Browsing by Author "Ahmed Kamal Bolad, Elham Omer Mahgoub"

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    Construction, expression and characterisation of a single chain variable fragment in the Escherichia coli periplasmic that recognise MCF-7 breast cancer cell line
    (Medknow Publications, 2014-04-01) Ahmed Kamal Bolad, Elham Omer Mahgoub
    Background: A functional  single-chain fragment variable (scFv) recognizing the  MCF-7 breast cancer carcinoma cell line was constructed from the C3A8 hybridoma using phage display technology. Aim of Study: This study was conducted to evaluate the binding activity of scFv antibody recognise MCF-7 breast cancer cells carcinoma, the scfv antibody constructed and expressed in Escherichia coli periplasmic. Materials and Methods: The scFv coding sequence was cloned in frame with the pIII phage coat protein. The signal sequence included in the C terminus directed the expression of the scFv in the Escherichia coli periplasm. Following several rounds of biopanning, colonies that expressed a scFv that recognized MCF-7 cells in Western blots, ELISAs, and flow cytometry test were isolated. Results: A 750-bp scFv gene was successfully isolated. Cloning and two rounds of biopanning isolated the candidate with the highest activity (clone B7), as screened by ELISA. Following poly-acrylamide gel electrophoresis (SDS-PAGE) of the purified product, a 32-kDa band was observed. A similar-sized band was observed following Western blot analysis with an E tag-specific antibody. Binding reactivity of scFv antibody with MCF cells was determined using indirect ELISA and compared with monoclonal antibodies' reactivity. Also, flow cytometry was useful in further characterization to the binding reactivity of scFv antibody with MCF-7 cells. Conclusions: The recombinant antibody technology used in this study is a rapid and effective approach that will aid in the development of the next generation of immunodiagnostic reagents.