe; Salazar, Juan C.; Cespedes, Sandra; Cádiz, Leandro; Arenas-Salinas, Mauricio; Reyes, José; Oñate, Ángel; Vidal, Roberto M.
Sep 2019
10.1101/783829
Abstract Shiga toxin-producing Escherichia coli (STEC) cause diarrhea and dysentery, which may progress to hemolytic uremic syndrome (HUS). Vaccination has been proposed as a preventive approach against STEC infection; however, there is no vaccine for humans and those used in animals reduce but do not eliminate the intestinal colonization of STEC. The OmpT, Cah and Hes proteins are widely distributed among clinical STEC strains and are recognized by serum IgG and IgA in patients with HUS. Here, we develop a vaccine formulation based on two chimeric antigens containing epitopes of OmpT, Cah and Hes proteins against STEC strains. Intramuscular and intranasal immunization of mice with these chimeric antigens elicited systemic and local long-lasting humoral responses. However, the class of antibodies generated was dependent on the adjuvant and the route of administration. Moreover, while intramuscular immunization with the combination of the chimeric antigens conferred protection against colonization by STEC O157:H7 and the intranasal conferred protection against renal damage caused by STEC O91:H21. This pre-clinical study supports the potential use of this formulation based on recombinant chimeric proteins as a preventive strategy against STEC infections.

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