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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.

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.

Active vaccination against interleukin-5 as long-term treatment for insect-bite hypersensitivity in horses

Fettelschoss-Gabriel, Antonia; Fettelschoss, Victoria; Olomski, Florian; Birkmann, Katharina; Thoms, Franziska; Bühler, Maya; Kummer, Martin; Zeltins, Andris; Kündig, Thomas M.; Bachmann, Martin F.
Allergy.
Mar 2019
10.1111/all.13659
Background Insect-bite hypersensitivity (IBH) in horses is a chronic allergic dermatitis caused by insect bites. Horses suffer from pruritic skin lesions, caused by type-I/type-IV allergic reactions accompanied by prominent eosinophil infiltration into the skin. Interleukin-5 (IL-5) is the key cytokine for eosinophils and we have previously shown that targeting IL-5 by vaccination reduces disease symptoms in horses. Objective Here, we analyzed the potential for long-term therapy by assessing a second follow-up year of the previously published study. Methods The vaccine consisted of equine IL-5 (eIL-5) covalently linked to a cucumber mosaic virus-like particle (VLP) containing a universal T cell epitope (CuMVTT) using a semi-crossover design to follow vaccinated horses during a second treatment season. Thirty Icelandic horses were immunized with 300 μg of eIL-5-CuMVTT without adjuvant. Results The vaccine was well tolerated and did not reveal any safety concerns throughout the study. Upon vaccination, all horses developed reversible anti-eIL-5 auto-antibody titers. The mean course of eosinophil levels was reduced compared to placebo treatment leading to significant reduction of clinical lesion scores. Horses in their second vaccination year showed a more pronounced improvement of disease symptoms when compared to first treatment year, most likely due to more stable antibody titers induced by a single booster injection. Hence, responses could be maintained over two seasons and the horses remained protected against disease symptoms. Conclusion Yearly vaccination against IL-5 may be a long-term solution for the treatment of IBH and other eosinophil-mediated diseases in horses and other species including humans.

ANTIBODY TARGETING CELL SURFACE DEPOSITED COMPLEMENT PROTEIN C3d AND USE THEREOF

Wiestner, Adrian U.; Skarzynski, Martin W.; Lindorfer, Margaret A.; Taylor, Ronald P.; Rader, Christoph; Vire, Berengere
Feb 2019
An anti-C3d antibody or antibody fragment; method for use thereof to kill cancer cells; and related methods and compositions.

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.

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.

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.

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.

Gdf-15 as a diagnostic marker to predict the clinical outcome of a treatment with immune checkpoint blockers

WISCHHUSEN, Jörg; Haake, Markus; DUMMER, Reinhard; MEHLING, Matthias
Aug 2018
The present invention relates to methods for predicting the probability of a treatment response of a human cancer patient to an immune checkpoint blocker treatment e.g. with anti PD-1, and to methods for predicting the probability of survival of a human cancer patient following an immune checkpoint blocker treatment, and to apparatuses and kits which can be used in these methods.

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