Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells

Recombinant antibody cloning and phage display technologies can be employed to produce and isolate single-chain antibodies (scFv) specifically against antigen of interest. The aims of this study are to construct single chain variable fragment (scFv) towards MCF-7 breast cancer cells and to characterize scFv antibodies that interacts with MCF-7. Initially, mRNA was extracted from previously well-characterized monoclonal antibody (C3A8) against MCF-7. The genes encoding heavy (VH) and light (VL) chains were amplified, linked in VH-VL orientation via PCR and cloned into a pCANTAB 5E phagemid vector. The protein was then expressed in a supE strain of E. coli TG1. Phage particles displaying scFv were panned against MCF-7 and the selected clones were further used for infecting non-suppressor strain, E. coli HB2151. The scFv antibodies expressed were characterized by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. As demonstrated by ELISA result, the scFv antibodies could strongly bind to MCF-7 breast cancer cells. It retained the binding capacity of its parental C3A8 monoclonal antibody. Clone B7 was expressed mainly as soluble periplasmic protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 32 kDa. Nucleotide sequence analysis of C3A8 scFv showed high homology (99%) with published single chain antibody against rice stripe virus protein P20 [synthetic construct]. In conclusion, the recombinant antibody technology is an effective approach in the development of scFv antibody for the next generation of immunotherapy reagents especially towards MCF-7 breast cancer cells

scFv Antibody: Principles and Clinical Application

To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper

Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells.

A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications