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

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Autoantibodies to a novel Rpp38 (Th/To) derived B-cell epitope are specific for systemic sclerosis and associate with a distinct clinical phenotype

Koenig, Martial; Bentow, Chelsea; Satoh, Minoru; Fritzler, Marvin J; Senécal, Jean-Luc; Mahler, Michael
Apr 2019
10.1093/rheumatology/kez123
Abstract Objective Detection of antinuclear antibodies and specific autoantibodies is important in the diagnosis and classification of SSc. Several proteins of the Th/To complex, including Rpp25, Rpp38 and hPop1 are the target of autoantibodies in SSc patients. However, very little is known about the epitope distribution of this autoantigen. Consequently, we screened Rpp25, Rpp38 and hPop1 for B cell epitopes and evaluated their clinical relevance. Methods Serum pools with (n = 2) and without (n = 1) anti-Th/To autoantibodies were generated and used for epitope discovery. Identified biomarker candidate sequences were then utilized to synthesize synthetic, biotinylated, soluble peptides. The peptides were tested to determine reactivity with sera from SSc cohorts (n = 202) and controls (n = 159) using a chemiluminescence immunoassay. Additionally, samples were also tested for antibodies to full-length recombinant Rpp25 antibodies by chemiluminescence immunoassay. Results Several immunodominant regions were found on the three proteins. The strongest reactivity was observed with an Rpp38 peptide (aa 229–243). Autoantibodies to the Rpp38 peptide were detected in 8/149 (5.4%) limited cutaneous SSc patients, but not in any of 159 controls (P = 0.003 by two-sided Fisher’s exact probability test). Although reactivity to the novel antigenic peptide was correlated with the binding to Rpp25 (rho = 0.44; P < 0.0001), subsets of patient sera either reacted strongly with Rpp25 or with the novel Rpp38-derived peptide. Conclusion A novel Rpp38 epitope holds promise to increase the sensitivity in the detection of anti-Th/To autoantibodies, thus enhancing the serological diagnosis of SSc.

In-depth serum proteomics reveals biomarkers of psoriasis severity and response to traditional Chinese medicine

Xu, Meng; Deng, Jingwen; Xu, Kaikun; Zhu, Tiansheng; Han, Ling; Yan, Yuhong; Yao, Danni; Deng, Hao; Wang, Dan; Sun, Yaoting; Chang, Cheng; Zhang, Xiaomei; Dai, Jiayu; Yue, Liang; Zhang, Qiushi; Cai, Xue; Zhu, Yi; Duan, Hu; Liu, Yuan; Li, Dong; Zhu, Yunping; Radstake, Timothy R. D. J.; Balak, Deepak M.W.; Xu, Danke; Guo, Tiannan; Lu, Chuanjian; Yu, Xiaobo
Theranostics.
Apr 2019
10.7150/thno.31144
Serum and plasma contain abundant biological information that reflect the body’s physiological and pathological conditions and are therefore a valuable sample type for disease biomarkers. However, comprehensive profiling of the serological proteome is challenging due to the wide range of protein concentrations in serum. Methods: To address this challenge, we developed a novel in-depth serum proteomics platform capable of analyzing the serum proteome across ~10 orders or magnitude by combining data obtained from Data Independent Acquisition Mass Spectrometry (DIA-MS) and customizable antibody microarrays. Results: Using psoriasis as a proof-of-concept disease model, we screened 50 serum proteomes from healthy controls and psoriasis patients before and after treatment with traditional Chinese medicine (YinXieLing) on our in-depth serum proteomics platform. We identified 106 differentially-expressed proteins in psoriasis patients involved in psoriasis-relevant biological processes, such as blood coagulation, inflammation, apoptosis and angiogenesis signaling pathways. In addition, unbiased clustering and principle component analysis revealed 58 proteins discriminating healthy volunteers from psoriasis patients and 12 proteins distinguishing responders from non-responders to YinXieLing. To further demonstrate the clinical utility of our platform, we performed correlation analyses between serum proteomes and psoriasis activity and found a positive association between the psoriasis area and severity index (PASI) score with three serum proteins (PI3, CCL22, IL-12B). Conclusion: Taken together, these results demonstrate the clinical utility of our in-depth serum proteomics platform to identify specific diagnostic and predictive biomarkers of psoriasis and other immune-mediated diseases.

High-density Peptide Arrays Help to Identify Linear Immunogenic B-cell Epitopes in Individuals Naturally Exposed to Malaria Infection

Jaenisch, Thomas; Heiss, Kirsten; Fischer, Nico; Geiger, Carolin; Bischoff, F. Ralf; Moldenhauer, Gerhard; Rychlewski, Leszek; Sié, Ali; Coulibaly, Boubacar; Seeberger, Peter H.; Wyrwicz, Lucjan S.; Breitling, Frank; Loeffler, Felix F.
Mol Cell Proteomics.
Apr 2019
10.1074/mcp.RA118.000992
High-density peptide arrays are an excellent means to profile anti-plasmodial antibody responses. Different protein intrinsic epitopes can be distinguished, and additional insights are gained, when compared with assays involving the full-length protein. Distinct reactivities to specific epitopes within one protein may explain differences in published results, regarding immunity or susceptibility to malaria. We pursued three approaches to find specific epitopes within important plasmodial proteins, (1) twelve leading vaccine candidates were mapped as overlapping 15-mer peptides, (2) a bioinformatical approach served to predict immunogenic malaria epitopes which were subsequently validated in the assay, and (3) randomly selected peptides from the malaria proteome were screened as a control. Several peptide array replicas were prepared, employing particle-based laser printing, and were used to screen 27 serum samples from a malaria-endemic area in Burkina Faso, West Africa. The immunological status of the individuals was classified as “protected” or “unprotected” based on clinical symptoms, parasite density, and age. The vaccine candidate screening approach resulted in significant hits in all twelve proteins and allowed us (1) to verify many known immunogenic structures, (2) to map B-cell epitopes across the entire sequence of each antigen and (3) to uncover novel immunogenic epitopes. Predicting immunogenic regions in the proteome of the human malaria parasite Plasmodium falciparum, via the bioinformatics approach and subsequent array screening, confirmed known immunogenic sequences, such as in the leading malaria vaccine candidate CSP and discovered immunogenic epitopes derived from hypothetical or unknown proteins.

High-throughput epitope profiling of antibodies in the plasma of Alzheimer’s disease patients using random peptide microarrays

Sim, Kyu-Young; Park, Sang-Heon; Choi, Kyu Yeong; Park, Jung Eun; Lee, Jung Sup; Kim, Byeong C.; Gwak, Jeonghwan; Song, Woo Keun; Lee, Kun Ho; Park, Sung-Gyoo
Sci Rep.
Mar 2019
10.1038/s41598-019-40976-x
The symptoms of Alzheimer’s disease (AD), a major cause of dementia in older adults, are linked directly with neuronal cell death, which is thought to be due to aberrant neuronal inflammation. Autoantibodies formed during neuronal inflammation show excellent stability in blood; therefore, they may be convenient blood-based diagnostic markers of AD. Here, we performed microarray analysis of 29,240 unbiased random peptides to be used for comprehensive screening of AD-specific IgG and IgM antibodies in the blood. The results showed that (1) sequence-specific and isotype-specific antibodies are regulated differentially in AD, and combinations of these antibodies showing high area under the receiver operating characteristic curve values (0.862–0.961) can be used to classify AD, (2) AD-specific IgG antibodies arise from IgM antibody-secreting cells that existed before disease onset and (3) target protein profiling of the antibodies identified some AD-related proteins, some of which are involved in AD-related signalling pathways. Therefore, we propose that these epitopes may facilitate the development of biomarkers for AD diagnosis and form the basis for a mechanistic study related to AD progression.

Automated laser-assisted synthesis of microarrays for infectious disease research

Paris, Grigori; Heidepriem, Jasmin; Tsouka, Alexandra; Mende, Marco; Eickelmann, Stephan; Loeffler, Felix F.
Mar 2019
10.1117/12.2516781
We developed a next-generation method for chemical in–situ combinatorial biomolecule array synthesis. This allows for an unprecedented combinatorial freedom in the automated chemical synthesis of molecule arrays with very high spot densities. Key feature of this new method is an automated positioning and laser transfer process: Small solid material spots are rapidly transferred from a donor film to an acceptor surface, requiring only minute amounts of materials. The transfer is performed with different and easy-to-produce donor slides. Each donor slide bears a thin polymer film, embedding one type of monomer. The coupling reaction occurs in a separate heating step, where the matrix becomes viscous and building blocks can diffuse within the material and couple to the acceptor surface. Since these transferred material spots are only several nanometers thin, this method allows for a consecutive multi-layer material deposition of e.g. activation reagents and amino acids. Subsequent heat-induced mixing facilitates an in–situ activation and coupling of the monomers. Positioning several of such resin spots, containing different chemical reagents, on top of each other, will enable for the first time in such small dimensions unique chemical synthesis strategies for each spot. Amount and concentration of the deposited materials can be adjusted with the laser parameters. Employing similar arrays, we can analyze the human immune response towards the proteome of different pathogens. We screened several peptide array replicas with different patient sera. The screenings resulted in significant hits in several proteins with interesting implications for future diagnostics and vaccine development.

Methods of Selecting Binding Reagents

Mallick, Parag; Egertson, Jarrett
Feb 2019
Methods and systems are provided herein for selecting an affinity reagent which binds a desired peptide epitope in a plurality of sequence contexts. The method relies on obtaining a peptide library, each peptide having the sequence αΧβ, wherein X is the desired peptide epitope, wherein each of a and β comprise an amino acid, using the peptide library to select an affinity reagent.

Patent infections with soil-transmitted helminths and Schistosoma mansoni are not associated with increased prevalence of antibodies to the Onchocerca volvulus peptide epitopes OvMP-1 and OvMP-23

Vlaminck, Johnny; Lagatie, Ole; Verheyen, Ann; Dana, Daniel; Van Dorst, Bieke; Mekonnen, Zeleke; Levecke, Bruno; Stuyver, Lieven J.
Parasites Vectors.
Jan 2019
10.1186/s13071-019-3308-z
Background Ov16 serology is considered a reference method for Onchocerca volvulus epidemiological mapping. Given the suboptimal sensitivity of this test and the fact that seroconversion takes more than a year after infection, additional serological tests might be needed to guide onchocerciasis elimination programmes. Recently, two linear epitopes encoded in OvMP-1 and OvMP-23 peptides were introduced as serological markers, but the observed antibody cross-reactivity in samples originating from Onchocerca volvulus non-endemic areas required further investigation. Methods We evaluated both peptide markers in an O. volvulus hypo-endemic setting in Jimma Town, Ethiopia using peptide ELISA. For all individuals (n = 303), the infection status with soil-transmitted helminths and Schistosoma mansoni was known. Results We found that 11 (3.6%) individuals were positive for anti-Ov16 IgG4 antibodies, while 34 (11.2%) and 15 (5.0%) individuals were positive for OvMP-1 and OvMP-23, respectively. Out of the 34 OvMP-1 positive samples, 33 were negative on the Ov16 IgG4 ELISA. Similarly, out of the 15 OvMP-23 positive samples, 14 scored negative on this reference method. No difference in seroprevalence for all three markers could be observed between uninfected individuals and individuals infected with different soil-transmitted helminths or S. mansoni. Moreover, the intensity of the response to OvMP-1, OvMP-23 or Ov16 was not significantly stronger in individuals carrying patent STH or S. mansoni infections, nor was there any correlation between the intensities of the responses to the three different antigens. Conclusions This study demonstrates that a patent infection with either soil-transmitted helminths or S. mansoni does not lead to increased antibody recognition of both OvMP-1 and OvMP23.

Molecular mimicry, genetic homology, and gene sharing proteomic “molecular fingerprints” using an EBV (Epstein-Barr virus)-derived microarray as a potential diagnostic method in autoimmune disease

Dreyfus, David H.; Farina, Antonella; Farina, Giuseppina Alessandra
Immunol Res.
Dec 2018
10.1007/s12026-018-9045-0
EBV (Epstein-Barr Virus) and other human DNA viruses are associated with autoimmune syndromes in epidemiologic studies. In this work, immunoglobulin G response to EBV-encoded proteins which share regions with human immune response proteins from the human host including ZEBRA (BZLF-1 encoded protein), BALF-2 recombinase expressed primarily during the viral lytic replication cycle, and EBNA-1 (Epstein-Barr Virus Nuclear Antigen) expressed during the viral latency cycle respectively were characterized using a laser-printed micro-array (PEPperprint.com). IgG response to conserved “A/T hooks” in EBV-encoded proteins such as EBNA-1 and the BALF-2 recombinase related to host DNA-binding proteins including RAG-1 recombinase and histones, and EBV-encoded virokines such as the IL-10 homologue BCRF-1 suggest further directions for clinical research. The author suggests that proteomic “molecular fingerprints” of the immune response to viral proteins shared with human immune response genes are potentially useful in early diagnosis and monitoring of autoantibody production and response to therapy in EBV-related autoimmune syndromes.

Soybean Allergy Related Epitopes

Kern, Karolin; Spiegel, Holger; Havenith, Heide; Szardenings, Michael
Nov 2018
The invention relates to a compilation comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. The invention further relates to an in vitro method for determining a patient’s immune status to soybean allergens, to a method for detecting at least one soybean allergen in a substance and to a method for determining the allergenicity of a soybean variety. Additionally, the invention relates to a kit comprising at least one composition containing a compound comprising at least five different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. Furthermore, the invention relates to the use of a peptide comprising a sequence element corresponding to an epitope for providing a molecule binding to a protein or peptide comprising the epitope.

Linear epitopes in Onchocerca volvulus vaccine candidate proteins and excretory-secretory proteins

Lagatie, Ole; Verheyen, Ann; Van Dorst, Bieke; Batsa Debrah, Linda; Debrah, Alex; Stuyver, Lieven J.
Parasite Immunol.
Nov 2018
10.1111/pim.12587
In our previous study, a proteome-wide screen was conducted to identify linear epitopes in this parasite’s proteome, resulting in the discovery of three immunodominant motifs. Here, we investigated whether such antigenic peptides were found in proteins that were already known as vaccine candidates and excretome/secretome proteins for Onchocerca volvulus This approach led to the identification of 71 immunoreactive stretches in 46 proteins. A deep-dive into the immunoreactivity profiles of eight vaccine candidates that were chosen as most promising candidates for further development (Ov-CPI-2, Ov-ALT-1, Ov-RAL-2, Ov-ASP-1, Ov-103, Ov-RBP-1, Ov-CHI-1, and Ov-B20), resulted in the identification of a poly-glutamine stretch in Ov-RAL-2 that has properties for use as a serodiagnostic marker for O. volvulus infection. A peptide ELISA was developed, and the performance of this assay was evaluated. Based on this assessment, it was found that this assay has a sensitivity of 75.0% [95% CI: 64.9%-83.5%] and a specificity of 98.5% [95% CI: 94.6%-99.8%]. Furthermore, 8.7% reactivity in Asian parasite-infected individuals (8 out of 92) was observed. Besides this identification of a linear epitope marker, the information on the presence of linear epitopes in vaccine candidate proteins might be useful in the study of vaccines for river blindness.

Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

Lee, Yeon-Ah; Hahm, Dae-Hyun; Kim, Jung Yeon; Sur, Bonjun; Lee, Hyun Min; Ryu, Chun Jeih; Yang, Hyung-In; Kim, Kyoung Soo
Arthritis Res Ther.
Oct 2018
10.1186/s13075-018-1736-3
Background Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. The purpose of this study was to generate monoclonal antibodies (mAbs) specific to different adiponectin isoforms and investigate whether these mAbs have potential as therapeutic agents for such diseases. Methods Hybridoma cells producing monoclonal antibodies were generated and screened using enzyme-linked immunosorbent assay and Western blotting for the production of mAbs recognizing human adiponectin isoforms. Results The mAb from hybridoma clone KH7–41 recognized both the middle molecular weight (MMW) (hexamer) and low molecular weight (LMW) (trimer) isoforms of adiponectin in human serum, whereas the KH7–33 mAb detected only MMW (hexamer) adiponectin. The KH4–8 clone recognized both the high molecular weight (HMW) (multimer) and MMW adiponectin isoforms. However, in mouse and rat sera, the abovementioned antibodies recognized only the MMW isomer. These mAbs also recognized adiponectin in various human tissues, such as lung, kidney, and adipose tissues, although the three mAbs had different staining intensities. The mAb from clone KH4–8 effectively inhibited increases in interleukin-6 (IL-6) and IL-8 expression in recombinant adiponectin-stimulated human osteoblasts and human umbilical vein endothelial cells. Also, the mAbs KH7–33 and KH4–8 significantly ameliorated rheumatic symptoms in a collagen-induced arthritis mouse model. This result suggests that these mAb treatments may ameliorate adiponectin-mediated inflammatory response. Conclusions mAbs against human adiponectin isomers can potentially be developed as therapeutic antibodies to target specific detrimental isoforms of adiponectin while maintaining the functions of beneficial isoforms.

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.

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