Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

• MeSH
• alkylace MeSH
• footprinting proteinů metody   MeSH
• halogenace MeSH
• imunokomplex chemie   MeSH
• lidé MeSH
• mapování epitopu * metody   MeSH
• monoklonální protilátky chemie   imunologie   MeSH
• receptor erbB-2 * chemie   imunologie   MeSH
• trastuzumab * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cells play a key role in the adaptive antiviral immune response by killing infected cells and facilitating the selection of virus-specific antibodies. However, neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T-cell response nor the diversity of resulting immune memory is well understood. In this study, we use longitudinal high-throughput T-cell receptor (TCR) sequencing to track changes in the T-cell repertoire following two mild cases of COVID-19. In both donors, we identified CD4+ and CD8+ T-cell clones with transient clonal expansion after infection. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones and show preferential occurrence of these motifs in publicly available large dataset of repertoires from COVID-19 patients. We show that in both donors, the majority of infection-reactive clonotypes acquire memory phenotypes. Certain T-cell clones were detected in the memory fraction at the pre-infection time point, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.

• Klíčová slova
• COVID-19, RepSeq, SARS-CoV-2, TCR, computational biology, human, immunology, inflammation, systems biology,
• MeSH
• COVID-19 imunologie   patofyziologie   MeSH
• genová knihovna MeSH
• imunologická paměť * MeSH
• lidé MeSH
• longitudinální studie MeSH
• mapování epitopu MeSH
• receptory antigenů T-buněk chemie   genetika   MeSH
• SARS-CoV-2 fyziologie   MeSH
• sekvence aminokyselin MeSH
• stupeň závažnosti nemoci MeSH
• T-lymfocyty imunologie   MeSH
• testování histokompatibility MeSH
• zkřížené reakce MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH

In current work, we used recombinant OspC protein derived from B. afzelii strain BRZ31 in the native homodimeric fold for mice immunization and following selection process to produce three mouse monoclonal antibodies able to bind to variable parts of up to five different OspC proteins. Applying the combination of mass spectrometry assisted epitope mapping and affinity based theoretical prediction we have localized regions responsible for antigen-antibody interactions and approximate epitopes' amino acid composition. Two mAbs (3F4 and 2A9) binds to linear epitopes located in previously described immunogenic regions in the exposed part of OspC protein. The third mAb (2D1) recognises highly conserved discontinuous epitope close to the ligand binding domain 1.

• Klíčová slova
• Borrelia burgdorferi, OspC, epitope mapping, immunoprecipitation, monoclonal antibody, outer surface protein C, protein alignment,
• MeSH
• antigeny bakteriální chemie   genetika   imunologie   MeSH
• Borrelia burgdorferi chemie   genetika   imunologie   MeSH
• mapování epitopu * MeSH
• multimerizace proteinu * MeSH
• myši inbrední BALB C MeSH
• myší monoklonální protilátky chemie   imunologie   MeSH
• myši MeSH
• proteiny vnější bakteriální membrány chemie   genetika   imunologie   MeSH
• sbalování proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• myší monoklonální protilátky MeSH
• OspC protein MeSH Prohlížeč
• proteiny vnější bakteriální membrány MeSH

The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD-antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD-scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.

• Klíčová slova
• CD44, Epitope mapping, MEM-85, Monoclonal antibody, NMR, SAXS,
• MeSH
• antigeny CD44 chemie   MeSH
• Jurkat buňky MeSH
• kyselina hyaluronová chemie   MeSH
• lidé MeSH
• mapování epitopu MeSH
• molekulární modely MeSH
• monoklonální protilátky chemie   MeSH
• mutace MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD44 MeSH
• kyselina hyaluronová MeSH
• monoklonální protilátky MeSH

BACKGROUND: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is generally recognized as a diagnostic and therapeutic cancer antigen and a molecular address for targeted imaging and drug delivery studies. Due to its significance in cancer research, numerous monoclonal antibodies (mAbs) against GCPII have been described and marketed in the past decades. Unfortunately, some of these mAbs are poorly characterized, which might lead to their inappropriate use and misinterpretation of the acquired results. METHODS: We collected the 13 most frequently used mAbs against GCPII and quantitatively characterized their binding to GCPII by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Using a peptide library, we mapped epitopes recognized by a given mAb. Finally, we assessed the applicability of these mAbs to routine experimental setups, including Western blotting, immunohistochemistry, and flow cytometry. RESULTS: ELISA and SPR analyses revealed that mAbs J591, J415, D2B, 107-1A4, GCP-05, and 2G7 bind preferentially to GCPII in native form, while mAbs YPSMA-1, YPSMA-2, GCP-02, GCP-04, and 3E6 bind solely to denatured GCPII. mAbs 24.4E6 and 7E11-C5.3 recognize both forms of GCPII. Additionally, we determined that GCP-02 and 3E6 cross-react with mouse GCPII, while GCP-04 recognizes GCPII and GCPIII proteins from both human and mouse. CONCLUSION: This comparative analysis provides the first detailed quantitative characterization of the most commonly used mAbs against GCPII and can serve as a guideline for the scientific community to use them in a proper and efficient way.

• Klíčová slova
• ELISA, NAALADase, Western blot, flow cytometry, folate hydrolase, glutamate carboxypeptidase II, immunohistochemistry, prostate-specific membrane antigen, surface plasmon resonance,
• MeSH
• adenokarcinom diagnóza   imunologie   metabolismus   MeSH
• ELISA MeSH
• imunohistochemie MeSH
• lidé MeSH
• mapování epitopu MeSH
• monoklonální protilátky analýza   imunologie   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty diagnóza   imunologie   metabolismus   MeSH
• prostatický specifický antigen imunologie   metabolismus   MeSH
• průtoková cytometrie MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• monoklonální protilátky MeSH
• prostatický specifický antigen MeSH

Microtubules formed by αβ-tubulin dimers represent cellular structures that are indispensable for the maintenance of cell morphology and for cell motility generation. Microtubules in intact cells are in highly regulated equilibrium with cellular pools of soluble tubulin dimers. Sensitive, reproducible and rapid assays are necessary to monitor tubulin changes in cytosolic pools after treatment with anti-mitotic drugs, during the cell cycle or activation and differentiation events. Here we describe new assays for α-tubulin quantification. The assays are based on sandwich ELISA, and the signal is amplified with biotinyl-tyramide or immuno-PCR. Matching monoclonal antibody pair recognizes phylogenetically highly conserved epitopes localized outside the C-terminal isotype-defining region. This makes it possible to detect α-tubulin isotypes in different cell types of various species. Biotinyl-tyramide amplification and immuno-PCR amplification enable detection of tubulin at concentrations 2.5ng/ml and 0.086ng/ml, respectively. Immuno-PCR detection shows enhanced sensitivity and wider dynamic range when compared to ELISA with biotinyl-tyramide detection. Our results on taxol-treated and activated bone marrow-derived mast cells demonstrate, that the assays allow sensitive quantification of tubulin in complex biological fluids.

• Klíčová slova
• 3,3′,5,5′-tetramethylbenzidine., BMMC, BSA, BSS, Biotinyl-tyramide, C(q), ELISA, Immuno-PCR, Mast cells, PBS, PCR, TBS, TBS containing 0.05% Tween 20, TBST, TMB, Tris buffer solution, bone marrow-derived mast cell, bovine serum albumin, buffered saline solution, enzyme-linked immunosorbent assay, phosphate buffer solution, polymerase chain reaction, quantification cycle, α-Tubulin isotypes,
• MeSH
• biotin analogy a deriváty   MeSH
• buněčné linie MeSH
• ELISA metody   statistika a číselné údaje   MeSH
• mapování epitopu MeSH
• mastocyty účinky léků   metabolismus   MeSH
• monoklonální protilátky MeSH
• myši MeSH
• paclitaxel farmakologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• protein - isoformy analýza   genetika   imunologie   MeSH
• thapsigargin farmakologie   MeSH
• tubulin analýza   genetika   imunologie   MeSH
• tyramin analogy a deriváty   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biotin MeSH
• biotinyltyramide MeSH Prohlížeč
• monoklonální protilátky MeSH
• paclitaxel MeSH
• protein - isoformy MeSH
• thapsigargin MeSH
• tubulin MeSH
• tyramin MeSH

The aim of this work is to identify antigenic regions within the ORF1 protein of Torque teno sus virus 1 (TTSuV1) and Torque teno virus sus 2 (TTSuV2) that could be used as antigens to detect virus-specific antibodies following infection in pigs. Protein sequences of TTSuV ORF1 genes were analysed to predict linear antigenic epitopes. Synthesized peptides were analysed for serological reactivity with swine sera. Such an antigenic region was identified at the C terminus of the ORF1 protein of both viruses and showed serological reactivity with 78 % (TTSuV1) and 88 % (TTSuV2) of swine sera. An ELISA with an immunodominant peptide as antigen was used to examine the sera of piglets, aged 4-20 weeks, and adults. Results indicated that TTSuV1- and TTSuV2-specific antibodies were detectable at 4 weeks. Antibody titres increased from week 10 and peaked at week 20. A relatively high antibody titre persisted to adulthood.

• MeSH
• infekce DNA virem imunologie   veterinární   virologie   MeSH
• mapování epitopu MeSH
• molekulární sekvence - údaje MeSH
• nemoci prasat imunologie   virologie   MeSH
• prasata MeSH
• protilátky virové imunologie   MeSH
• sekvence aminokyselin MeSH
• Torque teno virus chemie   genetika   imunologie   MeSH
• virové plášťové proteiny chemie   genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protilátky virové MeSH
• virové plášťové proteiny MeSH

High titers of anti-carbonic anhydrase I (anti-CA I) autoantibodies were detected in the sera of patients with malignancies who developed an aplastic anemia-like (AA-like) syndrome after a high-dose therapy (HDT) and autologous stem cell transplantation (ASCT). It was found, that the presence of these anti-CA I autoantibodies is associated with spontaneous tumor regression. The main immunodominant epitopes of carbonic anhydrase isoform I (CA I) have previously been identified using epitope extraction technique in combination with mass spectrometric detection and bioinformatic verification. Similarly, the sera of patients with bona fide aplastic anemia (AA) who poorly responded to immunosuppressive treatment with anti-thymocyte globulin (ATG) demonstrated high titers of anti-CA I antibodies. In order to reveal differences between these antibodies, we applied the same methodology of epitope mapping procedure. Surprisingly, the anti-CA I antibodies from the both groups of patients compatibly recognized the same four candidate CA I epitopes--DGLAV, NVGHS, SLKPI, SSEQL. This finding may indicate common pathophysiological mechanisms in these two syndromes. However, at this moment it remains unresolved if anti-CA I antibodies are implicated in marrow or tumor suppression or are just an epi-phenomenon.

• Klíčová slova
• Anti-CA I autoantibodies, Aplastic anemia, Carbonic anhydrase I, Epitope extraction, LC–MS/MS,
• MeSH
• aplastická anemie farmakoterapie   imunologie   MeSH
• autoprotilátky krev   imunologie   MeSH
• epitopy imunologie   MeSH
• imunoglobulin G imunologie   MeSH
• karboanhydrasa I imunologie   MeSH
• lidé MeSH
• mapování epitopu MeSH
• nádory farmakoterapie   imunologie   MeSH
• transplantace kmenových buněk škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoprotilátky MeSH
• epitopy MeSH
• imunoglobulin G MeSH
• karboanhydrasa I MeSH

BACKGROUND: The function of the cortical microtubules, composed of alphabeta-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited. RESULTS: Using homology modeling we have generated an Arabidopsis thaliana microtubule protofilament model that served for the prediction of surface exposure of five beta-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (beta1-10), TUB2.1 (beta426-435) and TU-14 (beta436-445). Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of beta-tubulins (behind beta425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that beta-tubulins could be one of the targets for tyrosine kinases. CONCLUSIONS: We predicted surface exposure of five beta-tubulin epitopes, as well as tyrosine residues, on the surface of A. thaliana microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.The results suggest that prediction of epitope exposure on microtubules by means of homology modeling combined with site-directed antibodies can contribute to a better understanding of the interactions of plant microtubules with associated proteins.

• MeSH
• Arabidopsis imunologie   MeSH
• epitopy imunologie   MeSH
• fluorescenční mikroskopie MeSH
• mapování epitopu metody   MeSH
• mikrotubuly imunologie   MeSH
• molekulární modely MeSH
• monoklonální protilátky imunologie   MeSH
• proteiny huseníčku imunologie   MeSH
• tubulin imunologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• monoklonální protilátky MeSH
• proteiny huseníčku MeSH
• tubulin MeSH

A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.

• MeSH
• bcr-abl fúzové proteiny genetika   imunologie   MeSH
• časové faktory MeSH
• chronická myeloidní leukemie genetika   imunologie   patologie   prevence a kontrola   MeSH
• DNA vakcíny genetika   imunologie   MeSH
• HL-60 buňky MeSH
• imunizace MeSH
• lidé MeSH
• mapování epitopu MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidové fragmenty imunologie   MeSH
• protinádorové vakcíny genetika   imunologie   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• abl-bcr fusion protein, human MeSH Prohlížeč
• bcr-abl fúzové proteiny MeSH
• DNA vakcíny MeSH
• peptidové fragmenty MeSH
• protinádorové vakcíny MeSH