Cod C
In order to make GENE silencing
Abstract
Over the past 20 years, a diverse group of ligands targeting surface biomarkers or receptors has been identified with several investigated to target siRNA to tumors. Many approaches to developing tumor-homing peptides, RNA and DNA aptamers, and single-chain variable fragment antibodies by using phage display, in vitro evolution, and recombinant antibody methods could not have been imagined by researchers in the 1980s. Despite these many scientific advances, there is no reason to expect that the ligand field will not continue to evolve. From development of ligands based on novel or existing biomarkers to linking ligands to drugs and gene and antisense delivery systems, several fields have coalesced to facilitate ligand-directed siRNA therapeutics. In this review, we discuss the major categories of ligand-targeted siRNA therapeutics for tumors, as well as the different strategies to identify new ligands.
Abstract
We have shown previously that detection of circulating antibodies against mimotopes selected by phage display were useful in neurocysticercosis diagnosis. However, circulating antigens may also be useful in patients' clinical follow-up. Therefore, we aimed to select novel combinatorial antibodies, single-chain variable fragment (scFv), which can be used for specific antigens with pre-defined affinity and specificity without prior immunization. A phage scFv antibody library was selected against Taenia solium mimotopes displayed on phages coupled in beads and total saline extract of T. solium metacestodes (S) immobilized on microtiter plate wells. After two rounds of selection, 96 phage clones were evolved and validated against each target by enzyme linked immunosorbent assay (ELISA), and dot-blot, and three specific antibodies (B6, G10 and A4) were further characterized by sequencing and indirect immunofluorescence (IFI) assays. IFI revealed tegument staining for the B6, while the others showed a non-uniform staining in the whole parasite. The selected scFvs were used to capture their antigen targets that were elucidated through mass spectrometry, and used for antibody detection in NC patients' sera by ELISA, which achieved sensitivities greater than 97% and specificities above 95%. We have successfully developed scFv antibodies against important mimotopes used in NC diagnosis, and can be further explored to detect circulating antigens for clinical follow-up of patients with NC. Our strategy also highlighted the possibility of using this combinatorial approach to select, capture and characterize specific antigens to better understand this intriguing parasite infection and disease evolution.
Protein Expression and Purification
Volume 171, July 2020, 105627
Selection, preparation and characterization of scFv against human lipocalin 6 by phage display technology
Author links open overlay panelJiongChenaWeiFenga
Show more
Outline
Share
Cite
https://doi.org/10.1016/j.pep.2020.105627Get rights and content
Highlights
•
A murine scFv library specific for hLCN6 was produced with a simple and rapid method using phage display technology.
•
Functional anti-hLCN6 scFv with good specificity and affinity was solubly expressed and purified from E.coli in high purity.
•
This scFv is a potencial tool for sperm separation from forensic mixed stains by immunomagnetic beads method at low cost.
Abstract
Human lipocalin 6 (hLCN6) is a newly discovered epididymal-specific secreted protein, capable of binding to the head and tail of spermatozoa and involved in sperm maturation. Anti-hLCN6 monoclonal antibody coupled immunomagnetic beads (IMBs) can be effectively used for the separation and forensic identification of sperm cells from mixed stains. But the source of monoclonal antibody is limited. In this study, an immunized mouse phage display antibody library was constructed and the single-chain variable fragments (scFvs) against hLCN6 were screened. The selection was performed using four rounds of biopanning and positive clones were validated by phage ELISA. Two anti-hLCN6 scFv clones with highest affinity were selected and sequencing result showed that the two sequences were identical. After prokaryotic expression and purification, the purified scFv could specifically recognize the hLCN6 in the lysate of human sperm cells and epididymis by western blot analysis, without any cross-reactivity with cellular antigens in female epithelial cells. The dissociation constant (Kd) of anti-hLCN6 scFv was 6.69 × 10−7 mol/L measured by indirect ELISA. Therefore, our work not only provides a useful tool for further exploration of the biological functions of hLCN6, but also opens up new research avenues for the separation of sperm cells from mixed stains based on immuno-binding reaction.