Cloning and Expression of a Recombinant Single-Chain Variable Fragment Antibody Specific to Hemoglobin Bart’s

Abstract

Hemoglobin Bart’s (γ4 ), an abnormal Hb, is a homotetramer of γ-globin chains . Τhe amount of this abnormal Hb in blood circulation can be used as an indicator for the presence of different genotypes of α-thalassemias. We successfully cloned and expressed a novel recombinant scFv antibody derived from mouse hybridoma producing monoclonal antibody highly specific to Hb Bart’s. The genes encoding variable regions of the heavy (VH) and light (VL) chains were cloned and identified by DNA sequencing. The VH and VL genes were connected via a short linker to form the full length VH-linker-VL construct and ligated into pET28a. The His tag-scFv fusion protein was expressed in E. coli and purified by affinity chromatography. The recombinant scFv antibody was mostly expressed as inclusion bodies with the predicted molecular weight of 28 kDa. This scFv antibody was very specific by reacting with Hb Bart’s (γ4) but not cross-react with HbA (α2β2), HbF (α2γ2), HbS (α2β2 S ), HbE (α2β2 E ), HbA2 (α2δ2 ), and HbH (β4 ), as determined by Western blot. The detection sensitivity of this scFv antibody was 5 µg/µl of Hb Bart’s by dot blot ELISA. The scFv antibody should be useful in development of an immunoassay with high sensitivity and specificity for the diagnosis of α-thalassemias.