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

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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.

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.

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies: Peptide Arrays for Antibody Selectivity Studies

Vernet, Thierry; Choulier, Laurence; Nominé, Yves; Bellard, Laure; Baltzinger, Mireille; Travé, Gilles; Altschuh, Danièle
J. Mol. Recognit..
Oct 2015
10.1002/jmr.2477
Antibody selectivity represents a major issue in the development of efficient immuno-therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high-throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross-reactive capacity of an antibody fragment using the PEPperCHIP® technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore® technology; GE-Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N-terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence 6TAMFQDPQER15) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross-reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity.

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.

Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen

Schmidt, Ronny; Jacak, Jaroslaw; Schirwitz, Christopher; Stadler, Volker; Michel, Gerd; Marmé, Nicole; Schütz, Gerhard J.; Hoheisel, Jörg D.; Knemeyer, Jens-Peter
J. Proteome Res..
Jan 2011
10.1021/pr101070j

Combinatorial Peptide Synthesis on a Microchip

Schirwitz, Christopher; Block, Ines; König, Kai; Nesterov, Alexander; Fernandez, Simon; Felgenhauer, Thomas; Leibe, Klaus; Torralba, Gloria; Hausmann, Michael; Lindenstruth, Volker; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Current Protocols in Protein Science.
Aug 2009
10.1002/0471140864.ps1802s57
Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom-built microchips can be equipped with an amino-modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer-coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high-complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm2 can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background-free immunostaining on the chip-based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high-density peptide arrays.

Particle-Based Synthesis of Peptide Arrays

Breitling, Frank; Felgenhauer, Thomas; Nesterov, Alexander; Lindenstruth, Volker; Stadler, Volker; Bischoff, F. Ralf
ChemBioChem.
Mar 2009
10.1002/cbic.200800735
Lithographic methods allow for the combinatorial synthesis of >50,000 oligonucleotides per cm(2), and this has revolutionized the field of genomics. High-density peptide arrays promise to advance the field of proteomics in a similar way, but currently lag behind. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. Combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A color laser printer or a chip addresses the different charged particles consecutively to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids to the support in one single coupling reaction. The method should allow for the translation of entire genomes into sets of overlapping peptides to be used in proteome research.

High-density peptide arrays

Breitling, Frank; Nesterov, Alexander; Stadler, Volker; Felgenhauer, Thomas; Bischoff, F. Ralf
Mol. BioSyst..
Jan 2009
10.1039/b819850k
Arrays promise to advance biology by allowing parallel screening for many different binding partners. Meanwhile, lithographic methods enable combinatorial synthesis of >50 000 oligonucleotides per cm2, an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This review discusses recent developments in the field with an emphasis on methods that lead to very high-density peptide arrays.

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