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

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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.

Anti-Trail Antibodies and Methods of Use

Boico, Olga; Tzaban, Salit; Oved, Kfir; Cohen-Dotan, Assaf; Eden, Eran
May 2019
An antibody comprising an antigen recognition domain that binds specifically the extracellular domain of TNF-related apoptosis-inducing ligand (TRAIL) between amino acids 95-155 and/or amino acids 190-210 is disclosed. Uses thereof are also disclosed.

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.

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.

An oligoclonal combination of human monoclonal antibodies able to neutralize tetanus toxin in vivo

Aliprandini, Eduardo; Takata, Daniela Yumi; Lepique, Ana; Kalil, Jorge; Boscardin, Silvia Beatriz; Moro, Ana Maria
Toxicon: X.
Jan 2019
10.1016/j.toxcx.2019.100006
The use of antibody-based therapy to treat a variety of diseases and conditions is well documented. The use of antibodies as an antidote to treat tetanus infections was one of the first examples of immunotherapy and remains the standard of care for cases involving potential infections. Plasma-derived immunoglobulins obtained from human or horse pose risks of infection from undetectable emergent viruses or may cause anaphylaxis. Further, there is a lack of consistency between lots. In the search for new formulations, we obtained a series of clonally related human monoclonal antibodies (mAbs) derived from B cells sorted from donors that presented anti-tetanus neutralizing titers. Donors were revaccinated prior to blood collection. Different strategies were used for single-cell sorting, since it was challenging to identify cells at a very low frequency: memory B cell sorting using fluorescent-labeled tetanus toxoid and toxin as baits, and plasmablast sorting done shortly after revaccination. Screening of the recombinant mAbs with the whole tetanus toxin allowed us to select candidates with therapeutic potential, since mAbs to different domains can contribute additively to the neutralizing effect. Because of selective binding to different domains, we tested mAbs individually, or in mixtures of two or three, in the neutralizing in vivo assay specified by Pharmacopeia for the determination of polyclonal hyperimmune sera potency. An oligoclonal mixture of three human mAbs completely neutralized the toxin injected in the animals, signaling an important step for clinical mAb development.

Novel targets of acinetobacter baumannii

Urwyler, Simon; Haake, Markus; Rudolf, Michael
Jan 2019
The present invention provides antigenic polypeptides expressed during an infection by a pathogenic organism, such as Acinetobacter and compositions comprising these polypeptides. The invention further provides compositions for use in treating, preventing or detecting a bacterial infection, in particular vaccine compositions using the antigenic polypeptides. The invention further provides antibodies directed to said antigenic polypeptides.

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.

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.

Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin

Aliprandini, Eduardo; Tavares, Joana; Panatieri, Raquel Hoffmann; Thiberge, Sabine; Yamamoto, Marcio Massao; Silvie, Olivier; Ishino, Tomoko; Yuda, Masao; Dartevelle, Sylvie; Traincard, François; Boscardin, Silvia Beatriz; Amino, Rogerio
Nat Microbiol.
Oct 2018
10.1038/s41564-018-0254-z
The circumsporozoite protein (CSP) is the major surface protein of malaria sporozoites (SPZs), the motile and invasive parasite stage inoculated in the host skin by infected mosquitoes. Antibodies against the central CSP repeats of different plasmodial species are known to block SPZ infectivity, but the precise mechanism by which these effectors operate is not completely understood. Here, using a rodent Plasmodium yoelii malaria model, we show that sterile protection mediated by anti-P. yoelii CSP humoral immunity depends on the parasite inoculation into the host skin, where antibodies inhibit motility and kill P. yoelii SPZs via a characteristic ‘dotty death’ phenotype. Passive transfer of an anti-repeat monoclonal antibody (mAb) recapitulates the skin inoculation-dependent protection, in a complement- and Fc receptor γ-independent manner. This purified mAb also decreases motility and, notably, induces the dotty death of P. yoelii SPZs in vitro. Cytotoxicity is species-transcendent since cognate anti-CSP repeat mAbs also kill Plasmodium berghei and Plasmodium falciparum SPZs. mAb cytotoxicity requires the actomyosin motor-dependent translocation and stripping of the protective CSP surface coat, rendering the parasite membrane susceptible to the SPZ pore-forming-like protein secreted to wound and traverse the host cell membrane. The loss of SPZ fitness caused by anti-P. yoelii CSP repeat antibodies is thus a dynamic process initiated in the host skin where SPZs either stop moving, or migrate and traverse cells to progress through the host tissues at the eventual expense of their own life.

Interaction of the Warsaw breakage syndrome DNA helicase DDX11 with the replication fork-protection factor Timeless promotes sister chromatid cohesion

Cortone, Giuseppe; Zheng, Ge; Pensieri, Pasquale; Chiappetta, Viviana; Tatè, Rosarita; Malacaria, Eva; Pichierri, Pietro; Yu, Hongtao; Pisani, Francesca M.
PLoS Genet.
Oct 2018
10.1371/journal.pgen.1007622
Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS.

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