1887

Abstract

Abstract AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody recognizing the human breast cancer cell line (MCF7). METHODS: mRNA was isolated from the hybridoma cell line producing C3A8 monoclonal antibody and the cDNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified Next, the ScFv DNA was ligated into the phagemid vector pCANTAB 5E and later transformed into E.coli TG1. Following transformation, the cells were then infected with M13KO7 helper. Then two biopanning step was carried ScFv-phages fusions antibodies were selected by ELISA. One selected clone was used to infect E.coli HB2151. The soluble ScFvs were then identified by Western blot, and their antigen-binding activity was assayed by ELISA. The scFv gene was then cloned into nova-blue host strain for cloning purposes and then into origami DE host strain for further characterization. The purified single-chain antibody expressed in origami DE3 was purified using Immobilized Metal Affinity Chromatography (IMAC). The purified scFv protein was characterized using western blot, flow Cytometery and immunofluorescence tests. Bioinformatics tools were also used and databases to gain specific functional insights into scFv anti-MCF-7. Finally, the VH and VL chains models were evaluated. RESULTS: The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. The strongest positive clone (B7) was used to proceed for larger scale antibody production. The DNA sequencing results show that the ScFv gene has a gene bank similarity of 99%. Further, immuno-fluorescence test clearly proved that the scFv recognized the MCF-7 antigen epitopes which is localized in MCF-7 nuclear. Also, 53% of the cells numbers were bound to scFv protein as measured by flow cytometery analysis. Finally, the predicted structures of heavy and light chains were connected with peptide linker to build the full scFv protein structure. CONCLUSION: The soluble ScFv of is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Loading

Article metrics loading...

/content/papers/10.5339/qfarc.2014.HBPP0705
2014-11-18
2020-08-10
Loading full text...

Full text loading...

http://instance.metastore.ingenta.com/content/papers/10.5339/qfarc.2014.HBPP0705
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error