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

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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.

Performance evaluation of 3 serodiagnostic peptide epitopes and the derived multi-epitope peptide OvNMP-48 for detection of Onchocerca volvulus infection

Lagatie, Ole; Verheyen, Ann; Nijs, Erik; Batsa Debrah, Linda; Debrah, Yaw A.; Stuyver, Lieven J.
Parasitol Res.
Jul 2019
10.1007/s00436-019-06345-3
Current diagnostic tools to determine infection with the helminth parasite Onchocerca volvulus have limited performance characteristics. In previous studies, a proteome-wide screen was conducted to identify linear epitopes in this parasite’s proteome, resulting in the discovery of 1110 antigenic peptide fragments. Here, we investigated three of these peptides using peptide ELISA’s and evaluated their sensitivity and specificity. Epitope mapping was performed, and peptides were constructed that contained only the minimal epitope, flanked by a linker. Investigation of the performance of these minimal epitope peptides demonstrated that all three of them have a specificity (as defined by lack of response in non-helminth-infected individuals) of 100%, low cross-reactivity (5.6%, 5.6%, and 9.3%, respectively), but low sensitivity (36.9%, 46.5%, and 41.2%, respectively). Some cross-reactivity was observed in samples from individuals infected with soil-transmitted helminths or Brugia malayi. Combining these three minimal epitopes in a single peptide, called OvNMP-48, resulted in a performance that exceeded the sum of the individual epitopes, with a sensitivity of 76.0%, a specificity of 97.4%, and a cross-reactivity of 11.1%. Cross-reactivity was observed in some STH and Brugia malayi-infected individuals. This work opens the opportunity to start exploring how these novel linear epitope markers might become part of the O. volvulus diagnostic toolbox.

E2814: A NOVEL ANTI-TAU THERAPEUTIC ANTIBODY FOR ALZHEIMER’S DISEASE

Roberts, Malcolm; Sevastou, Ioanna; Imaizumi, Yoichi; Mistry, Kavita; Talma, Sonia; Dey, Madhurima; Gartlon, Jane; Ochiai, Hiroshi; Zhou, Zhi; Akasofu, Shigeru; Tokuhara, Naoki; Ogo, Makoto; Strand, Catherine; Agarwala, Kishan; Spidel, Jared; Albone, Earl; Horie, Kanta; Staddon, James; De Silva, Rohan
Alzheimer's & Dementia.
Jul 2019
10.1016/j.jalz.2019.09.059

LRPAP1 is a frequent proliferation-inducing antigen of BCRs of mantle cell lymphomas and can be used for specific therapeutic targeting

Thurner, Lorenz; Hartmann, Sylvia; Fadle, Natalie; Kemele, Maria; Bock, Theresa; Bewarder, Moritz; Regitz, Evi; Neumann, Frank; Nimmesgern, Anna; von Müller, Lutz; Pott, Christiane; Kim, Yoo-Jin; Bohle, Rainer Maria; Wasik, Mariusz; Schuster, Stephen J.; Hansmann, Martin-Leo; Preuss, Klaus-Dieter; Pfreundschuh, Michael
Leukemia.
Jun 2019
10.1038/s41375-018-0182-1
The predominant usage of VH4-34 and V3-21 and reports of stereotyped CDR3s suggest a shared antigenic target of B-cell receptors (BCR) from mantle cell lymphomas (MCL). To identify the target antigens of MCL–BCRs, BCRs from 21 patients and seven MCL cell lines were recombinantly expressed and used for antigen screening. The BCRs from 8/21 patients and 2/7 MCL cell lines reacted specifically with the autoantigen low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1). High-titered and light chain-restricted anti-LRPAP1 serum antibodies were found in MCL patients, but not in controls. LRPAP1 induced proliferation by BCR pathway activation, while an LRPAP1–ETA′ toxin-conjugate specifically killed MCL cells with LRPAP1-specific BCRs. Our results suggest a role of LRPAP1 in lymphomagenesis and maintenance of a considerable proportion of MCL cases by chronic autoantigenic stimulation, likely evolving from a chronic autoreactive B-cell response. Importantly, LRPAP1 can be used for a novel therapeutic approach that targets MCL with LRPAP1-reactive BCRs with high specificity.

Miniaturized and Automated Synthesis of Biomolecules—Overview and Perspectives

Mattes, Daniela S.; Jung, Nicole; Weber, Laura K.; Bräse, Stefan; Breitling, Frank
Adv. Mater..
Jun 2019
10.1002/adma.201806656
Chemical synthesis is performed by reacting different chemical building blocks with defined stoichiometry, while meeting additional conditions, such as temperature and reaction time. Such a procedure is especially suited for automation and miniaturization. Life sciences lead the way to synthesizing millions of different oligonucleotides in extremely miniaturized reaction sites, e.g., pinpointing active genes in whole genomes, while chemistry advances different types of automation. Recent progress in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging could match miniaturized chemical synthesis with a powerful analytical tool to validate the outcome of many different synthesis pathways beyond applications in the life sciences. Thereby, due to the radical miniaturization of chemical synthesis, thousands of molecules can be synthesized. This in turn should allow ambitious research, e.g., finding novel synthesis routes or directly screening for photocatalysts. Herein, different technologies are discussed that might be involved in this endeavor. A special emphasis is given to the obstacles that need to be tackled when depositing tiny amounts of materials to many different extremely miniaturized reaction sites.

Clinical expression and antigenic profiles of a Plasmodium vivax vaccine candidate: merozoite surface protein 7 (PvMSP-7)

Cheng, Chew Weng; Jongwutiwes, Somchai; Putaporntip, Chaturong; Jackson, Andrew P.
Malar J.
Jun 2019
10.1186/s12936-019-2826-7
Background Vivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates. Methods Merozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs. Results Ten patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients. Conclusions These results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A.

Identification of Two Distinct Linear B Cell Epitopes of the Matrix Protein of the Newcastle Disease Virus Vaccine Strain LaSota

Bi, Youkun; Jin, Zhongyuan; Wang, Yanhong; Mou, Sujing; Wang, Wenbin; Wei, Qiaolin; Huo, Na; Liu, Siqi; Wang, Xinglong; Yang, Zengqi; Chen, Hongjun; Xiao, Sa
Viral Immunology.
Jun 2019
10.1089/vim.2019.0007
Matrix (M) protein of Newcastle disease virus (NDV) is an abundant protein that can induce a robust humoral immune response. However, its antigenic epitopes remain unknown. In this study, we used a pepscan approach to map linear B cell immunodominant epitopes (IDEs) of M protein with NDV-specific chicken antisera. The six epitopes with the highest reactivity by peptide scanning were obtained as IDE candidates. Among them, aa71–85 and aa349–363 were identified by immunological assays with NDV-specific or IDE-specific antisera. The minimal antigenic epitopes of the two IDEs were further characterized as 77MIDDKP82 and 354HTLAKYNPFK363. Moreover, an amino acid sequence alignment and immunoblot analysis revealed the conservation of the two IDEs in the M protein of strains of different genotypes. These two IDEs of M protein could be genetically eliminated as negative markers in recombinant NDV for serologically differential diagnosis in the development of marker vaccines.

A high-sensitivity enzyme immunoassay for the quantification of soluble human semaphorin 4D in plasma

Laber, Anna; Gadermaier, Elisabeth; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
Analytical Biochemistry.
Jun 2019
10.1016/j.ab.2019.03.004
Human semaphorin 4D (SEMA4D), a type I integral membrane glycoprotein, regulates key cellular functions (e.g. cell-cell communication, platelet activation). Its 120 kDa extracellular region can be shed from the membrane to release soluble SEMA4D (sSEMA4D). Studies on circulating sSEMA4D levels are mostly performed with poorly characterized assays and use serum and plasma as matrix. We developed and validated a sandwich ELISA utilizing two monoclonal antibodies with resolved epitopes and determined affinities. Human serum and plasma samples were analyzed, and the influence of protease activity on sSEMA4D concentration was tested by collecting samples in the presence of the protease inhibitor TAPI-1. Both antibodies recognize conformational epitopes in the sema domain. Validation for plasma (EDTA, citrate, heparin) showed valid specificity, precision, accuracy, dilution linearity, and robustness. The assay shows a calibration range from 62.5 to 2000 pmol/L with a quantification limit of 31 pmol/L. sSEMA4D was significantly higher in serum than in plasma, whereas serum and plasma levels from samples collected in the presence of TAPI-1 showed no statistical difference. This ELISA provides a reliable tool for the quantification of sSEMA4D in human plasma. Serum is not recommended as matrix due to the accumulation of shed SEMA4D during blood coagulation altering serum sSEMA4D levels.

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.

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