Academic literature on the topic 'CSF single chain library'
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Journal articles on the topic "CSF single chain library"
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Tapryal, Suman, Yogender Pal khasa, and K. J. Mukherjee. "Single chain Fv fragment specific for human GM-CSF: Selection and expression using a bacterial expression library." Biotechnology Journal 5, no. 10 (September 3, 2010): 1078–89. http://dx.doi.org/10.1002/biot.201000043.
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Piper, Clinton, Achia Khatun, Yao Chen, Ryan Zander, Weiguo Cui, and William R. Drobyski. "Single Cell Immune Profiling of Colitogenic T Cells during Acute Graft Versus Host Disease Reveals Two Novel Transcriptionally Distinct CD4+ GM-CSF+ Lineages." Blood 134, Supplement_1 (November 13, 2019): 197. http://dx.doi.org/10.1182/blood-2019-127095.
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Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft versus host disease (GVHD) and pathological damage is largely attributable to inflammatory cytokine production. Recently, we and others identified GM-CSF as a cytokine that is produced primarily by donor-derived CD4+ T cells and mediates inflammation in the GI tract. However, the precise mechanism by which GM-CSF induces pathological damage and the transcriptional profile of this novel colitogenic CD4+ GM-CSF+ T cell population have not been defined. To address these questions, we employed a well-defined murine model of GVHD [C57BL/6 (H-2b)→Balb/c (H-2d)] and demonstrated that GM-CSF induces inflammation by enhancing the activation of donor-derived dendritic cells in the colon as evidenced by increased expression of costimulatory molecules (i.e. CD80 and CD86) and the production of IL-23. In addition, GM-CSF linked adaptive to innate immunity by promoting indirect alloantigen presentation in the mesenteric lymph nodes which was IL-23 dependent and characterized by an increased number of CD103+ CD11b+ dendritic cells and donor CD4+ T cells with a proinflammatory cytokine phenotype. Unexpectedly, we observed two distinct CD4+ GM-CSF+ populations in the GI tract that were distinguishable by the presence or absence of IFN-γ production by intracellular cytokine staining (i.e. CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-). Notably, CD4+ GM-CSF+ IFN-γ- cells were largely absent from other target organs (e.g. liver, lung), suggesting that this population had a unique role in the biology of GVHD in the GI tract. To determine whether these two populations represented transcriptionally distinct lineages or reflected TH1-biased lineage plasticity, we performed single cell RNA sequencing and immunological profiling on donor-derived sort-purified T cells from the colons of GVHD mice one and three weeks post-transplant using the 10X Genomics platform. After selecting only high quality reads, we recovered 6315 unique barcodes corresponding to individual cells and identified several transcriptionally distinct cell clusters that spatially segregated following Seurat and UMAP analysis. Colonic T cells obtained on days 7 and 21 post transplantation completely separated, indicating that the transcriptional profile of these cells changes dramatically between early and later time points. Detectable transcription of GM-CSF was observed in two distinct populations of CD4+ T cells only at the 21-day timepoint. Notably, only one of these clusters co-expressed IFN-γ, confirming our flow-based results, and indicating that CD4+ GM-CSF+ IFN-γ+ and GM-CSF+ IFN-γ- T cells represented distinct populations. Further analysis revealed that CD4+ GM-CSF+ IFN-γ+ T cells were T-bet+ and differentially expressed high levels of costimulatory molecules (CD137, OX40, and CD81) and PD-1, indicative of an activated T cell phenotype. In contrast, CD4+ GM-CSF+ IFN-γ- T cells were distinguishable by the co-expression of T-bet and Gata-3, which is a TH2-defining transcription factor, as well as by the IL-7R and a series of interferon stimulated genes (IFITM1, IFITM2, and IFITM3), supporting the premise that these cells constitute a discrete TH cell lineage. To further characterize these CD4+ T cell populations, we examined the T cell repertoire (TCR) using a targeted sequencing analysis approach of our barcoded cDNA library. We identified 444 unique clonotypes among CD4+ GM-CSF+ T cells based on sequencing of CDR3 regions of TCR alpha and beta chains. Notably, only 5 clonotypes were shared between CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-T cells, representing 58 of 1154 (~5%) of the total cells in both clusters. Thus, this minimal overlap suggested that these T cells were responding to non-overlapping antigens within the GI tract. Analysis of V beta TCR gene usage revealed that CD4+ GM-CSF+ IFN-γ- cells had a highly-biased repertoire with approximately half of cells utilizing a single V beta gene, Vbeta3. In contrast, CD4+ GM-CSF+ IFN-γ+ T cells had a much more evenly distributed Vbeta receptor profile with no predominant Vbeta usage. Collectively, these studies demonstrate the existence of two transcriptionally distinct CD4+ GM-CSF+ T cell populations that accumulate within the GI tract, possess non-overlapping T cell repertoires, promote indirect alloantigen presentation, and mediate pathological damage during GVHD. Disclosures No relevant conflicts of interest to declare.
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Ning, Liangxia, and Bin Wang. "Neurofilament light chain in blood as a diagnostic and predictive biomarker for multiple sclerosis: A systematic review and meta-analysis." PLOS ONE 17, no. 9 (September 14, 2022): e0274565. http://dx.doi.org/10.1371/journal.pone.0274565.
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Background Neurofilament light chain (NfL) in cerebrospinal fluid (CSF) is a biomarker of multiple sclerosis (MS). However, CSF sampling is invasive and has limited the clinical application. With the development of highly sensitive single-molecule assay, the accurate quantification of the very low NfL levels in blood become feasible. As evidence being accumulated, we performed a meta-analysis to evaluate the diagnostic and predictive value of blood NfL in MS patients. Methods We performed literature search on PubMed, EMBASE, Web of Science and Cochrane Library from inception to May 31, 2022. The blood NfL differences between MS vs. controls, MS vs. clinically isolated syndrome (CIS), progressive MS (PMS) vs. relapsing-remitting MS (RRMS), and MS in relapse vs. MS in remission were estimated by standard mean difference (SMD) and corresponding 95% confidence interval (CI). Pooled hazard ratio (HR) and 95%CI were calculated to predict time to reach Expanded Disability Status Scale (EDSS) score≥4.0 and to relapse. Results A total of 28 studies comprising 6545 MS patients and 2477 controls were eligible for meta-analysis of diagnosis value, and 5 studies with 4444 patients were synthesized in analysis of predictive value. Blood NfL levels were significantly higher in MS patients vs. age-matched controls (SMD = 0.64, 95%CI 0.44–0.85, P<0.001), vs. non-matched controls (SMD = 0.76, 95%CI 0.56–0.96, P<0.001) and vs. CIS patients (SMD = 0.30, 95%CI 0.18–0.42, P<0.001), in PMS vs. RRMS (SMD = 0.56, 95%CI 0.27–0.85, P<0.001), and in relapsed patients vs. remitted patients (SMD = 0.54, 95%CI 0.16–0.92, P = 0.005). Patients with high blood NfL levels had shorter time to reach EDSS score≥4.0 (HR = 2.36, 95%CI 1.32–4.21, P = 0.004) but similar time to relapse (HR = 1.32, 95%CI 0.90–1.93, P = 0.155) compared to those with low NfL levels. Conclusion As far as we know, this is the first meta-analysis evaluating the diagnosis and predictive value of blood NfL in MS. The present study indicates blood NfL may be a useful biomarker in diagnosing MS, distinguishing MS subtypes and predicting disease worsening in the future.
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Tashiro, Haruko, Ryosuke Shirasaki, Yoko Oka, Tadashi Yamamoto, Nobu Akiyama, Kazuo Kawasugi, and Naoki Shirafuji. "Interleukin-1β Promotes the Expression of CD34 and Granulocyte Colony-Stimulating Factor-Receptor in Adult Dermal Fibroblasts." Blood 118, no. 21 (November 18, 2011): 4815. http://dx.doi.org/10.1182/blood.v118.21.4815.4815.
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Abstract Abstract 4815 Background and Aims: We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and also in a non-obese diabetes/ severe combined immunodeficiency (NOD/SCID) murine bone-marrow in vivo. In normal hematopoiesis, hematopoietic stem cell (HSC) and stromal immature mesenchymal stem cell (MSC) are speculated to have a cross-talk, and some reports indicate that the HSC generates MSC, and also a specific fraction of MSC shares similar molecular expressions to that of HSC. We made a hypothesis that HSC might be generated from MSC. To make clear this issue, expression cloning was performed to isolate a molecule that stimulated bone-marrow stromal myofibroblasts to express hematopoietic stem cell marker, CD34. And, we also observed the effect of the isolated molecule to an adult human dermal fibroblast (HDF). Materials and Methods: cDNA-expression library was constructed using PHA-P-stimulated normal human blood lymphocytes, and the prepared plasmids were transfected to COS7 cells. After 3 days of culture, supernatants were added to the normal human bone-marrow-derived myofibroblasts (final 10%), and cells were further cultured for one week. RNA was extracted from the cultured myofibroblasts, and cDNA was synthesized. Positive clones were selected on CD34-expression with reverse transcription-polymerase chain reaction, and a single clone was isolated. The purified protein from the isolated single clone was added to HDF-culture, and the morphological changes and the expression of specific hematopoiesis-related proteins were analyzed. Results and Discussion: Isolated single clone was human interleukin 1β (IL-1β). When the purified IL-1β protein was added to the bone-marrow-derived myofibroblast cultures, cell growth was increased, and up-regulation of the expression of several hematopoietic specific proteins, including cytokine receptors and transcription factor SCL, was observed. Based on these observations, we determined the effect of IL-1β to HDF. When HDFs were cultured with human IL-1β for 3 weeks, the expression of granulocyte colony-stimulating factor (G-CSF)-receptor, and SCL was increased. When these IL-1β-stimulated cells were cultured in a non-coated dish, cells were floating, and budding of the cells was also observed. When HDF were cultured with IL-1β for 3 weeks, and then G-CSF and erythropoietin were added to the cultures, expression of transcription factor GATA-1 and CEBPA was significantly increased after one week. These observations indicate that IL-1β can stimulate to induce HDF toward hematopoietic cells. Now we determine the precise actions of human IL-1β to HDF using NOD/SCID transplantation model in vivo. Disclosures: No relevant conflicts of interest to declare.
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Smith, Eric L., Sham Mailankody, Arnab Ghosh, Reed Masakayan, Mette Staehr, Terence J. Purdon, Elizabeth Halton, et al. "Development and Evaluation of a Human Single Chain Variable Fragment (scFv) Derived Bcma Targeted CAR T Cell Vector Leads to a High Objective Response Rate in Patients with Advanced MM." Blood 130, Suppl_1 (December 7, 2017): 742. http://dx.doi.org/10.1182/blood.v130.suppl_1.742.742.
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Abstract Patients with relapsed/refractory MM (RRMM) rarely obtain durable remissions with available therapies. Clinical use of BCMA targeted CAR T cell therapy was first reported in 12/2015 for RRMM, and based on small numbers, preliminary results appear promising. Given that host immune anti-murine CAR responses have limited the efficacy of repeat dosing (Turtle C. Sci Trans Med 2016), our goal was to develop a human BCMA targeted CAR T cell vector for clinical translation. We screened a human B cell derived scFv phage display library containing 6x1010 scFvs with BCMA expressing NIH 3T3 cells, and validated results on human MM cell lines. 57 unique and diverse BCMA specific scFvs were identified containing light and heavy chain CDR's each covering 6 subfamilies, with HCDR3 length ranges from 5-18 amino acids. 17 scFvs met stringent specificity criteria, and a diverse set was cloned into CAR vectors with either a CD28 or a 4-1BB co-stimulatory domain. Donor T cells transduced with BCMA targeted CAR vectors that conveyed particularly desirable properties over multiple in vitro assays, including: cytotoxicity on human MM cell lines at low E:T ratios (>90% lysis, 1:1, 16h), robust proliferation after repeat antigen stimulation (up to 700 fold, stimulation q3-4d for 14d), and active cytokine profiling, were selected for in vivo studies using a marrow predominant human MM cell line model in NSG mice. A single IV injection of CAR T cells, either early (4d) or late (21d) after MM engraftment was evaluated. In both cases survival was increased when treated with BCMA targeted CAR T cells vs CD19 targeted CAR T cells (median OS at 60d NR vs 35d p<0.05). Tumor and CAR T cells were imaged in vivo by taking advantage of luciferase constructs with different substrates. Results show rapid tumor clearance, peak (>10,000 fold) CAR T expansion at day 6, followed by contraction of CAR T cells after MM clearance, confirming the efficacy of the anti-BCMA scFv/4-1BB containing construct. Co-culture with primary cells from a range of normal tissues did not activate CAR T cells as noted by a lack of IFN release. Co-culture of 293 cells expressing this scFv with those expressing a library of other TNFRSF or Ig receptor members demonstrated specific binding to BCMA. GLP toxicity studies in mice showed no unexpected adverse events. We generated a retroviral construct for clinical use including a truncated epithelial growth factor receptor (EGFRt) elimination gene: EGFRt/hBCMA-41BBz. Clinical investigation of this construct is underway in a dose escalation, single institution trial. Enrollment is completed on 2/4 planned dose levels (DL). On DL1 pts received cyclophosphamide conditioning (3g/m2 x1) and 72x106 mean CAR+ T cells. On DL2 pts received lower dose cyclophosphamide/fludarabine (300/30 mg/m2 x3) and 137x106 mean CAR+ T cells. All pts screened for BCMA expression by IHC were eligible. High risk cytogenetics were present in 4/6 pts. Median prior lines of therapy was 7; all pts had IMiD, PI, high dose melphalan, and CD38 directed therapies. With a data cut off of 7/20/17, 6 pts are evaluable for safety. There were no DLT's. At DL1, grade 1 CRS, not requiring intervention, occurred in 1/3 pts. At DL2, grade 1/2 CRS occurred in 2/3 pts; both received IL6R directed Tocilizumab (Toci) with near immediate resolution. In these 2 pts time to onset of fever was a mean 2d, Tmax was 39.4-41.1 C, peak CRP was 25-27mg/dl, peak IL6 level pre and post Toci were 558-632 and 3375-9071 pg/ml, respectively. Additional serum cytokines increased >10 fold from baseline in both pts include: IFNg, GM CSF, Fractalkine, IL5, IL8, and IP10. Increases in ferritin were limited, and there were no cases of hypofibrinogenemia. There were no grade 3-5 CRS and no neurotoxicities or cerebral edema. No pts received steroids or Cetuximab. Median time to count recovery after neutropenia was 10d (range 6-15d). Objective responses by IMWG criteria after a single dose of CAR T cells were observed across both DLs. At DL1, of 3 pts, responses were 1 VGPR, 1 SD, and 1 pt treated with baseline Mspike 0.46, thus not evaluable by IMWG criteria, had >50% reduction in Mspike, and normalization of K/L ratio. At DL2, 2/2 pts had objective responses with 1 PR and 1 VGPR (baseline 95% marrow involvement); 1 pt is too early to evaluate. As we are employing a human CAR, the study was designed to allow for an optional second dose in pts that do not reach CR. We have treated 2 pts with a second dose, and longer follow up data is pending. Figure 1 Figure 1. Disclosures Smith: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: BCMA targeted CAR T cells, Research Funding. Almo: Cue Biopharma: Other: Founder, head of SABequity holder; Institute for Protein Innovation: Consultancy; AKIN GUMP STRAUSS HAUER & FELD LLP: Consultancy. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xu: Eureka Therapeutics, Inc: Employment, Equity Ownership. Park: Amgen: Consultancy. Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Dogan: Celgene: Consultancy; Peer Review Institute: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
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Short, M. K., T. Housel, and intro by P. T. Jubinsky. "Selection of single chain antibody fragments against the gm-csf receptor." Experimental Hematology 28, no. 7 (July 2000): 86. http://dx.doi.org/10.1016/s0301-472x(00)00355-6.
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Hanna, Rachel, Lia Cardarelli, Nish Patel, Levi L. Blazer, Jarrett J. Adams, and Sachdev S. Sidhu. "A phage‐displayed single‐chain Fab library optimized for rapid production of single‐chain IgGs." Protein Science 29, no. 10 (September 15, 2020): 2075–84. http://dx.doi.org/10.1002/pro.3931.
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Schneider, Ruth, Barbara Bellenberg, Barbara Gisevius, Sarah Hirschberg, Roman Sankowski, Marco Prinz, Ralf Gold, Carsten Lukas, and Aiden Haghikia. "Chitinase 3–like 1 and neurofilament light chain in CSF and CNS atrophy in MS." Neurology - Neuroimmunology Neuroinflammation 8, no. 1 (November 10, 2020): e906. http://dx.doi.org/10.1212/nxi.0000000000000906.
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ObjectiveTo investigate cross-sectional associations of CSF levels of neurofilament light chain (NfL) and of the newly emerging marker chitinase 3–like protein 1 (CHI3L1) with brain and spinal cord atrophy, which are established MRI markers of disease activity in MS, to study CHI3L1 and NfL in relapsing (RMS) and progressive MS (PMS), and to assess the expression of CHI3L1 in different cell types.MethodsIn a single-center study, 131 patients with MS (42 RMS and 89 PMS) were assessed for NfL and CHI3L1 concentrations in CSF, MRI-based spinal cord and brain volumetry, MS subtype, age, disease duration, and disability. We included 42 matched healthy controls receiving MRI. CHI3L1 expression of human brain cell types was examined in 2 published single-cell RNA sequencing data sets.ResultsCHI3L1 was associated with spinal cord volume (B = −1.07, 95% CI −2.04 to −0.11, p = 0.029) but not with brain volumes. NfL was associated with brain gray matter (B = −7.3, 95% CI −12.0 to −2.7, p = 0.003) but not with spinal cord volume. CHI3L1 was suitable to differentiate between progressive or relapsing MS (p = 0.015, OR 1.0103, CI for OR 1.002–1.0187), and its gene expression was found in MS-associated microglia and macrophages and in astrocytes of MS brains.ConclusionsNfL and CHI3L1 in CSF were differentially related to brain and spinal cord atrophy. CSF CHI3L1 was associated with spinal cord volume loss and was less affected than NfL by disease duration and age, whereas CSF NfL was associated with brain gray matter atrophy. CSF NfL and CHI3L1 measurement provides complementary information regarding brain and spinal cord volumes.Classification of evidenceThis study provides Class II evidence that CSF CHI3L1 is associated with spinal cord volume loss and that CSF NfL is associated with gray matter atrophy.
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Kamiya, Toshio, Osamu Saitoh, and Hiroyasu Nakata. "Functional Expression of Single-Chain Heterodimeric G-Protein-Coupled Receptors for Adenosine and Dopamine." Cell Structure and Function 29, no. 5,6 (2005): 139–45. http://dx.doi.org/10.1247/csf.29.139.
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Rojas, Julio C., Jee Bang, Iryna V. Lobach, Richard M. Tsai, Gil D. Rabinovici, Bruce L. Miller, and Adam L. Boxer. "CSF neurofilament light chain and phosphorylated tau 181 predict disease progression in PSP." Neurology 90, no. 4 (December 27, 2017): e273-e281. http://dx.doi.org/10.1212/wnl.0000000000004859.
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ObjectiveTo determine the ability of CSF biomarkers to predict disease progression in progressive supranuclear palsy (PSP).MethodsWe compared the ability of baseline CSF β-amyloid1–42, tau, phosphorylated tau 181 (p-tau), and neurofilament light chain (NfL) concentrations, measured by INNO-BIA AlzBio3 or ELISA, to predict 52-week changes in clinical (PSP Rating Scale [PSPRS] and Schwab and England Activities of Daily Living [SEADL]), neuropsychological, and regional brain volumes on MRI using linear mixed effects models controlled for age, sex, and baseline disease severity, and Fisher F density curves to compare effect sizes in 50 patients with PSP. Similar analyses were done using plasma NfL measured by single molecule arrays in 141 patients.ResultsHigher CSF NfL concentration predicted more rapid decline (biomarker × time interaction) over 52 weeks in PSPRS (p = 0.004, false discovery rate–corrected) and SEADL (p = 0.008), whereas lower baseline CSF p-tau predicted faster decline on PSPRS (p = 0.004). Higher CSF tau concentrations predicted faster decline by SEADL (p = 0.004). The CSF NfL/p-tau ratio was superior for predicting change in PSPRS, compared to p-tau (p = 0.003) or NfL (p = 0.001) alone. Higher NfL concentrations in CSF or blood were associated with greater superior cerebellar peduncle atrophy (fixed effect, p ≤ 0.029 and 0.008, respectively).ConclusionsBoth CSF p-tau and NfL correlate with disease severity and rate of disease progression in PSP. The inverse correlation of p-tau with disease severity suggests a potentially different mechanism of tau pathology in PSP as compared to Alzheimer disease.
Dissertations / Theses on the topic "CSF single chain library"
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Cortini, Andrea. "Serum profiling and autoantibodies identification in Multiple Sclerosis using epitope and CSF IgG phage display libraries." Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3073.
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2007/2008Multiple sclerosis (MS) is considered the prototype of inflammatory autoimmune diseases of the central nervous system (CNS). The typical feature of the disease is the plaques of demyelination. The evolution of the plaque lesion in MS implicates an inflammatory phase followed by a recovery of functional myelin; a second step is the chronic progressive disease with axonal loss. The earlier phase of MS may be mediated by an autoimmune reaction. Whereas the role of T cells in MS pathogenesis is well established, the role of B cells and autoantibodies in demyelination and plaque formation is still unresolved. However several evidences suggest a contribute of autoantibodies in MS pathogenesis. B cells and myelin specific autoantibodies are present in the sclerosis plaques, and there is an increased production of immunoglobulin (Ig) in the cerebrospinal fluid (CSF) of more than 90% of MS patients . Typically these Ig present an oligoclonal pattern and sequencing of oligoclonal IgG showed extensive somatic mutations suggesting B cell clonal expansion and a specific antigen-driven immune response. The most extensively studied putative autoantigens are components of CNS myelin (myelin basic protein MBP, proteolipid protein PLP, myelin oligodendrocyte glycoprotein MOG). The autoantibodies in MS recognize both linear and conformational epitopes, but at present the conformational epitopes of myelin proteins have not been identified. For example, in MS, the T-cell receptors of autoreactive T lymphocytes recognize various peptides of the MBP, and, in EAE, the anti-MOG antibodies recognize only conformational epitopes. Furthermore, the progression of MS is accompanied by the decline of primary T-cell autoreactivity and by the concurrent emergence of neo-autoreactivity (epitope spreading). However recent investigation have showed that no myelin antigens, like neuron-specific enolase (NSE), retinal arrestin, beta-arrestin, may also have a role in MS pathogenesis. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis. Several strategies, involving the phage display technology, have been employed in the attempt to discover the antigen that drives the immune response in MS. A first strategy depends on the cloning of IgG repertoire of MS patients in a phage display library screened with brain sections or known antigens. Another strategy involves large phage display libraries of random peptides screened with IgG of CSF in order to identify peptides recognized by antibodies present in CSF of MS patients. Phage display is a technique which involves the coupling of phenotype to genotype in a selectable format. It has been extensively used in molecular biology to study protein-protein interactions and to select antibodies against a wide range of different antigens. In this project we have proposed: 1. to study the autoimmune response in MS by using the phage display for the expression of antibodies involved in the disease. We wanted to make a ScFv library from B cells of CSF of different multiple sclerosis patients, to employ as tool to select a phage display Human Brain cDNA library for the identification of new antigens recognized by the immune sistem in MS patients. 2. To produce single gene mini library of putative antigens (MBP,PLP, MOG) for the generation of epitope chips to use for serotyping the immune response in different patients 3. To investigate the feasibility to use a single gene phage display mini-library as tool for epitope mapping (both linear and conformational) of novels autoantigens 4. To investigate the role of NSE(neuron specific enolase), a new possible no myelin autoantigen in multiple sclerosis, in the pathogenesis of the disease and the usefulness as possible diagnostic marker. Results: Scope 1 B-cells from liquor of two MS patients were centrifuged and the total RNA was extracted from the pellets. Total RNA was retrotranscripted and variable region of heavy and light chain of the antibodies were amplified by PCR. Heavy chain and light chain were assorted and assembled before to be cloned in the phagemid vector pDAN 5. A 2x104 independent clones library was obtained and analyzed by PCR and fingerprinting. A diversity of 30,8% for heavy chain and 72,7% for light chain was established. ScFv library was used to select a phage display Human Brain cDNA library. 17 clones with an high reactivity were obtained and after sequencing 6 clones on 17 have shown to be the same antigen(antigen A ); the reactivity on other two antigens obtained with the selection (antigen B and C) of CSF from 18 MS patients and 16 patients with other neurological disease (OND) was tested by ELISA to evaluate diagnostic value of this protein. The results shown that SM response was statistically different from OND response; the ELISA test gave a specificity of 94,12% and a significance of 53,85 %. The reactivity for the antigen B was also evaluated on sera of MS patients and controls. The MS response was statistically different from OND response and shown a specificity of 97,44% and a significance of 58,62 %. Scope 2&3 We have generated three single gene mini libraries of the major antigens in MS (MBP, MOG and PLP); cDNA of each gene was obtained by RT-PCR and after fragmentation cloned in a phagemid vector (pEP1) to obtain a mini-library for each gene. We have obtained a 2x105 for MBP, 2.4x104 for MOG and 1.6x106 for PLP independent clones library. MBP and MOG libraries were characterized by PCR and fingerprinting. Sequencing analysis shown that the entire MBP transcript variant 7 mRNA (664-1177 nt) and MOG isoform alpha 1 mRNA (262-918 nt) were represented in the respective library. To testing the capacity of selecting a single epitope from our libraries, we have performed a selection test with a commercial monoclonal antibody that recognize MBP 82-98 epitope; after three selection panning all selected clones contain the nucleotidic sequence 906- 956 nt (MBP transcript variant 7 mRNA) which encodes the immunogenic epitope recognized by the monoclonal antibody. Scope 4 The reactivity of sera from 31 MS patients and 14 healthy controls was tested by ELISA on NSE ; statistical analysis of the results shown that the two populations were significantly different.
XXI Ciclo
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O'Brien, Siobhan Helen. "A single chain antibody bacteriophage display library from a patient with active uveoretinitis." Thesis, University of Aberdeen, 1999. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU123996.
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Studies suggest that natural autoantibodies may be part of an immunological network which maintains the normal homeostatic response seen in controls. Any defect in this network leading to autoimmunity may be represented in the anti-retinal antibody response observed in patients. Characterisation of the humoral autoimmune response occurring during active uveitis may provide valuable information on the immune mechanisms, both humoral and cellular, involved in uveitis. Serum titres and ELISA based tests can only partially describe an antibody response, a more complete description requires access to the B-cell repertoire constituting the response. In the past hybridoma technology has generated a wealth of vital information on antibody responses in animals, but with limited success when applied to humans, producing unstable cell lines with poor antigen affinity. Using scFv phage display antibody technology we attempted to isolate the immune response occurring during active uveitis using a phage display library derived from peripheral blood lymphocyte mRNA of a patient with active uveitis. In this study, we report the isolation and characterisation of human autoimmune recombinant scFv's from two libraries, a uveitis patient derived library and a healthy non uveitis donor derived library. Anti-IRBP and S-Antigen autoantibodies were successfully selected from both libraries. Sequence analysis of these selected autoantibodies revealed possible differential epitope targeting of disease associated anti-S-Ag autoantibodies, and exclusive use of the VH segment, DP49 was revealed among selected anti-S-Ag scFv's. In addition ELISA studies using the selected scFv's, and both patient and control serum, indicated that it may be possible to distinguish the 'natural' and disease associated anti-S-Ag responses at the idiotype/anti-idiotype network level.
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Bosompem, Amma N. "Isolation of an anti-CD20 single chain variable fragment from a naïve human phage-scFv library." Connect to this title online, 2007. http://etd.lib.clemson.edu/documents/1202410076/.
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Roberts, Anthony Simon. "The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv." Thesis, Queensland University of Technology, 2004. https://eprints.qut.edu.au/15914/1/Anthony_Roberts_Thesis.pdf.
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This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering.
The variable domain genes were isolated from the hybridoma cell line producing the monoclonal
antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE.
Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab
version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short
CDR2 was due to deletions generated during the somatic mutation process and a model for this is
described.
A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I.
A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE
and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding
chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to
increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a
selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified
and are discussed.
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Roberts, Anthony Simon. "The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv." Queensland University of Technology, 2004. http://eprints.qut.edu.au/15914/.
Full textAbstract:
This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonal antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE. Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short CDR2 was due to deletions generated during the somatic mutation process and a model for this is described. A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I. A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified and are discussed.
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Pilger, Franziska [Verfasser]. "Construction of an equine antibody library in the single-chain-Fragment-variable format (scFv) to express equine immunoglobulins / Franziska Pilger." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1230824510/34.
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Li, Weiyi. "Protein Engineering Hydrophobic Core Residues of Computationally Designed Protein G and Single-Chain Rop: Investigating the Relationship between Protein Primary structure and Protein Stability through High-Throughput Approaches." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398956266.
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Ndlovu, Siphumelele. "The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana." Master's thesis, Faculty of Science, 2020. http://hdl.handle.net/11427/32303.
Full textAbstract:
Monoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents.
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Shahsavarian, Melody. "Genesis of immune diversity and selection of catalytic antibodies : a new investigation." Thesis, Compiègne, 2015. http://www.theses.fr/2015COMP2215/document.
Full textAbstract:
Les anticorps catalytiques (ou abzyme) ont fait l’objet de nombreuses recherches et ont été produits pour réaliser de nombreuses réactions. Ces protéines ont été ensuite découvertes dans le sérum d’individus sains ou atteints de pathologies, dont les pathologies autoimmunes. Les études suggèrent que ces abzymes peuvent avoir des effets bénéfiques ou délétères sur la santé des individus. L’origine des anticorps catalytiques et leur rôle restent ambigus et doivent être approfondis. Nous avons développé une nouvelle stratégie visant à étudier les abzymes, basée sur la technologie du phage display. Nous avons construit 4 banques de fragments d’anticorps, chacune présentant un répertoire immun différent (fond génétique et état d’immunitaire) : saine et naïve, saine et immunisée, autoimmune et naïve, et autoimmune et immunisée. Les stratégies d’amplification et de clonage des régions variables des immunoglobulines ont été conçues afin d’optimiser la taille et la diversité des banques. Nous avons rassemblé les 4 banques en une banque unique élargie contenant 2.7×109 séquences. L’analyse des séquences a mis en évidence des différences dans les profils d’expression des sous-groupes de gènes selon la banque. Nous avons ensuite procédé à la sélection d’abzymes à activité β-lactamase en utilisant deux cibles : un peptide cyclique, et un dérivé de sulfone pénam, inhibiteurs de l’enzyme. Nous avons sélectionné 5 abzymes. Chacun de ces immunoglobulines ont des séquences protéiques propres, incluant un potentiel site actif. Ces résultats montrent que différents motifs peuvent assurer la fonction catalytique de la β-lactamase, confirmant la flexibilité moléculaire de cette enzymeCatalytic antibodies (or abzymes) have been the focus numerous studies for some decades and have been produced with the ability to catalyze a wide range of reactions. They have also been discovered naturally in normal physiological and pathological conditions, notably on the background of autoimmune disease. Some have beneficial effects and others are detrimental to individual’s health. Hence, the origin of abzymes and their role in the immune response are ambiguous and must be enhanced. We have developed a novel strategy for the study of abzymes based on the phage display technology. We have constructed 4 libraries representing 4 murine immune repertoires with different genetic backgrounds and immunological states : healthy and naïve, healthy and immunized, autoimmune and naïve , and autoimmune and immunized. The strategies for the amplification and cloning of the immunoglobulin (lg) variable regions have been designed to optimize the size and diversity of the libraries. We have been able to pool the four libraries to create a large repertoire of size 2.7x109. After sequence analysis, we have found a number of statistically significant differences between the libraries. We have then used two strategically chosen targets to select for antibodies endowed with β lactamase activity : a cycle peptide and a penam sulfone, both inhibitors of the enzyme. We have selected for a total of 5 lgs endowed with β lactamase activity. The selected abzymes have different amino acid sequences. 3D modeling has provides insights on potential active sites demonstrating the ability of different structures to maintain the β lactamase activity and confirming the flexibility of the active site
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"Construction and Characterization of a Single-Chain Variable Fragment Antibody Library against Fusobacterium nucleatum." 2012. http://hdl.handle.net/10222/15184.
Full textAbstract:
Dental plaque forms sequentially, with Fusobacterium nucleatum facilitating the adhesion of pathogenic late colonizers. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help elucidate the molecular mechanisms of coaggregation between F. nucleatum and other bacteria. A 4X10^8 clones scFv phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum 6 times and 292 individual clones tested by ELISA reacted strongly to F. nucleatum. Sixty-two of those clones inhibited F. nucleatum coaggregation with Streptococcus sanguinus. Analysis of select clones revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein RadD (Fn1526), as determined by mass spectrometry.Farhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.
Book chapters on the topic "CSF single chain library"
1
Nahary, Limor, and Itai Benhar. "Design of a Human Synthetic Combinatorial Library of Single-Chain Antibodies." In Therapeutic Antibodies, 61–80. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-554-1_3.
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Becker, Richard C., and Frederick A. Spencer. "Novel Anticoagulants." In Fibrinolytic and Antithrombotic Therapy. Oxford University Press, 2006. http://dx.doi.org/10.1093/oso/9780195155648.003.0023.
Full textAbstract:
While all anticoagulants have, to a certain extent, novel properties, the development of agents that inhibit specific coagulation proteases through structural affinity and can be inhibited themselves by the concomitant production of antidotes (drug–antidote pair construct) has the potential to revolutionize the field. With the evolution of our thinking toward hemostasis and thrombosis has come new pharmacologic constructs for safe and effective treatment. Aptamers are single-stranded nucleic acids that inhibit a protein’s function by folding into a specific three-dimensional structure that defines high-affinity binding to the target protein (White et al., 2000). The term aptamer (from the Latin aptus, “to fit”) was coined by Ellington and Szostak (1990) following their pioneering work published originally in Nature. Based on iterative selection techniques, aptamers that bind essentially any protein or small molecule can be generated. A high-affinity, specific inhibitor that interacts with functional groups (on both the nucleic acid and the protein) can be constructed if a small amount of pure target is available. The initiation point for aptamer development is a combinatorial library composed of single-stranded nucleic acids (RNA, DNA, or modified RNA), typically containing 20 to 40 randomized positions (1024 different sequences). Isolation of high-affinity nucleic acid ligands involves a process known as SELEX (systemic evolution of ligands by exponential enrichment). The starting library is incubated with the protein of interest. Nucleic acid molecules that adopt conformations that allow target protein binding are subsequently partitioned from other sequences (that do not bind the protein). The bound sequences are removed and amplified by reverse transcription and polymerase chain reaction (PCR) (for RNA-based libraries) or PCR alone (for DNA-based libraries). After repeating the process several times, the selected ligands are secured and evaluated for binding affinity and ability to inhibit activity (of the target protein). Postselection optimization steps typically include (1) reduction in aptamer length (from a starting molecule of 80–100 nucleotides to 40 nucleotides); (2) enhanced stability in biologic systems (achieved by substitution of ribonucleotides with 2-amino, 2´-fluoro, or 2´-0-alkyl nucleotides and protection from exonuclease digestion by 3´ end capping); and (3) reduced renal clearance (achieved by increasing the molecules’ mo lecular weight through site-specific addition of polyethylene glycol moieties or other hydrophobic groups.
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Georgiou, George, and Barrett R. Harvey. "Applications of Flow Cytometry in Protein Engineering." In Flow Cytometry for Biotechnology. Oxford University Press, 2005. http://dx.doi.org/10.1093/oso/9780195183146.003.0017.
Full textAbstract:
In recent years, the application of evolutionary methods for protein engineering has created tremendous optimism regarding our ability to generate proteins with tailored functional properties such as ligand binding, improved stability, allostery, and catalytic activity. The power of directed protein evolution lies in its simplicity : First, a gene encoding a polypeptide is subjected to mutagenesis, and the resulting ensemble of mutated genes is expressed in a suitable cellular host. Second, the population of expressed proteins is subjected to a screening process. Often, multiple rounds of screening are required to isolate the rare clones within the population that can satisfy the functional screen. Third, DNA is isolated from the enriched clones and subjected to additional rounds of mutagenesis and screening under increasingly stringent conditions. This iterative process is repeated several times until either little functional improvement is observed between sequential rounds or proteins that satisfy the chosen criteria have been generated. There is a plethora of methods for generating an ensemble of mutated genes. Specifically, sequence diversity can be created by random mutagenesis, typically accomplished using error-prone polymerase chain reaction techniques ; by homologous in vitro recombination ; or by nonhomologous recombination. The latter involves two families of methods collectively known as incremental truncation for the creation of hybrid enzymes and sequence-homology independent protein recombination. Regardless of the means for generating sequence diversity, the next and by far the more technically challenging step in directed evolution is the screening of the resulting library of protein-expressing cells to isolate those that are expressing a protein variant that exhibits the desired function. It is fair to say that evolutionary protein design has been hampered by limitations in screening technologies. The quantitative determination of protein function for each and every clone in a library in a high-throughput fashion is a difficult and technically demanding task. In broad terms, there are four general strategies suitable for the screening of combinatorial protein libraries: phage display; biological assays that include selections and assays that use reporter enzymes [e.g., two-hybrid-like techniques for detecting interacting proteins ]; single-well assays using high-density microtiter well plates; and flow cytometry (FC) methods. Each of these methods has a different set of advantages and shortcomings.
Conference papers on the topic "CSF single chain library"
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Timofeev, S. A., A. A. Tsarev, V. S. Zhuravlev, A. V. Konarev, and V. V. Dolgikh. "Construction of scFv-antibodies to the active center of the Sunn bug (Eurygaster integriceps Put) gluten-hydrolyzing protease GHP3." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.247.
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This work describes the preparation of immune library and selection of recombinant single-chain (scFv) antibodies to the active center of the gluten-destroying proteinase GHP3 of the Eurygaster integriceps Put. bug.
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Dahlbäck, B., and A. LundWall. "ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR HUMAN FACTOR V." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643886.
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Coagulation factor V is a single chain, 330 kDa glycoprotein functioning as a cofactor to factor Xa in the activation of prothrombin. Thrombin cleaves factor V into four major fragments, out of which the N-terminal (105kDa) and the C-terminal (71-74kDa) fragments together constitute the active factor V species. To isolate cDNA clones a λ-gt 11 liver library was screened with a polyclonal, monospecific antiserum against human factor V. Four positive clones (two "weak", Aland A2 and two "strong", A3 and A4) were identified and isolated. Al(0.7kb), A2 (1.25kb) and A4 (0.85kb) reacted strongly with an antiserum against the 105 kDa, N-terminal fragment (heavy chain of factor Va), whereas A3 (1.25kb) gave the best signal with an antiserum against the 71-74 kDa, C-terminal fragment (light chain of factor Va). A1 hybridized with A2 and A4, whereas A2 only hybridized with Al. A3, which did not hybridize to any of the other clones, was used to rescreen the library and 9 positive clones (Bl-9) were isolated. B9 (3kb) coded for the entire C-terminal factor V fragment and the 3' noncoding sequence. B8 (1.8kb) partially overlapped B9 but extented the 5' sequence with 0.8kb. In a third screening round Al was used in combination with B8 and a 1.1 kb clone (CIO) was identified which hybridized to both. C10 did not hybridize with A2. The following overlapping cDNA clones can be orderedfrom the 5´end: A2-A1-C10-B8-B9 and together they cover 6 kb of coding sequence
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LOFTUS, J. C., E. F. Plow, A. L. Frelinger III, M. A. Smith, S. D’ouza, and M. H. Ginsberg. "LOCALIZATION AND CHEMICAL SYNTHESIS OF A DIVALENT CATION REGULATED EPITOPE IN PLATELET MEMBRANE GPIIb." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643959.
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Platelet membrane glycoprotein (GP)IIb-IIIa is a component of a common adhesive protein receptor for fibrinogen, fibronectin, and von Willebrand factor. A monoclonal antibody, PMI-1, defines a divalent cation dependent regulation of the surface orientation of the heavy chain of GPIIb. Exposure of the PMI-1 epitope inversely correlates with the capacity of platelets to bind fibrinogen and aggregate. We have now localized and chemically synthesized this epitope. A 1.1 Kb cDNA clone which directs the synthesis of a fusion protein which bears the PMI-1 epitope was isolated from a lambda gt 11 expression library constructed from mRNA from the human erythroleukemia (HEL) cell line. The position of the N-terminal sequence of the light chain of GPIIb in the deduced amino acid sequence of the clone defined the orientation of the light and heavy chains of GPIIb. Analysis of the amino acid sequence corresponding to the heavy chain of GPIIb identified a single region with a high likelihood of containing a continuous epitope. A synthetic 17 residue peptide, corresponding to the predicted antigenic site, inhibited the binding of PMI-1 to platelets. Two uM peptide was required to inhibit binding 50% in the presence of 1 uM PMI-1, indicating an approximate dissociation constant of 1.5 uM for the peptide-antibody complex. This figure should be compared to a Kd of 0.95 uM (JCI 78:1103, 1986) for PMI-1 binding to GPIIb. A second peptide, corresponding to the region immediately adjacent to the predicted antigenic site, failed to inhibit PMI-1 binding. Neither peptide inhibited the binding of two other monoclonal anti GPIIb-IIIa’s to platelets. The peptides had similar effects on PMI-1 interaction with purified GPIIb-IIIa in detergent solution. These data localize the PMI-1 epitope to a 17 amino acid region located near the carboxyl terminal of the heavy chain of GPIIb. Thus, they chemically define a region of GPIIb whose surface expression reflects the competence of GPIIb-IIIa as a component of a platelet receptor for adhesive proteins.
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Koide, T. "CHARACTERIZATION OF THE GENE FOR HUMAN HISTIDINE-RICH GLYCOPROTEIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643599.
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Human histidine-rich glycoprotein (HRG) is a single-chain glycoprotein in plasma which is considered to modulate a coagulation and fibrinolysis system with the ability to bind to heparin, plasminogen, fibrinogen, thrombospondin, etc. Recently we have elucidated the primary structure of HRG by determining the nucleotide sequence of its cDNA, and showed that HRG is composed of several different types of internal repeats, each one of which shows considerable homology with the functional and/or structural domains of other proteins including high molecular weight kininogen, antithrombin III, cystatins, and proline-rich protein and peptide. Thus, the multifunctional property of HRG was suggested to be due to its multi-domain structure. In the present studies, a human genomic DNA library, cloned in the bacteriophage vector Charon 4A, was screened for HRG gene using a full-length cDNA coding for human IMI as a probe. A total of 7 clones were isolated from 6 × 105 phage and each was plaque purified. The entire HRG gene is represented in 3 genomic inserts with overlapping sequences that carry human DNA spanning 30 kb. Overlapping gene fragments were subcloned into pUC9 and characterized by Southern blot hybridization using 5’ and 3’ end probes isolated from human HRG cDNA and by DNA sequencing. These studies have shown that the gene for human HRG spans about 9 kb and consists of at least 5 exons and 4 introns. The putative histidine-rich region consisted of 12 tandemly repeated sequences of a 5 amino acid segment and 2 proline-rich regions contiguous to it are likely to be involved within one exon.
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Butler-Zimrin, A. E., J. S. Bennett, M. Poncz, E. Schwartz, S. Surrey, R. Eisman, R. A. Heidenreich, and G. Vilaire. "ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR THE PLATELET MEMBRANE GLYCOPROTEINS IIb and IIIa." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643961.
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The platelet membrane GPIIb/GPIIIa complex on activated platelets contains receptors for fibrinogen, von Willebrand factor, and fibronectin. GPIIb and GPIlia also appear to be members of a family of membrane receptors involved in cell-cell and cell-matrix interactions. To study the structure of GPIIb and GPIIIa, we have constructed an expression library in the vector lambda gtll using mRNA from the HEL cell line and screened it with polyclonal antibody against each platelet protein. HEL cells constitutively express proteins similar to platelet GPIIb and GPIIIa. A 3.2kb GPIIb cDNA clone was identified that encodes for all 1008 amino acids of GPIIb including the known N-terminal amino acids of the α Cand βsubunits. This confirms that GPIIb is synthesized as a single chain polypeptide that is cleaved into two disulfide-linked subunits posttranslation. Analysis of the amino acid sequence revealed a major C-terminal transmembrane domain in the βsubunit, two potential transmembrane domains near the N-terminus of the αsubunit, and four possible N-linked glycosylation sites. Approximately 30% amino acid identity was found between GPIIb and the available amino acid sequences for the larger chains of the fibronectin and vitronectin receptors. Initial sequence analysis of a 3.8kb cDNA for GPIIIa included the known N-terminal amino acids of the platelet protein. Northern blot analysis was performed using HEL cell total RNA. The GPIIb cDNA hybridized to a 4.1kb mRNA while the GPIIIa cDNA hybridized to a 5.8kb mRNA. This indicates that the two cDNAs do not cross-hybridize and suggests that GPIIb and GPIIIa are encoded by separate genes. The availability of these cDNA for GPIIb and GPIIIa will facilitate study of the structure and function of the proteins and will aid in clarifying their relationship to other adhesive protein receptors.