Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

<p>Abstract</p> <p>Background</p> <p>A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent <it>in vitro </it>and <it>in vivo </it>studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions.</p> <p>In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP).</p> <p>Results</p> <p>We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv) phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells.</p> <p>Conclusion</p> <p>Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.</p

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

<p>Abstract</p> <p>Background</p> <p>The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in <it>in vitro </it>and <it>in vivo </it>detection of CD expression in GDEPT/ADEPT studies.</p> <p>Results</p> <p>An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.</p> <p>Conclusion</p> <p>The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.</p

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

BACKGROUND: CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. METHODS: The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. RESULTS: The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. CONCLUSION: The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance

Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N) SARS-CoV protein using a phage display approach

BACKGROUND: Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. METHODS: The human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. RESULTS: Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. CONCLUSION: The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells

Uso di librerie fagiche per isolare in vitro anticorpi monoclonali ricombinanti di predeterminata specificità

The biotechnological generation of monoclonal antibodies of predetermined specificity has traditionally involved the production of hybridomas obtained by somatic cellular fusion of splenocytes from immunized animals with myeloma cell lines bearing selectable markers. Now, monoclonal antibodies could be genetically engineered thus bypassing all the natural systems for making antibodies. Filamentous bacteriophages provides a means to display and select large single chain fragments variable (scFv) repertoires created by cloning the natural rearranged V-immunoglobulin genes or introducing predetermined level of randomization into germline V-gene segments. In this article we demonstrated that by using a well characterized scFv phage synthetic library it is possible to generate in vitro recombinant human monoclonal antibodies directed to a large array of antigens showing different molecular weights, conformations and origins.</p

Flexible Structure of a Thermally Stable Hybrid Aluminosilicate Built with Only the Three-Ring Unit

Organic–inorganic aluminosilicate hybrids are an attractive new class of materials that add organic functionalities to conventional properties of solid inorganic catalysts. ECS-17, a novel crystalline hybrid, was synthesized using 1,4-bis-(triethoxysilyl)-benzene as the sole silicon source. Its structure was solved by direct methods starting from high-resolution synchrotron X-ray diffraction data and is composed of inorganic layers, characterized by 10 rings, held together by phenylene rings. ECS-17 is the first aluminosilicate built from only the three-ring secondary building unit. This new material shows intriguing reversible collapsibility upon dehydration/rehydration. Mild thermal treatment under vacuum causes its crystalline structure to collapse due to facile elimination of the water molecules around the cations. Successive exposure to ambient atmospheric moisture gives back the hydrated crystalline form. ECS-17 shows remarkably high thermal stability for a hybrid, being stable up to 450 °C under vacuum and breaking down at 350 °C in air. Structural, thermal, and optical properties were examined by X-ray powder diffraction, thermogravimetric analysis, nuclear magnetic resonance, and ultraviolet–visible-near-infrared reflectance and fluorescence spectroscopies

Identification by phage display of the linear continuous MRPr1 epitope in the multidrug resistance-associated protein (MRP1)

In order to study the structure of the multidrug resistance-associated protein (MRP1), which is one of the most important members of the ATP-binding cassette (ABC) protein family acting as drug-efflux systems, we have developed an epitope mapping-based strategy. By means of the mAb MRPr1, we have immunoselected clones from two distinct random peptide libraries displayed on phages and have identified several peptide sequences mimicking the internal conformation of this 190 kDa multidrug transporter protein. Phage clones able to block the immunolabeling of the MRPr1 antibody to MRP1-overexpressing multidrug resistance (MDR) H69/AR cells were isolated and, after sequencing the corresponding inserts, their amino acid sequence was compared to that of MRP1. This analysis led to the identification of the consensus sequence L.SLNWED, corresponding to the MRP1 segment LWSLNKED (residues 241-248). This MRP1 sequence is partially overlapping with the MRPr1 epitope GSDLWSLNKE (residues 238-247) previously mapped using peptide scanning techniques. These results demonstrate the high reliability of phage display technology to study not only the topography of complex integral membrane proteins such as MRP1, but also to help identify critical residues participating in the formation of the epitope structure.</p

Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells-0

<p><b>Copyright information:</b></p><p>Taken from "Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells"</p><p>http://www.biomedcentral.com/1472-6750/7/38</p><p>BMC Biotechnology 2007;7():38-38.</p><p>Published online 2 Jul 2007</p><p>PMCID:PMC1933425.</p><p></p>y populations with CCRF-CEM cells are shown after each round of biopanning selection. In the inserted boxes, the ELISA reactivity of the ETH-2 library (A') and selected phage antibody populations (B', C', D') with the rHaPrP are shown in parallel with the irrelevant phage antibody anti glucose oxidase and anti tetanus toxoid (A", B", C" and D"). Experiments were repeated at least twice and mean ± SD from representative experiments (triplicate samples) is shown. The cut-off value separating positive from negative sample was calculated as 3 standard deviation above the mean of the value obtained from irrelevant phage antibodies (0,085 OD). In the lower part of the figure, the nucleotide composition and the corresponding amino acid sequences in the CDR3 regions of the selected scFv antibodies MA3.B4 and MA3.G3 are shown. A schematic representation of the scFv antibody gene as M13 pIII fusion protein is also illustrated

Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells-3

<p><b>Copyright information:</b></p><p>Taken from "Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells"</p><p>http://www.biomedcentral.com/1472-6750/7/38</p><p>BMC Biotechnology 2007;7():38-38.</p><p>Published online 2 Jul 2007</p><p>PMCID:PMC1933425.</p><p></p> at two different magnification, in C and D the reactivity of CCRF-CEM cells with two distinct irrelevant phage antibodies (anti glucose oxidase and anti tetanus toxoid, respectively) are shown