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Southeast Asian J Trop Med Public Health. 2017 Jan;48(1):45-55.
CONSTRUCTION AND EXPRESSION OF H5N1 INFLUENZA VIRUS HEMAGGLUTININ-SPECIFIC scFv-Fc MONOCLONAL ANTIBODIES IN HEK293T CELLS.
Pipattanaboon C, Boonha K, Benjathummarak S, Pitaksajjakul P, Ramasoota P.
Abstract
Monoclonal antibody (MAb) is a key element in the development of rapid test kits for many infectious diseases. Our group previously developed two antigen-binding fragment (Fab) MAbs, H5Fab-6 and H5Fab-9, specific to hemagglutinin (H5 HA) of influenza A virus H5N1, but these Fabs do not have a constant fragment (Fc) portion with which to bind with gold particles in a strip test. In order to overcome this impediment, we joined a single-chain variable fragment (scFv) with an Fc region to produce a scFv-Fc MAb, which was expressed in mammalian HEK293T cells. Specificity and sensitivity of each generated scFv-Fc MAb for H5 HA was tested using western blotting and dot-enzyme-linked immunosorbent assay (dot-ELISA), respectively. Two scFv-Fcs (designated H5scFvFc-6 and H5scFvFc-9) were constructed and purified to near homogeneity with a yield of 12.87 mg/l and 33.56 mg/l, respectively. Western blotting indicated that both scFv-Fcs reacted as expected with H5 HA with a sensitivity of 60 pg of H5 HA. These scFv-Fc MAbs should prove useful in the development of antibody-based diagnostic tools.
CONSTRUCTION AND EXPRESSION OF H5N1 INFLUENZA VIRUS HEMAGGLUTININ-SPECIFIC scFv-Fc MONOCLONAL ANTIBODIES IN HEK293T CELLS.
Pipattanaboon C, Boonha K, Benjathummarak S, Pitaksajjakul P, Ramasoota P.
Abstract
Monoclonal antibody (MAb) is a key element in the development of rapid test kits for many infectious diseases. Our group previously developed two antigen-binding fragment (Fab) MAbs, H5Fab-6 and H5Fab-9, specific to hemagglutinin (H5 HA) of influenza A virus H5N1, but these Fabs do not have a constant fragment (Fc) portion with which to bind with gold particles in a strip test. In order to overcome this impediment, we joined a single-chain variable fragment (scFv) with an Fc region to produce a scFv-Fc MAb, which was expressed in mammalian HEK293T cells. Specificity and sensitivity of each generated scFv-Fc MAb for H5 HA was tested using western blotting and dot-enzyme-linked immunosorbent assay (dot-ELISA), respectively. Two scFv-Fcs (designated H5scFvFc-6 and H5scFvFc-9) were constructed and purified to near homogeneity with a yield of 12.87 mg/l and 33.56 mg/l, respectively. Western blotting indicated that both scFv-Fcs reacted as expected with H5 HA with a sensitivity of 60 pg of H5 HA. These scFv-Fc MAbs should prove useful in the development of antibody-based diagnostic tools.