Characterization of Chicken-Derived Single Chain Antibody Fragments against Venom of Naja Naja Atra

Traditional, horse-derived antivenin is currently the most efficient treatment against snake bites. However, it is costly and has unpredictable side effects. Thus, alternative, cost-effective strategies for producing antivenin are needed. In this study, we immunized hens with inactivated NNA venom proteins from the cobra Naja naja atra (NNA). Purified yolk IgY antibodies showed specific anti-NNA binding activity comparable to that of the equine-derived antivenin. We used phage display technology to generate two antibody libraries containing 9.0 × 108 and 8.4 × 108 clones with a short or long linker, respectively. The phage ELISA indicated that anti-NNA clones displaying single-chain variable fragments (scFv) were significantly enriched after biopanning. The nucleotide sequences of the light and heavy chain genes of 30 monoclonal scFv antibodies were determined and classified into six groups with the short linker and nine groups with the long linker. These scFv clones specifically bound to NNA proteins but not to venom proteins from other snakes. Their binding affinities were further determined by competitive ELISA. Animal model studies showed that anti-NNA IgY antibodies exhibited complete protective effects, while a combination of scFv antibodies raised the survival rates and times of mice challenged with lethal doses of NNA venom proteins.

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Purification, characterization and binding interactions of the Chinese-cobra (Naja naja atra) serum antitoxic protein CSAP

Biochemical Journal ◽

10.1042/bj2930559 ◽

1993 ◽

Vol 293 (2) ◽

pp. 559-566 ◽

Cited By ~ 16

Author(s):

J Shao ◽

H Shen ◽

B Havsteen

Keyword(s):

Protective Effect ◽

Binding Capacity ◽

Sequence Similarity ◽

Deae Cellulose ◽

Single Chain ◽

Rat Serum ◽

Binding Interactions ◽

Naja Naja ◽

Naja Atra

The characterization of the single-chain protein in Chinese-cobra (Naja naja atra) blood serum, which yields strong specific protection against the venom of the same snake, is reported. The protein, CSAP (cobra serum antitoxic protein), was purified to electrophoretic homogeneity. Over the pH range 5-9 it formed stable complexes with the neuro- and the cardio-toxin of the snake. The molecular size of the CSAP was estimated to be 70.3 +/- 0.3 kDa. Tryptic hydrolysis of CSAP yielded several peptides that were able to bind to the toxin. The native CSAP maximally bound 8 +/- 1 toxin molecules/molecule. Six tryptic fragments, containing 5-39 residues, were sequenced. The longest of these displayed sequence similarity to rat serum albumin. The protective effect of the CSAP was demonstrated in vivo on mice and in vitro by measurement of the rate of haemolysis. Kinetic and thermodynamic parameters of the binding interactions of the neurotoxin and the CSAP were determined from the rates of displacement of 125I-labelled toxin from its complexes with the CSAP by unlabelled toxin by using a DEAE-cellulose filter assay for CSAP-toxin complexes. The toxin molecules rapidly dissociated from one type of site and slowly from a second. The binding capacity and concentration of the CSAP suffice to explain the protective effect of the latter against the toxin.

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Selection of Linkers for a Catalytic Single-chain Antibody Using Phage Display Technology

Journal of Biological Chemistry ◽

10.1074/jbc.271.26.15682 ◽

1996 ◽

Vol 271 (26) ◽

pp. 15682-15686 ◽

Cited By ~ 46

Author(s):

Ying Tang ◽

Ning Jiang ◽

Cushrow Parakh ◽

Donald Hilvert

Keyword(s):

Phage Display ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Technology ◽

Display Technology ◽

Selection Of

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Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108330113 ◽

2009 ◽

Vol 14 (3) ◽

pp. 282-293 ◽

Cited By ~ 38

Author(s):

Laura Turunen ◽

Kristiina Takkinen ◽

Hans Söderlund ◽

Timo Pulli

Keyword(s):

Phage Display ◽

High Efficiency ◽

Magnetic Bead ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Technology ◽

Antibody Phage ◽

Antibody Phage Display ◽

Display Technology

Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human γ-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 γ-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and β-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 β-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency. ( Journal of Biomolecular Screening 2009:282-293)

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Development of a Novel Human Single Chain Antibody Against EGFRVIII Antigen by Phage Display Technology

Advanced Pharmaceutical Bulletin ◽

10.15171/apb.2016.070 ◽

2016 ◽

Vol 6 (4) ◽

pp. 563-571 ◽

Cited By ~ 1

Author(s):

Leila Rahbarnia ◽

Safar Farajnia ◽

Hossein Babaei ◽

Jafar Majidi ◽

Bahman Akbari ◽

...

Keyword(s):

Phage Display ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Technology ◽

Display Technology

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Nectin-like Protein 2 Defines a Subset of T-cell Zone Dendritic Cells and Is a Ligand for Class-I-restricted T-cell-associated Molecule

Journal of Biological Chemistry ◽

10.1074/jbc.m502095200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 21955-21964 ◽

Cited By ~ 136

Author(s):

Laurent Galibert ◽

Geoffrey S. Diemer ◽

Zhi Liu ◽

Richard S. Johnson ◽

Jeffrey L. Smith ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Antibody Fragment ◽

Class I ◽

Cytotoxic Lymphocytes ◽

Immune Functions ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Technology

Dendritic cells (DCs) are a phenotypically and functionally heterogenous population of leukocytes with distinct subsets serving a different set of specialized immune functions. Here we applied an in vitro whole cell panning approach using antibody phage display technology to identify cell-surface epitopes specifically expressed on human blood BDCA3+ DCs. A single-chain antibody fragment (anti-1F12 scFv) was isolated that recognizes a conserved surface antigen expressed on both human BDCA3+ DCs and mouse CD8α+ DCs. We demonstrate that anti-1F12 scFv binds Nectin-like protein 2 (Necl2, Tslc1, SynCaM, SgIGSF, or Igsf4), an adhesion molecule involved in tumor suppression, synapse formation, and spermatogenesis. Thus, Necl2 defines a specialized subset of DCs in both mouse and human. We further show that Necl2 binds Class-I-restricted T-cell-associated molecule (CRTAM), a receptor primarily expressed on activated cytotoxic lymphocytes. When present on antigen presenting cells, Necl2 regulates IL-22 expression by activated CD8+ T-cells. We propose that Necl2/CRTAM molecular pair could regulate a large panel of cell/cell interactions both within and outside of the immune system.

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A Novel Fully Human Recombinant Antibody Neutralizing the α-hemolysin of Staphylococcus Aureus

10.21203/rs.3.rs-88302/v1 ◽

2020 ◽

Author(s):

Lei Peng ◽

Chun Li ◽

Tong Wu ◽

Haiyan Zhang ◽

Han Chen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Recombinant Antibody ◽

A549 Cells ◽

Human Antibody ◽

Protective Effects ◽

Single Chain ◽

Suspension Cells ◽

Single Chain Antibody ◽

Human Scfv ◽

Mrsa Infection

Abstract BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is resistant to almost all β-lactam antibiotics. Hence, new ways to control MRSA infection, such as antibacterial antibodies, need to be explored. α-Hemolysin is the most important virulence factor widely expressed in S. aureus. This study aimed to develop a new fully human antibody against α-hemolysin of S. aureus and explore its neutralizing effect. ResultsThe single-chain antibody fragments（scFvs）against S. aureus were screened from a fully human scFv library using phage display technology with purified α-hemolysin protein. The selected scFvs had good binding affinities to α-hemolysin and were highly specific to S. aureus. The IgG-like scFv-Fc inserted into the pcDNA3.1 or pMH3 vector was expressed in HEK293F suspension cells to extend the antibody's in vivo half-life and restore Fc function. The size of purified scFv-Fc was about 55 kDa. The functions of expressed scFv-Fcs against α-hemolysin were validated. The results of A549 cytotoxicity assays showed that scFv10-Fc and scFv555-Fc had better protective effects than other scFv-Fcs on A549 cells. The results of anti-rabbit erythrocyte lysis assay confirmed that scFv555-Fc had a significant neutralizing effect on toxins. The scFv555-Fc was used to construct the docking model of antigen–antibody complexes using Discovery Studio software. It predicted that the key binding sites of α-hemolysin were TYR28, LYS37, PHE39, ARG56, and LYS58, which might be the key toxic sites of α-hemolysin.ConclusionsA novel fully human scFv-Fc antibody neutralizing the α-hemolysin toxin of S. aureus was successfully developed. The findings might provide a new theoretical basis and treatment method for preventing MRSA infection.

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Purification of a Novel Anti-VEGFR2 Single Chain Antibody Fragmentand Evaluation of Binding Affinity by Surface Plasmon Resonance

Advanced Pharmaceutical Bulletin ◽

10.15171/apb.2019.008 ◽

2019 ◽

Vol 9 (1) ◽

pp. 64-69 ◽

Cited By ~ 3

Author(s):

Shirafkan Kordi ◽

Mohammad Rahmati-Yamchi ◽

Mehdi Asghari Vostakolaei ◽

Abolfazl Barzegari ◽

Jalal Abdolalizadeh

Keyword(s):

Surface Plasmon Resonance ◽

Phage Display ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Sodium Dodecyl ◽

Scfv Antibody ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Technology ◽

Sds Page

Purpose: The single-chain variable fragment (scFv) domain of antibodies is now considered asone of the therapeutic tools that can be produced by phage display technology (PDT). Antibodypurification is one of the most important steps in antibodies production. The aim of study waspurification and characterization of anti-VEGFR2 scFv antibody fragments.Methods: After the coating of vascular endothelial growth factor receptor 2 (VEGFR2) peptidein ELISA microplates, the phage display library of Tomlinson was used for antibody isolation.The targeted scFv was purified by chromatography using a zeolite-based column. The purity andfunctional assessment of purified scFv were evaluated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and western blotting techniques, respectively. Affinity bindingwas evaluated by surface plasmon resonance (SPR).Results: The desired scFv was selected after four stages of biopanning. SDS-PAGE analysisshowed a 28 kDa scFv with high purity (>90%). The western bloting analysis confirmed thebinding of produced scFv antibody to the desired peptide. The affinity binding of scFv antibodyanalyzed by SPR was about 60 μM.Conclusion: In this study, the novel scFv antibody against VEGFR2 peptide was purified bychromatography column containing zeolite. Based on our findings the produced antibody maybe applied for diagnosis or targeting of VEGFR2 in antibody-based therapy strategies.

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