scholarly journals Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

2005 ◽  
Vol 37 (6) ◽  
pp. 415-420 ◽  
Author(s):  
Mei-Yun Liu ◽  
Wei Han ◽  
Yan-Li Ding ◽  
Tian-Hong Zhou ◽  
Rui-Yang Tian ◽  
...  
Keyword(s):  
B Lymphocyte ◽  
Neutralization Assay ◽  
Binding Activity ◽  
Scfv Antibody ◽  
Nucleotide Sequencing ◽  
Single Chain ◽  
Scfv Gene ◽  
Scfv Library ◽  
Soluble Scfv

Abstract B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS. After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

scholarly journals Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

2020 ◽  
Author(s):  
Matthew D. Beasley ◽  
Sanja Aracic ◽  
Fiona M. Gracey ◽  
Ruban Kannan ◽  
Avisa Masarati ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Single Chain ◽  
Single Chain Antibody ◽  
High Affinity ◽  
Scfv Library ◽  
Antibody Libraries ◽  
Prophylactic Use

AbstractAntibodies with high affinity against the receptor binding domain (RBD) of the SARS-CoV-2 S1 ectodomain were identified from screens using the Retained Display™ (ReD) platform employing a 1 × 1011 clone single-chain antibody (scFv) library. Numerous unique scFv clones capable of inhibiting binding of the viral S1 ectodomain to the ACE2 receptor in vitro were characterized. To maximize avidity, selected clones were reformatted as bivalent diabodies and monoclonal antibodies (mAb). The highest affinity mAb completely neutralized live SARS-CoV-2 virus in cell culture for four days at a concentration of 6.7 nM, suggesting potential therapeutic and/or prophylactic use. Furthermore, scFvs were identified that greatly increased the interaction of the viral S1 trimer with the ACE2 receptor, with potential implications for vaccine development.

Heparin-Induced Thrombocytopenia and Thrombosis: Therapeutic Strategy Using a Single-Chain Variable Fragment (scFv) Antibody

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3346-3346
Author(s):  
Jaa Yien New ◽  
Jose Perdomo ◽  
Xing-Mai Jiang ◽  
Beng Chong
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Heparin Therapy ◽  
Human Platelets ◽  
Binding Activity ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Heparin Induced Thrombocytopenia ◽  
E Coli

Abstract Abstract 3346 Introduction and Aim Heparin-Induced Thrombocytopenia and Thrombosis (HIT) is a life threatening disorder that affects 1–5% of patients receiving heparin therapy. A low platelet count is usually recorded (<150,000 per cubic millimetre) with a decrease of 50% or more from the baseline. The occurrence of HIT is due to the presence of an IgG antibody that recognizes the immune complex formed between Platelet Factor 4 (PF4) and heparin. The antibody/PF4/Heparin complex binds to the FcγRIIa receptor on platelets, leading to platelet activation and thrombotic complications in patients receiving heparin. IV.3 is a murine monoclonal antibody that was raised against the FcγRIIa receptor and has been used as an inhibitor in specificity assays to confirm HIT in patients. We have developed a humanized single-chain variable fragment (scFv) antibody based on the IV.3 monoclonal antibody that binds to the FcγRIIa receptor on platelets and prevents platelet aggregation induced by HIT antibodies. Methods The variable heavy chain (VH) and light chain (VL) of the IV.3 antigen binding fragment (Fab) moiety were amplified using polymerase chain reaction (PCR). These two fragments were then coupled with a linker (Glycine4 and Serine)6. This was followed by introduction of several components including fusion tags (FLAG and c-Myc) at both termini for cloning, detection and purification purposes. The construct was transformed into E. coli (strain-BL21) for protein expression of the scFv. The presence of the protein was detected via immunostaining using anti-FLAG and anti-c-Myc antibodies. The scFv was purified by affinity chromatography and the binding activity was detected using flow cytometry and confocal microscopy. The functional activity was determined using Platelet Aggregation Assay. The scFv was then humanized to minimize potential immunogenicity. Humanization was achieved by introducing specific mutations that rendered the molecule human-like but did not affect binding specificity. The humanized scFv was also expressed in E. coli, purified and tested as before. Results The scFv protein (32kDa) was expressed, purified and confirmed via immunostaining. The created humanized scFv exhibits binding activity against the FcγRIIa on human platelets as determined by flow cytometry and confocal microscopy. In addition, the protein successfully inhibits platelet aggregation at micro molar concentrations in aggregation assays conducted in vitro in the presence of HIT antibodies. Conclusions The humanized scFv was successful in recapitulating the properties of the IV.3 murine monoclonal antibody. It demonstrated binding activity against the FcgRIIa on human platelets and exhibited functional activity by inhibiting platelet activation and aggregation in vitro. This implies that our scFv is able to stop binding of the antibody/PF4/Heparin immune complex to platelets, thus hindering one of the critical initial steps in HIT. The scFv described here may be able to ameliorate the unwanted side effects of heparin therapy and could serve as a potential therapeutic drug for HIT patients. Disclosures: No relevant conflicts of interest to declare.

A Single-Chain Variable Fragment Antibody Inhibits Aggregation of Phosphorylated Tau and Ameliorates Tau Toxicity in vitro and in vivo

2021 ◽  
Vol 79 (4) ◽  
pp. 1613-1629
Author(s):  
Sen Li ◽  
Yushan Yi ◽  
Ke Cui ◽  
Yanqiu Zhang ◽  
Yange Chen ◽  
...  
Keyword(s):  
High Capacity ◽  
Scfv Antibody ◽  
Phosphorylated Tau ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Potential Applications ◽  
Tau Aggregation ◽  
Transgenic Drosophila

Background: Alzheimer’s disease (AD) is a common cause of dementia among elderly people. Hyperphosphorylation and aggregation of tau correlates with the clinical progression of AD; therefore, therapies targeting the aggregation of tau may have potential applications for anti-AD drug development. Several inhibitors of tau aggregation, including small molecules and antibodies, have been found to decrease the aggregation of tau and the corresponding pathology. Objective: To screen one kind of single-chain variable fragment (scFv) antibody which could inhibit the aggregation of tau and ameliorate its cytotoxicity. Methods/Results: Using phosphorylated tau (pTau) as an antigen, we obtained a scFv antibody via the screening of a high-capacity phage antibody library. Biochemical analysis revealed that this scFv antibody (scFv T1) had a strong ability to inhibit pTau aggregation both in dilute solutions and under conditions of macromolecular crowding. ScFv T1 could also depolymerize preformed pTau aggregates in vitro. Furthermore, scFv T1 was found to be able to inhibit the cytotoxicity of extracellular pTau aggregates and ameliorate tau-mediated toxicity when coexpressed with a hTauR406W mutant in the eye of transgenic Drosophila flies. Conclusion: This scFv T1 antibody may be a potential new therapeutic agent against AD. Our methods can be used to develop novel strategies against protein aggregation for the treatment of neurodegenerative diseases.

Ribosome display and selection of human anti-CD22 scFvs derived from an acute lymphocytic leukemia patient

2008 ◽  
Vol 389 (4) ◽  
pp. 433-439 ◽  
Author(s):  
Achim Rothe ◽  
Anne Nathanielsz ◽  
Frank Oberhäuser ◽  
Elke Pogge von Strandmann ◽  
Andreas Engert ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Specific Binding ◽  
Binding Activity ◽  
Lymphocytic Leukemia ◽  
Target Antigen ◽  
Single Chain ◽  
Ribosome Display ◽  
Cell Assays ◽  
Human Scfv

AbstractNovelin vitromethods for the display of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) to isolate specific binding reagents to these antigens. Here, we present the isolation of human scFv against the immunotherapeutic target antigen CD22 from a patient-derived human scFv library using ribosome display technology. The ribosome complexes were enriched against the extra-cellular domain of human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific binding activity was demonstrated by surface plasmon resonance and validated usingin vitrocell assays. The isolated human anti-CD22 scFvs can provide a basis for the development of new immunotherapeutic strategies in CD22-expressing malignant diseases.

scholarly journals Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus

2004 ◽  
Vol 36 (8) ◽  
pp. 541-547 ◽  
Author(s):  
Hui Liu ◽  
Yan-Li Ding ◽  
Wei Han ◽  
Mei-Yun Liu ◽  
Rui-Yang Tian ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Binding Activity ◽  
Scfv Antibody ◽  
Respiratory Syndrome Virus ◽  
Single Chain ◽  
N Protein ◽  
Recombinant Scfv ◽  
E Protein ◽  
Equilibrium Dissociation ◽  
Site Binding

Abstract Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C20, could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, Kd) of SARS-CoV E protein was 5.7×10−8 M for B10 and 8.9×10−8 M for C20. The affinity of A17 for N protein was 2.1×10−6 M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

scholarly journals Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

2021 ◽  
Author(s):  
Fangyu Wang ◽  
Ning Li ◽  
Yunshang Zhang ◽  
Xuxefeng Sun ◽  
Yali Zhao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Scfv Antibody ◽  
Phage Library ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Relative Standard ◽  
Directional Evolution ◽  
Scfv Library

Abstract A recombinant anti-enrofloxacin single-chain antibody (scFv) was produced for the detection of enrofloxacin. An immunized mouse phage display scFv library with a capacity of 2.35×109 CFU/mL was constructed and used for anti-enrofloxacin scFv screening. After four rounds of bio-panning, 10 positives were isolated and identified successfully. The highest positive scFv was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residues Leu121 were the key residues for the binding of ScFv to ENR. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Leu121 to Asn. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant ScFv were established for enrofloxacin respectively. The IC50 value of the assay established with the ScFv mutant was 1.63 ng/mL, while the parental ScFv was 21.08 ng/mL, this result showed highly increased affinity with up to 12.9-folds improved sensitivity. The mean recovery for ENR ranged from 71.80% to 117.35% with 10.46% relative standard deviation between the intra-assay and the inter-assay. The results indicate that we have obtained a highly sensitive anti-ENR scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of ENR residue in animal derived edible tissues and milk.

Recognition of endothelial cells byCandida albicans: role of complement-binding proteins

1995 ◽  
Vol 73 (S1) ◽  
pp. 1154-1159 ◽  
Author(s):  
Richard A. Calderone
Keyword(s):  
Cell Surface ◽  
B Lymphocyte ◽  
Binding Activity ◽  
Surface Proteins ◽  
Host Cells ◽  
Peptide Sequence ◽  
Complement C3 ◽  
Systemic Infections ◽  
Surface Adhesins

Candida albicans, a commensal of humans, can cause either mucosal or systemic infections. The virulence properties of the organism include cell-surface adhesins that recognize ligands of host cells. Hyphal forms of the organism possess a 60-kDa mannoprotein that recognizes a variety of host-cell ligands including the complement C3 conversion products, C3bi and C3d. In addition, a protein of similar molecular mass also binds to endothelial extracellular matrix proteins such as laminin and fibronectin. While the 60-kDa protein is associated with the cell surface of hyphal forms of the organism, a protein of 50 kDa with similar ligand-binding activities is associated with the plasma membrane of blastoconidia. This protein cross reacts with antibodies to the 60-kDa protein. Isolation of the gene(s) encoding these cell-surface proteins is underway using both a human B-lymphocyte CR2 gene fragment or oligonucleotides based upon peptide sequence to screen libraries of C. albicans. Mutants of the organism with reduced expression of either C3d or C3bi-binding activity have been isolated. These strains are less virulent and also less adherent in vitro. Studies are currently underway to define the contribution of these proteins to the virulence of the organism. Key words: adherence, complement receptor, mannoprotein, virulence, ligand recognition.

scholarly journals In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16

2021 ◽  
Vol 22 (8) ◽  
pp. 4146
Author(s):  
Pharaoh Fellow Mwale ◽  
Chi-Hsin Lee ◽  
Peng-Nien Huang ◽  
Sung-Nien Tseng ◽  
Shin-Ru Shih ◽  
...  
Keyword(s):  
Viral Infection ◽  
Clinical Symptoms ◽  
Binding Protein ◽  
Foot And Mouth Disease ◽  
Binding Activity ◽  
In Vitro Assays ◽  
Single Chain ◽  
Coxsackievirus A16 ◽  
Causative Agents

Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.

Selection of Human Anti-CD22 scFvs from an Acute Lymphocytic Leukemia Related scFv Library with Ribosome Display.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4527-4527
Author(s):  
Achim Rothe ◽  
Ralf J. Hosse ◽  
Anne Nathanielsz ◽  
Frank Oberhaeuser ◽  
Elke Pogge von Strandmann ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Specific Binding ◽  
Lymphocytic Leukemia ◽  
Target Antigen ◽  
Single Chain ◽  
Ribosome Display ◽  
Cell Assays ◽  
Scfv Library ◽  
Human Scfv

Abstract Novel in vitro methods for screening of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Ribosome display represents an emerging and innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) in order to isolate specific binding reagents. Here we present the isolation of human scFv against the validated immunotherapeutic target antigen CD22 from a leukaemia patient-derived human scFv library using ribosome display technology. The ribosome-displayed scFv were enriched against the human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific target recognition was demonstrated by surface plasmon resonance and validated using in vitro cell assays.

scholarly journals Intrabodies targeting the Kaposi sarcoma–associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3797-3802 ◽  
Author(s):  
Sofia Corte-Real ◽  
Chris Collins ◽  
Frederico Aires da Silva ◽  
J. Pedro Simas ◽  
Carlos F. Barbas ◽  
...  
Keyword(s):  
Primary Effusion Lymphoma ◽  
Kaposi Sarcoma ◽  
Artificial Chromosome ◽  
Binding Activity ◽  
Nuclear Antigen ◽  
In Vitro Assays ◽  
Single Chain ◽  
Lymphoma Cells ◽  
Phage Display Technology

Kaposi sarcoma–associated herpesvirus (KSHV) latency-associated nuclear antigen-1 (LANA1) is essential for the maintenance and segregation of viral episomes in KSHV latently infected B cells. We report development of intracellular, rabbit-derived antibodies generated by phage display technology, which bind to N-terminal LANA1 epitopes and neutralize the chromosome-binding activity of LANA1. Although these cloned single-chain variable fragments (scFvs) show relatively low binding affinities for the LANA1 viral antigen in in vitro assays, they nonetheless outcompete KSHV-seropositive human sera for LANA1 epitope binding. In heterologous cells, intracellular intrabody expression inhibits LANA1-dependent plasmid maintenance of both an artificial plasmid containing KSHV LANA1 binding sequences and a bacterial artificial chromosome containing the entire KSHV genome. In KSHV naturally infected primary effusion lymphoma cells, intracellular intrabody expression causes a reduction or loss of the typical LANA1 punctate, nuclear pattern. This morphologically apparent LANA1 dispersion correlates to loss of viral episome by molecular analysis. These data suggest a novel approach to antiherpes viral therapy and confirm LANA1 is critical target for neutralization of KSHV viral latency.

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