scholarly journals Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Shukra M. Aavula ◽  
Sridevi V. Nimmagadda ◽  
Neelakantam Biradhar ◽  
Samuel Sula ◽  
Dev Chandran ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Specific Binding ◽  
Recombinant Antibody ◽  
Antigenic Site ◽  
Antibody Fragment ◽  
Single Chain ◽  
Phage Display Technology ◽  
Single Chain Fv ◽  
Rabies Glycoprotein

Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv) A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain) using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

scholarly journals Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2311
Author(s):  
Kohei Yumoto ◽  
Tomoaki Arisaka ◽  
Kazuma Okada ◽  
Kyosuke Aoki ◽  
Toyoyuki Ose ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Expression System ◽  
Case Fatality ◽  
Baculovirus Expression System ◽  
Amino Acid Sequences ◽  
Single Chain ◽  
Neutralizing Activity ◽  
Binding Characteristics ◽  
Single Chain Fv

Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15–13 and 12–22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15–13 and 12–22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm–baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15–13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12–22 Fab has a weaker binding affinity (KD ~ M) with the RABVG compared to the 15–13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15–13 and 12–22 antibodies as another potential biopharmaceutical for targeting rabies. The 15–13 and 12–22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the µM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

scholarly journals Expression, Purification, and Characterization of Anti-Zika virus Envelope Protein: Polyclonal and Chicken-Derived Single Chain Variable Fragment Antibodies

2020 ◽  
Vol 21 (2) ◽  
pp. 492 ◽  
Author(s):  
Pharaoh Fellow Mwale ◽  
Chi-Hsin Lee ◽  
Liang-Tzung Lin ◽  
Sy-Jye Leu ◽  
Yun-Ju Huang ◽  
...  
Keyword(s):  
Zika Virus ◽  
Envelope Protein ◽  
Therapeutic Potential ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Virus Envelope ◽  
Phage Display Technology ◽  
High Level

Zika virus (ZIKV) is a new and emerging virus that has caused outbreaks worldwide. The virus has been linked to congenital neurological malformations in neonates and Guillain–Barré syndrome in adults. Currently there are no effective vaccines available. As a result, there is a great need for ZIKV treatment. In this study, we developed single chain variable fragment (scFv) antibodies that target the ZIKV envelope protein using phage display technology. We first induced an immune response in white leghorn laying hens against the ZIKV envelope (E) protein. Chickens were immunized and polyclonal immunoglobulin yolk (IgY) antibodies were extracted from egg yolks. A high-level titer of anti-ZIKV_E IgY antibodies was detected using enzyme-linked immunosorbent assay (ELISA) after the third immunization. The titer persisted for at least 9 weeks. We constructed two antibody libraries that contained 5.3 × 106 and 4.5 × 106 transformants. After biopanning, an ELISA phage assay confirmed the enrichment of specific clones. We randomly selected 26 clones that expressed ZIKV scFv antibodies and classified them into two groups, short-linker and long-linker. Of these, four showed specific binding activities toward ZIKV_E proteins. These data suggest that the polyclonal and monoclonal scFv antibodies have the diagnostic or therapeutic potential for ZIKV.

scholarly journals Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e15783 ◽  
Author(s):  
Nives Škrlj ◽  
Tanja Vranac ◽  
Mara Popović ◽  
Vladka Čurin Šerbec ◽  
Marko Dolinar
Keyword(s):  
Specific Binding ◽  
Antibody Fragment ◽  
Single Chain
Sign in / Sign up

Export Citation Format

Share Document