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Phage Display Technology (phage + display_technology)
Selected AbstractsIdentification and Repair of Positive Binding Antibodies Containing Randomly Generated Amber Codons from Synthetic Phage Display LibrariesBIOTECHNOLOGY PROGRESS, Issue 3 2006Warren D. Marcus Phage display technology allows for the rapid isolation and characterization of monoclonal antibodies that have vast potential for therapeutic and diagnostic applications. However, the panning process, which utilizes a host strain that suppresses termination by the amber codon, has an inherent bias toward clones containing randomly generated amber stop codons, complicating identification of positive binding antibodies when the antibody genes are finally expressed in a nonsupressor host. Here, we perform biopanning against a Histone 2A peptide using streptavidin- or anti-biotin-coated beads. After four rounds, a dominant clone is characterized but contains a spurious amber stop codon. A protocol is given that readily corrects the amber codon, allowing for soluble antibody production once the phagemid is transformed into a nonsuppressor bacterial strain. This work also highlights the ability to isolate antibodies against a protein antigen by using only a small peptide (15 amino acids) representing a portion of the antigen. [source] Developments in the prediction of type 1 diabetes mellitus, with special reference to insulin autoantibodiesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2005Bernd Franke Abstract The prodromal phase of type 1 diabetes is characterised by the appearance of multiple islet-cell related autoantibodies (Aab). The major target antigens are islet-cell antigen, glutamic acid decarboxylase (GAD), protein-tyrosine phosphatase-2 (IA-2) and insulin. Insulin autoantibodies (IAA), in contrast to the other autoimmune markers, are the only ,-cell specific antibodies. There is general consensus that the presence of multiple Aab (, 3) is associated with a high risk of developing diabetes, where the presence of a single islet-cell-related Aab has usually a low predictive value. The most commonly used assay format for the detection of Aab to GAD, IA-2 and insulin is the fluid-phase radiobinding assay. The RBA does not identify or measure Aab, but merely detects its presence. However, on the basis of molecular studies, disease-specific constructs of GAD and IA-2 have been employed leading to somewhat improved sensitivity and specificity of the RBA. Serological studies have shown epitope restriction of IAA that can differentiate diabetes-related from unrelated IAA, but current assays do not distinguish between disease-predictive and non-predictive IAA or between IAA and insulin antibodies (IA). More recently, phage display technology has been successful in identifying disease-specific anti-idiotopes of insulin. In addition, phage display has facilitated the in vitro production of antibodies with high affinity. Identification of disease-specific anti-idiotopes of insulin should enable the production of a high affinity reagent against the same anti-idiotope. Such a development would form the basis of a disease-specific radioimmunoassay able to identify and measure particular idiotypes, rather than merely detect and titrate IAA. Copyright © 2005 John Wiley & Sons, Ltd. [source] Exploring the idiotypes of insulin antibodies as markers for remission in Type 1 diabetesDIABETIC MEDICINE, Issue 12 2004D. Devendra Abstract Aims Complete or partial remission can occur in newly diagnosed Type 1 diabetes patients. We created idiotype-specific reagents to explore the idiotypes of insulin antibodies (IA) in a patient in remission, and to compare with a patient who was not. Methods Phage display was used to create a library of phagotopes specific to insulin binding in four sera. Sera from a Type 1 diabetes subject deemed to have undergone remission were taken at diagnosis and again during remission. Sera from a non-remitter were taken at diagnosis and after 3 months on insulin. Phagotopes from the four sera were randomly selected and tested for insulin specificity in a radiobinding assay by using sera from remitters and non-remitters. Results IA-binding phagotope selected from serum during remission displaced insulin binding in all nine IA+ remitters and all 10 IA+ non-remitters. IA-binding phagotope selected from the non-remission patient (3 months after insulin therapy) displaced insulin binding in 8/9 IA+ remitters and 8/10 IA+ non-remitters. The consensus peptide sequences adduced from the phages were identical for both these phagotopes. Phagotopes derived from insulin autoantibody-positive individuals at diagnosis were unable to displace insulin binding in the IA+ sera 3 months later, whether in remission or not. Conclusions We have established the principle of using phage display in the investigation of insulin antibodies during remission in Type 1 diabetes. The immunological characteristics of IA 3 months after the introduction of insulin treatment were different from those at diagnosis of Type 1 diabetes (IAA). Using phage display technology, it was not possible to distinguish insulin antibodies according to remission status. [source] Development of an inhibitory antibody fragment to human tissue factor using phage display technologyDRUG DEVELOPMENT RESEARCH, Issue 3 2009S.M. Meiring Abstract Tissue factor is involved in the etiology of thrombotic diseases initiating the thrombosis associated with the inflammation that occurs during infection. The prevention of blood coagulation and inflammation is of primary importance in a number of pathological situations. A single-chain variable antibody fragment of molecular weight of 26,kD that inhibits the action of human tissue factor was selected by phage display technology, purified and tested for its tissue factor inhibitory effect, purified on a protein A column, and its purity evaluated on SDS-PAGE. The effects of the antibody fragment on prothrombin times, Factor Xa production, and thrombin generation were assessed with increasing fragment concentrations, using chromogenic and fluorometric substrates. The antibody fragment dose-dependently prolonged the prothrombin time (IC50=0.5,,M) and delayed the lag phase before the thrombin generation burst and the peak thrombin concentration in the thrombin generation assay. The effect on thrombin generation was more pronounced in thrombophilic plasma than in normal plasma. Antibody-based tissue factor inhibitors therefore may provide an effective treatment for thrombotic disease without serious bleeding complications. Drug Dev Res 2009. © 2009 Wiley-Liss, Inc. [source] The clinical pharmacology of therapeutic monoclonal antibodiesDRUG DEVELOPMENT RESEARCH, Issue 3 2004Lorin K. Roskos Abstract Seventeen monoclonal antibodies are currently approved in the United States for therapeutic use in organ transplantation, percutaneous coronary intervention, prophylaxis of respiratory syncytial virus disease, rheumatoid arthritis, Crohn's disease, asthma, chronic lymphocytic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, and colorectal cancer. All approved antibodies are of the IgG class. Thirteen are unconjugated intact antibodies, three are intact immunoconjugates, and one is a Fab fragment. Three of the antibodies are murine, five are chimeric, eight are humanized, and one is a fully human antibody generated by phage display technology. The antigen target and the structural and binding characteristics of the antibody determine the antibody's mechanism of action, pharmacokinetics, safety, and immunogenicity. Antibodies act through multiple mechanisms that include functional modulation of the antigen, recruitment of ADCC and CDC, and delivery of radionuclide or toxin payloads to target cells. Antibody half-life is usually governed by interaction with the FcRn receptor. In some cases, the antigen may act as a sink for antibody elimination. Safety profiles are determined by the pharmacology and tissue distribution of the target antigen, antibody isotype, the antibody payload, cytokine release, hypersensitivity reactions to xenogeneic protein, and immunogenicity. Fully human antibody technology may allow development of antibodies that have reduced risks of hypersensitivity reactions and immunogenicity, thereby enhancing safety and efficacy. The exquisite target specificity of antibodies, improvements in antibody engineering technology, and the wide availability of novel and validated therapeutic targets provide many current and future opportunities for the clinical development of therapeutic antibodies. Drug Dev. Res. 61:108,120, 2004. © 2004 Wiley-Liss, Inc. [source] Characterization of prostate-specific antigen binding peptides selected by phage display technologyJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2006Catherine Ferrieu-Weisbuch Abstract Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82,87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer. Copyright © 2005 John Wiley & Sons, Ltd. [source] Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2PROTEIN SCIENCE, Issue 11 2006Zsolt Keresztessy Abstract Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q6, Q8, and Q22 are modified by TG2. Kinetic parameters of SnQ1 transamidation (KMapp = 250 ,M, kcat = 18.3 sec,1, and kcat/KMapp = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research. [source] REVIEW ARTICLE: Status of Contraceptive VaccinesAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2009Rajesh K. Naz Problem, This is a review of anti-sperm contraceptive vaccines (CV), and synthesis of human scFv antibodies that can be used as immunocontraceptives. Method of study, Various methods of proteomics and genomics, peptide synthesis, phage display technology, and antibody engineering were used to obtain multi-epitope vaccines and human scFv antibodies from immunoinfertile and vasectomized men. The present review primarily focuses on the effect of multi-epitope vaccines and Izumo on fertility, and synthesis and characterization of sperm specific human scFv antibodies. Results, The immunization with Izumo peptides causes a contraceptive effect in female mice. The efficacy is enhanced by combination vaccination, including peptides based on other sperm antigens. Using phage display technology, we were able to synthesize at least four novel scFv antibodies with unique complementarity determining regions (CDRs) that reacted with specific fertility-related sperm antigens. These antibodies inhibited human sperm function in vitro, and their immunocontraceptive effect in vivo by these antibodies is currently being investigated. Conclusion, The multi-epitope vaccines may provide an efficacious and viable approach to contraception. The human scFv antibodies, if they block fertility in vivo, may provide unique and novel immunocontraceptives, the first of its kind for human use. The multi-epitope CV and preformed engineered antibodies of defined specificity may obliterate the concern related to inter-individual variability of the immune response. [source] Affibody-mediated transferrin depletion for proteomics applicationsBIOTECHNOLOGY JOURNAL, Issue 11 2007Caroline Grönwall Abstract An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses. [source] | ||||||||||||||||