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

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Identification of novel antigens contributing to autoimmunity in cardiovascular diseases

Müller, Anna-Maria; Bockstahler, Mariella; Hristov, Georgi; Weiß, Christel; Fischer, Andrea; Korkmaz-Icöz, Sevil; Giannitsis, Evangelos; Poller, Wolfgang; Schultheiss, Heinz-Peter; Katus, Hugo A.; Kaya, Ziya
Clinical Immunology.
Dec 2016
10.1016/j.clim.2016.09.003
In myocarditis and dilated cardiomyopathy (DCM) patients the immune system may play an important role in disease progression. In this study, we aimed to identify new antigens as a target for autoimmune response that might play a crucial role in these diseases. Therefore, a peptide-array was used to investigate antibody binding profiles in patients with autoimmune myocarditis or DCM compared to healthy controls and thus to identify disease relevant antigens. To analyze the pathogenicity of the identified antigens, an experimental autoimmune myocarditis (EAM) model was used. Hereby, 3 peptide sequences, derived from myosin-binding-protein-C (MYBPC) fast-type, RNA-binding-protein 20 (RBM20), and dystrophin, showed pathogenic effects on the myocardium of mice. In summary, 3 potentially cardiopathogenic peptides (MYBPC fast-type, RBM20, dystrophin) were identified. Thus, this study could serve as a basis for future investigations aimed at determining further antigens leading to pathogenic effects on the myocardium of DCM as well as myocarditis patients.

Properties of Two Enterovirus Antibodies that are Utilized in Diabetes Research

Maccari, Giuseppe; Genoni, Angelo; Sansonno, Silvia; Toniolo, Antonio
Sci Rep.
Apr 2016
10.1038/srep24757
Human enteroviruses (EVs) comprise >100 different types. Research suggests a non-chance association between EV infections and type 1 diabetes. Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects. When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings. To address this issue, properties of EV antibodies 5D-8.1 and 9D5 have been investigated using peptide microarrays, peptide substitution scanning, immunofluorescence of EV-infected cells, EV neutralization assays, bioinformatics analysis. Evidence indicates that the two antibodies bind to distinct non-neutralizing linear epitopes in VP1 and are specific for a vast spectrum of EV types (not for other human viruses). However, their epitopes may align with a few human proteins at low expected values. When tested by immunofluorescence, high concentrations of 5D-8.1 yelded faint cytoplasmic staining in uninfected cells. At reduced concentrations, both antibodies produced dotted staining only in the cytoplasm of infected cells and recognized both acute and persistent EV infection. Thus, the two monoclonals represent distinct and independent probes for hunting EVs in tissues of patients with diabetes or other endocrine conditions.

Serum peptide reactivities may distinguish neuromyelitis optica subgroups and multiple sclerosis

Metz, Imke; Beißbarth, Tim; Ellenberger, David; Pache, Florence; Stork, Lidia; Ringelstein, Marius; Aktas, Orhan; Jarius, Sven; Wildemann, Brigitte; Dihazi, Hassan; Friede, Tim; Brück, Wolfgang; Ruprecht, Klemens; Paul, Friedemann
Neurol Neuroimmunol Neuroinflamm.
Apr 2016
10.1212/NXI.0000000000000204
Objective: To assess in an observational study whether serum peptide antibody reactivities may distinguish aquaporin-4 (AQP4) antibody (Ab)–positive and -negative neuromyelitis optica spectrum disorders (NMOSD) and relapsing-remitting multiple sclerosis (RRMS). Methods: We screened 8,700 peptides that included human and viral antigens of potential relevance for inflammatory demyelinating diseases and random peptides with pooled sera from different patient groups and healthy controls to set up a customized microarray with 700 peptides. With this microarray, we tested sera from 66 patients with AQP4-Ab-positive (n = 16) and AQP4-Ab-negative (n = 19) NMOSD, RRMS (n = 11), and healthy controls (n = 20). Results: Differential peptide reactivities distinguished NMOSD subgroups from RRMS in 80% of patients. However, the 2 NMOSD subgroups were not well-discriminated, although those patients are clearly separated by their antibody reactivities against AQP4 in cell-based assays. Elevated reactivities to myelin and Epstein-Barr virus peptides were present in RRMS and to AQP4 and AQP1 peptides in AQP4-Ab-positive NMOSD. Conclusions: While AQP4-Ab-positive and -negative NMOSD subgroups are not well-discriminated by peptide antibody reactivities, our findings suggest that peptide antibody reactivities may have the potential to distinguish between both NMOSD subgroups and MS. Future studies should thus concentrate on evaluating peptide antibody reactivities for the differentiation of AQP4-Ab-negative NMOSD and MS.

Identification of Equine arteritis virus immunodominant B cell epitopes using a peptide microarray

Mayers, J.; Westcott, D.; Steinbach, F.
Journal of Equine Veterinary Science.
Apr 2016
10.1016/j.jevs.2016.02.028

Non-invasive glioblastoma immunoprofiling by printed peptide arrays

Mock, Andreas; Herold-Mende, Christel
OncoImmunology.
Feb 2016
10.1080/2162402X.2015.1069941
Immune monitoring assays for patient stratification and treatment efficacy in clinical trials are in demand. We have recently described a cost-effective non-invasive assay to determine the immune status of glioblastoma patients. Profiling antitumor serum antibodies by customized printed peptide arrays identified response against a tenascin-C (TNC) peptide as a robust prognostic biomarker.

Autoantibodies specific to estrogen receptor alpha act as estrogen agonists and their levels correlate with breast cancer cell proliferation

Maselli, Angela; Capoccia, Sara; Pugliese, Patrizia; Raggi, Carla; Cirulli, Francesca; Fabi, Alessandra; Malorni, Walter; Pierdominici, Marina; Ortona, Elena
OncoImmunology.
Feb 2016
10.1080/2162402X.2015.1074375
Estrogen receptors have recently been demonstrated at the cell surface. Unlike nuclear receptors, they are able to trigger rapid responses inside the cells. In this study, we evaluated the presence and the possible role of autoantibodies specific to estrogen receptor (anti-ER Abs) in the peripheral blood of breast cancer patients. Anti-ERα Abs were detectable in 22/48 (46%) patients’ sera and their levels positively correlated with the percentage of Ki-67-positive breast cancer cells. Anti-ERα Abs purified from breast cancer patients’ sera were able: (i) to recognize ERα epitopes expressed at the cell surface of ER-positive breast cancer cells, (ii) to trigger rapid extracellular signal-regulated kinase (ERK) phosphorylation, and (iii) to induce cell proliferation. Our results suggest that anti-ERα Abs can act as estrogen agonists playing a pathogenetic role as breast cancer-promoting factors. These autoantibodies could also be considered as possible peripheral blood biomarkers indicative of the breast cancer growth potential.

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.

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.

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.

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