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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

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.

High-resolution mapping of linear epitopes from LiNTPDase2: Advancing leishmaniasis detection using optimized protein and peptide antigens

Castro, Raissa Barbosa De; Badaró De Moraes, João Victor; De Souza, Anna Cláudia Alves; Favarato, Evandro Silva; Voorwald, Fabiana Azevedo; Dos Santos, Fabiane Matos; Bressan, Gustavo Costa; Vasconcellos, Raphael De Souza; Fietto, Juliana Lopes Rangel
Diagnostic Microbiology and Infectious Disease.
Oct 2024
10.1016/j.diagmicrobio.2024.116448
Visceral Leishmaniasis, caused by Leishmania infantum, is a tropical neglected disease and the most dangerous form of Leishmaniasis. It occurs zoonotically, with domestic transmission posing risks to humans as dogs have high susceptibility and are natural reservoirs of the parasite. Given their epidemiological role, improvements are needed in diagnosing Canine Visceral Leishmaniasis (CVL). Thus, we mapped linear epitopes from the rLiNTPDase2 antigen through peptide microarray and identified six positive epitopes. Validation through peptide ELISA revealed three promising peptides with accuracies of 78.6%, 85.92%, and 79.59%. Their combination yielded 97.58% accuracy. Negative epitopes were also found, which interacted with CVL-negative and Chagas Disease positive samples. Their removal from the rLiNTPDase2 sequence resulted in the rNT2.neg, which obtained enhanced specificity over rLiNTPDase2. The rNT2.neg validation achieved 87.50% sensitivity, 90.55% specificity, and 93.5% accuracy within 127 CVL-positive and 96 CVL-negative samples. Therefore, three peptides and rNT2.neg show significant promise for CVL diagnosis.

Identification of Schistosoma haematobium and Schistosoma mansoni linear B-cell epitopes with diagnostic potential using in silico immunoinformatic tools and peptide microarray technology

Vengesai, Arthur; Manuwa, Marble; Midzi, Herald; Mandeya, Masimba; Muleya, Victor; Mujeni, Keith; Chipako, Isaac; Mduluza, Takafira
PLoS Negl Trop Dis.
Aug 2024
10.1371/journal.pntd.0011887
Introduction: Immunoinformatic tools can be used to predict schistosome-specific B-cell epitopes with little sequence identity to human proteins and antigens other than the target. This study reports an approach for identifying schistosome peptides mimicking linear B-cell epitopes using in-silico tools and peptide microarray immunoassay validation. Method: Firstly, a comprehensive literature search was conducted to obtain published schistosome-specific peptides and recombinant proteins with the best overall diagnostic performances. For novel peptides, linear B-cell epitopes were predicted from target recombinant proteins using ABCpred, Bcepred and BepiPred 2.0 in-silico tools. Together with the published peptides, predicted peptides with the highest probability of being B-cell epitopes and the lowest sequence identity with proteins from human and other pathogens were selected. Antibodies against the peptides were measured in sera, using peptide microarray immunoassays. Area under the ROC curve was calculated to assess the overall diagnostic performances of the peptides. Results: Peptide AA81008-19-30 had excellent and acceptable diagnostic performances for discriminating S. mansoni and S. haematobium positives from healthy controls, with AUC values of 0.8043 and 0.7326 respectively for IgG. Peptides MS3_10186-123-131, MS3_10385-339-354, SmSPI-177-193, SmSPI-379-388, MS3-10186-40-49 and SmS-197-214 had acceptable diagnostic performances for discriminating S. mansoni positives from healthy controls with AUC values ranging from 0.7098 to 0.7763 for IgG. Peptides SmSPI-359-372, Smp126160-438-452 and MS3 10186-25-41 had acceptable diagnostic performances for discriminating S. mansoni positives from S. mansoni negatives with AUC values of 0.7124, 0.7156 and 0.7115 respectively for IgG. Peptide MS3-10186-40-49 had an acceptable diagnostic performance for discriminating S. mansoni positives from healthy controls, with an AUC value of 0.7413 for IgM. Conclusion: One peptide with a good diagnostic performance and nine peptides with acceptable diagnostic performances were identified using the immunoinformatic approach and peptide microarray validation. There is need for evaluation of the peptides with true negatives and a good standard positive reference.

Alzheimer’s disease risk associated with changes in Epstein-Barr virus nuclear antigen 1-specific epitope targeting antibody levels

Sim, Kyu-Young; An, Jaekyeung; Bae, So-Eun; Yang, Taewoo; Ko, Gwang-Hoon; Hwang, Jeong-Ryul; Choi, Kyu Yeong; Park, Jung Eun; Lee, Jung Sup; Kim, Byeong C.; Lee, Kun Ho; Park, Sung-Gyoo
Journal of Infection and Public Health.
Jul 2024
10.1016/j.jiph.2024.05.050
*Background* Alzheimer’s disease (AD) is a neurodegenerative disorder influenced by age, sex, genetic factors, immune alterations, and infections. Multiple lines of evidence suggest that changes in antibody response are linked to AD pathology. *Methods* To elucidate the mechanisms underlying AD development, we investigated antibodies that target autoimmune epitopes using high-resolution epitope microarrays. Our study compared two groups: individuals with AD (n = 19) and non-demented (ND) controls (n = 19). To validate the results, we measured antibody levels in plasma samples from AD patients (n = 96), mild cognitive impairment (MCI; n = 91), and ND controls (n = 97). To further explore the invlovement of EBV, we performed epitope masking immunofluorescence microscopy analysis and tests to induce lytic replication using the B95–8 cell line. *Results* In this study, we analyzed high-resolution epitope-specific serum antibody levels in AD, revealing significant disparities in antibodies targeting multiple epitopes between the AD and control groups. Particularly noteworthy was the significant down-regulation of antibody (anti-DG#29) targeting an epitope of Epstein-Barr virus nuclear antigen 1 (EBNA1). This down-regulation increased AD risk in female patients (odds ratio up to 6.6), but not in male patients. Our investigation further revealed that the down-regulation of the antibody (anti-DG#29) is associated with EBV reactivation in AD, as indicated by the analysis of EBV VCA IgG or IgM levels. Additionally, our data demonstrated that the epitope region on EBNA1 for the antibody is hidden during the EBV lytic reactivation of B95–8 cells. *Conclusion* Our findings suggest a potential relationship of EBV in the development of AD in female. Moreover, we propose that antibodies targeting the epitope (DG#29) of EBNA1 could serve as valuable indicators of AD risk in female.

Potent Adjuvanticity of a Pure TLR7-Agonistic Imidazoquinoline Dendrimer

Shukla, Nikunj M.; Salunke, Deepak B.; Balakrishna, Rajalakshmi; Mutz, Cole A.; Malladi, Subbalakshmi S.; David, Sunil A.
PLoS ONE.
Aug 2012
10.1371/journal.pone.0043612
Engagement of toll-like receptors (TLRs) serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. We synthesized a dendrimeric molecule bearing six units of a potent TLR7/TLR8 dual-agonistic imidazoquinoline to explore if multimerization of TLR7/8 would result in altered activity profiles. A complete loss of TLR8-stimulatory activity with selective retention of the TLR7-agonistic activity was observed in the dendrimer. This was reflected by a complete absence of TLR8-driven proinflammatory cytokine and interferon (IFN)-γ induction in human PBMCs, with preservation of TLR7-driven IFN-α induction. The dendrimer was found to be superior to the imidazoquinoline monomer in inducing high titers of high-affinity antibodies to bovine α-lactalbumin. Additionally, epitope mapping experiments showed that the dendrimer induced immunoreacti

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 Synthesis of Peptide Arrays onto a Microchip

Beyer, M.; Nesterov, A.; Block, I.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F.
Science.
Dec 2007
10.1126/science.1149751
Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.

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