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

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Replacing antibodies with modified DNA aptamers in vaccine potency assays

Trausch, Jeremiah J.; Shank-Retzlaff, Mary; Verch, Thorsten
Vaccine.
Oct 2017
10.1016/j.vaccine.2017.04.003
Vaccine in vitro potency assays are vital regulatory tests that are used to confirm the presence and concentration of an antigen of interest in a form that directly or indirectly relates to protective activity in patients. Current assays come in many forms, but they almost exclusively use antibody reagents for selective detection of the target antigen. Antibodies provide specific recognition of vaccine antigens but also exhibit drawbacks such as stability limitations, cost, and lot-to-lot variation, which can make it challenging to maintain the reagent throughout the lifetime of the vaccine. We explored replacing antibodies with aptamers. Aptamers are macromolecules, such as nucleic acids, which can bind to their targets with high specificity and affinity, similar to that of antibodies. Some of the advantages of using aptamers over antibodies is that aptamers can be more stable, smaller, less expensive to produce, synthesized in vitro, and logistically easier to supply throughout the multi-decade lifespan of a commercial vaccine. We created modified DNA aptamers against the common vaccine carrier protein, CRM197. Several aptamers were discovered and one was chosen for further characterization. The binding kinetics of the aptamer revealed an off-rate 16-fold slower than anti-CRM197 antibodies used for comparison. The aptamers were more sensitive than available antibodies in some assay formats and comparable in others. The aptamer epitope was mapped to the receptor-binding domain of CRM197, a site adjacent to a known antibody binding site. These data address some key aspects for a path forward in replacing antibodies with aptamers for use as critical reagents in vaccine assays. We further highlight the possibility of using nucleic acid reagents to develop next generation potency assays.

Anti-CYP4Z1 autoantibodies detected in breast cancer patients

Nunna, Venkatrao; Jalal, Nasir; Bureik, Matthias
Cell Mol Immunol.
Jun 2017
10.1038/cmi.2017.21

Peptide array functionalization via the Ugi four-component reaction

Ridder, B.; Mattes, D. S.; Nesterov-Mueller, A.; Breitling, F.; Meier, M. A. R.
Chem. Commun..
May 2017
10.1039/C7CC01945A
The Ugi four-component reaction was investigated as a tool for the functionalization of peptide arrays via post-synthetic side-chain modification, mimicking post-translational processes. Additionally, as a proof of concept for the synthesis of peptidomimetics on arrays, the integration of an Ugi unit into a growing peptide chain was demonstrated.

Acquired Factor XIII inhibitor associated with mantle cell lymphoma: ACQUIRED FXIII INHIBITOR

Nixon, Christian P.; Prsic, Elizabeth H.; Guertin, Christine A.; Stevenson, Ryan L.; Sweeney, Joseph D.
Transfusion.
Mar 2017
10.1111/trf.13947
BACKGROUND Acquired Factor (F)XIII deficiency is a very rare bleeding diathesis with a potentially fatal outcome, previously described in the context of autoimmune disorders and leukemias. There is minimal information on autoantibody characterization and the role of antifibrinolytic therapy in patient management. CASE REPORT A 79-year-old woman with a 3-month history of bruising and heavy menorrhagia presented with ongoing vaginal bleeding, symptomatic anemia, and a right thigh hematoma. Initial management included an axillary lymph node biopsy and coagulation evaluation. Pathologic examination of the biopsy specimen revealed mantle cell lymphoma. Clot solubility assay was consistent with a FXIII activity of less than 3%. An anti-FXIII inhibitor was suspected, the epitope specificity of which was mapped by micropeptide array analysis to regions in the β-sandwich and catalytic core domain of the FXIII-A subunit. Management with cryoprecipitate, steroids, rituximab, and antifibrinolytic therapy resolved the bleeding diathesis and suppressed the inhibitor. CONCLUSION This is the first reported case of an acquired FXIII inhibitor associated with mantle cell lymphoma in which the epitope specificity of the pathologic autoantibody was accurately defined. Antifibrinolytic therapy played a prominent role in the prevention of bleeding complications in the window period between initiation of immunosuppression and disappearance of the pathologic anti-FXIII autoantibody.

Autoantikehad täppismeditsiinis

Jaks, Viljar; Uibo, Raivo
Feb 2017
10.15157/EA.V0I0.13344
Immuuntolerantsi häirumine, mille üheks väljundiks on antikehade teke organismile omaste biomolekulide vastu, on oluline patogeneetiline mehhanism mitmete laialdaselt levinud haiguste puhul ja seetõttu on autoantikehade määramine kujunenud oluliseks diagnostiliseks vahendiks. Artiklis on käsitletud autoantikehade esinemise olulisust haiguste tekke ja kulu prognoosimisel. Kuigi sellekohane info on veel üsna napp, on selge, et organismi immuunstaatuse muutus eelneb aastaid haiguse ilmnemisele ning autoimmuunset komponenti sisaldava haiguse kulg ja prognoos on seotud patsiendil esinevate kindlate autoantikehadega. Sellest tulenevalt võime loota, et organismi immuunstaatuse uurimine, eriti aga autoantikehade spektri iseloomustamine, on tulevikus geneetilise info analüüsimise kõrval üks täppismeditsiini olulisemaid tööriistu.

Antibody repertoire profiling with mimotope arrays

Pashova, Shina; Schneider, Christoph; von Gunten, Stephan; Pashov, Anastas
Human Vaccines & Immunotherapeutics.
Jan 2017
10.1080/21645515.2017.1264786
Large-scale profiling and monitoring of antibody repertoires is possible through next generation sequencing (NGS), phage display libraries and microarrays. These methods can be combined in a pipeline, which ultimately maps the antibody reactivities onto defined arrays of structures – peptides or carbohydrates. The arrays can help analyze the individual specificities or can be used as complex patterns. In any case, the targets recognized should formally be considered mimotopes unless they are proven to be epitopes driving the antibody synthesis. Here, the advantages and disadvantages of the major profiling techniques as well as their current and future application in disease prediction and vaccination are discussed.

Non-invasive glioblastoma immunoprofiling by printed peptide arrays

Mock, Andreas; Herold-Mende, Christel
OncoImmunology.
Feb 2016
10.1080/2162402X.2015.1069941
Immune monitoring assays for patient stratification and treatment efficacy in clinical trials are in demand. We have recently described a cost-effective non-invasive assay to determine the immune status of glioblastoma patients. Profiling antitumor serum antibodies by customized printed peptide arrays identified response against a tenascin-C (TNC) peptide as a robust prognostic biomarker.

Autoantibodies specific to estrogen receptor alpha act as estrogen agonists and their levels correlate with breast cancer cell proliferation

Maselli, Angela; Capoccia, Sara; Pugliese, Patrizia; Raggi, Carla; Cirulli, Francesca; Fabi, Alessandra; Malorni, Walter; Pierdominici, Marina; Ortona, Elena
OncoImmunology.
Feb 2016
10.1080/2162402X.2015.1074375
Estrogen receptors have recently been demonstrated at the cell surface. Unlike nuclear receptors, they are able to trigger rapid responses inside the cells. In this study, we evaluated the presence and the possible role of autoantibodies specific to estrogen receptor (anti-ER Abs) in the peripheral blood of breast cancer patients. Anti-ERα Abs were detectable in 22/48 (46%) patients’ sera and their levels positively correlated with the percentage of Ki-67-positive breast cancer cells. Anti-ERα Abs purified from breast cancer patients’ sera were able: (i) to recognize ERα epitopes expressed at the cell surface of ER-positive breast cancer cells, (ii) to trigger rapid extracellular signal-regulated kinase (ERK) phosphorylation, and (iii) to induce cell proliferation. Our results suggest that anti-ERα Abs can act as estrogen agonists playing a pathogenetic role as breast cancer-promoting factors. These autoantibodies could also be considered as possible peripheral blood biomarkers indicative of the breast cancer growth potential.

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.

A Novel Combinatorial Approach to High-Density Peptide Arrays

Beyer, Mario; Block, Ines; König, Kai; Nesterov, Alexander; Fernandez, Simon; Felgenhauer, Thomas; Schirwitz, Christopher; Leibe, Klaus; Bischoff, Ralf F.; Breitling, Frank; Stadler, Volker
Jan 2009
10.1007/978-1-60327-394-7_16
Combinatorial synthesis of peptides on solid supports (1), either as spots on cellulose membranes (2) or with split-pool-libraries on polymer beads (3), substantially forwarded research in the field of peptide-protein interactions. Admittedly, these concepts have specific limitations, on one hand the number of synthesizable peptide sequences per area, on the other hand elaborate decoding/encoding strategies, false-positive results and sequence limitations. We recently established a method to produce high-density peptide arrays on microelectronic chips (4). Solid amino acid microparticles were charged by friction and transferred to defined pixel electrodes onto the chip’s surface, where they couple to a functional polymer coating simply upon melting (Fig. 16.1 A-D,F). By applying standard Fmoc chemistry according to Merrifield, peptide array densities of up to 40,000 spots per square centimetre were achieved (Fig. 16.1G). The term Merrifield synthesis describes the consecutive linear coupling and deprotecting of L-amino acids modified with base-labile fluorenylmethoxy (Fmoc) groups at the N-terminus and different acid-sensitive protecting groups at their side chains. Removing side chain protecting groups takes place only once at the very end of each synthesis and generates the natural peptide sequence thereby.

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