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

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Antibody Properties Associate with Clinical Phenotype in LGI1 Encephalitis

Ludewig, Susann; Salzburger, Leonie; Goihl, Alexander; Rohne, Jana; Leypoldt, Frank; Bittner, Daniel; Düzel, Emrah; Schraven, Burkhart; Reinhold, Dirk; Korte, Martin; Körtvélyessy, Péter
Cells.
Jan 2023
10.3390/cells12020282
Autoimmune encephalitis (AE) associated with autoantibodies against leucine-rich glioma-inactivated protein-1 (LGI1) can present with faciobrachial dystonic seizures (FBDS) and/or limbic encephalitis (LE). The reasons for this heterogeneity in phenotypes are unclear. We performed autoantibody (abs) characterization per patient, two patients suffering from LE and two from FBDS, using isolated antibodies specified with single amino acid epitope mapping. Electrophysiological slice recordings were conducted alongside spine density measurements, postsynaptic Alpha-amino-3-hydoxy-5-methyl-4-isoaxole-proprionate-receptors (AMPA-R) and N-methyl-D-aspartate-receptors receptor (NMDA-R) cluster counting. These results were correlated with the symptoms of each patient. While LGI1 abs from LE patients mainly interacted with the Leucine-rich repeat section of LGI1, abs from both FBDS patients also recognized the Epitempin section as well. Six-hour incubation of mouse hippocampal slices with LE patients autoantibodies but not from the FBDS patients resulted in a significant decline in long-term potentiation (p = 0.0015) or short-term plasticity at CA3-CA1 neurons and in decreased hippocampal synaptic density. Cluster differentiation showed no decrease in postsynaptic AMPA-R and NMDA-R. LGI1 autoantibodies selected by phenotype show an almost distinct epitope pattern, elicit disparate functional effects on hippocampal neurons, and cause divergent effects on spine density. This data illuminates potential pathomechanisms for disease heterogeneity in LGI1 AE.

Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape

Oliveira, Jamille Ramos; Ruiz, Cesar Manuel Remuzgo; Machado, Rafael Rahal Guaragna; Magawa, Jhosiene Yukari; Daher, Isabela Pazotti; Urbanski, Alysson Henrique; Schmitz, Gabriela Justamante Händel; Arcuri, Helen Andrade; Ferreira, Marcelo Alves; Sasahara, Greyce Luri; de Medeiros, Giuliana Xavier; Júnior, Roberto Carlos Vieira Silva; Durigon, Edison Luiz; Boscardin, Silvia Beatriz; Rosa, Daniela Santoro; Schechtman, Deborah; Nakaya, Helder I.; Cunha-Neto, Edecio; Gadermaier, Gabriele; Kalil, Jorge; Coelho, Verônica; Santos, Keity Souza
Front. Immunol..
Jan 2023
10.3389/fimmu.2022.1010105
Introduction Considering the likely need for the development of novel effective vaccines adapted to emerging relevant CoV-2 variants, the increasing knowledge of epitope recognition profile among convalescents and afterwards vaccinated with identification of immunodominant regions may provide important information. Methods We used an RBD peptide microarray to identify IgG and IgA binding regions in serum of 71 COVID-19 convalescents and 18 vaccinated individuals. Results We found a set of immunodominant RBD antibody epitopes, each recognized by more than 30% of the tested cohort, that differ among the two different groups and are within conserved regions among betacoronavirus. Of those, only one peptide, P44 (S415-429), recognized by 68% of convalescents, presented IgG and IgA antibody reactivity that positively correlated with nAb titers, suggesting that this is a relevant RBD region and a potential target of IgG/IgA neutralizing activity. Discussion This peptide is localized within the area of contact with ACE-2 and harbors the mutation hotspot site K417 present in gamma (K417T), beta (K417N), and omicron (K417N) variants of concern. The epitope profile of vaccinated individuals differed from convalescents, with a more diverse repertoire of immunodominant peptides, recognized by more than 30% of the cohort. Noteworthy, immunodominant regions of recognition by vaccinated coincide with mutation sites at Omicron BA.1, an important variant emerging after massive vaccination. Together, our data show that immune pressure induced by dominant antibody responses may favor hotspot mutation sites and the selection of variants capable of evading humoral response.

Deciphering the Autoantibody Response to the OJ Antigenic Complex

Fritzler, Marvin J.; Bentow, Chelsea; Satoh, Minoru; McHugh, Neil; Ghirardello, Anna; Mahler, Michael
Diagnostics.
Jan 2023
10.3390/diagnostics13010156
(1) Background: Myositis specific antibodies (MSA) are important diagnostic biomarkers. Among the rarest and most challenging MSA are anti-OJ antibodies which are associated with anti-synthetase syndrome (ASS). In contrast to the other tRNA synthetases that are targets of ASS autoantibodies (e.g Jo-1, PL-7, PL-12, EJ, KS, Zo), OJ represents a macromolecular complex with several ribonucleoprotein subunits. Therefore, the choice of the antigen in autoantibody assays can be challenging. (2) Methods: We collected two independent cohorts with anti-OJ antibodies, one based on a commercial line immunoassay (LIA) (n = 39), the second based on protein immunoprecipitation (IP) (n = 15). Samples were tested using a particle-based multi-analyte technology (PMAT) system that allows for the simultaneous detection of antibodies to various autoantigens. For the detection of anti-OJ antibodies, two different antigens were deployed (KARS, IARS) on PMAT. The reactivity to the two antigens KARS and IARS was analyzed individually and combined in a score (sum of the median fluorescence intensities). (3) Results: In the cohort selection based on LIA, 3/39 (7.7%) samples were positive for anti-KARS and 7/39 (17.9%) for anti-IARS and 14/39 (35.9%) when the two antigens were combined. In contrast, in samples selected by IP the sensitivity of anti-KARS was higher: 6/15 (40.0%) samples were positive for anti-KARS, 4/15 (26.7%) for anti-IARS and 12/15 (80.0%) for the combination of the two antigens. 18/39 (46.2%) of the LIA samples generated a cytoplasmic IIF pattern (compatible with anti-synthetase antibodies), but there was no association with the antibody levels, neither with LIA nor with PMAT. (4) Conclusions: The combination of IARS and KARS might represent a promising approach for the detection of anti-OJ antibodies on a fully automated platform.

Potent Adjuvanticity of a Pure TLR7-Agonistic Imidazoquinoline Dendrimer

Shukla, Nikunj M.; Salunke, Deepak B.; Balakrishna, Rajalakshmi; Mutz, Cole A.; Malladi, Subbalakshmi S.; David, Sunil A.
PLoS ONE.
Aug 2012
10.1371/journal.pone.0043612
Engagement of toll-like receptors (TLRs) serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. We synthesized a dendrimeric molecule bearing six units of a potent TLR7/TLR8 dual-agonistic imidazoquinoline to explore if multimerization of TLR7/8 would result in altered activity profiles. A complete loss of TLR8-stimulatory activity with selective retention of the TLR7-agonistic activity was observed in the dendrimer. This was reflected by a complete absence of TLR8-driven proinflammatory cytokine and interferon (IFN)-γ induction in human PBMCs, with preservation of TLR7-driven IFN-α induction. The dendrimer was found to be superior to the imidazoquinoline monomer in inducing high titers of high-affinity antibodies to bovine α-lactalbumin. Additionally, epitope mapping experiments showed that the dendrimer induced immunoreacti

Sensing Immune Responses with Customized Peptide Microarrays

Schirwitz, Christopher; Loeffler, Felix F.; Felgenhauer, Thomas; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Biointerphases.
Aug 2012
10.1007/s13758-012-0047-5
The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding. A new technology to produce peptide microarrays with a laser printer provides access to affordable and highly complex peptide microarrays. Such a peptide microarray can contain up to 775 peptide spots per cm², whereby the position of each peptide spot and, thus, the amino acid sequence of the corresponding peptide, is exactly known. Compared to other techniques, such as the SPOT synthesis, more features per cm² at lower costs can be synthesized which paves the way for laser printed peptide microarrays to take on roles as efficient and affordable biomedical sensors. Here, we describe the laser printer-based synthesis of peptide microarrays and focus on an application involving the blood sera of tetanus immunized individuals, indicating the potential of peptide arrays to sense immune responses.

Physical Characterization of the “Immunosignaturing Effect”

Stafford, Phillip; Halperin, Rebecca; Legutki, Joseph Bart; Magee, Dewey Mitchell; Galgiani, John; Johnston, Stephen Albert
Mol Cell Proteomics.
Apr 2012
10.1074/mcp.M111.011593
Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer’s disease display a characteristic and reproducible immunosignature on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.

Combinatorial Synthesis of Peptide Arrays onto a Microchip

Beyer, M.; Nesterov, A.; Block, I.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F.
Science.
Dec 2007
10.1126/science.1149751
Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.

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