title: Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing.
abstract: An expression system for mono- and bivalent single-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli. The cDNA encoding the Fv fragment of the anti-D monoclonal antibody D10 was cloned using the polymerase chain reaction and expressed in E.coli by fusing with a peptide tag link in the C-terminus of the light chain variable region. The scFv fragment expressed by the bacteria produced specific agglutination of human D positive red cells in the presence of an anti-peptide tag antibody. Flow cytometric analysis clearly indicated that the bacterially prepared scFv showed high specificity and affinity for D antigen, which was identical with that of the parental IgG. In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, bacterial alkaline phosphatase (BAP). The fusion protein produced significant agglutination of human red blood cells with D antigen, confirming that the bacterially expressed fusion protein is a functional bivalent antibody fragment. Specific agglutination of D positive red cells by D10 scFv-BAP was enhanced in the presence of anti-BAP antibody, suggesting that further multimerization of scFv led to highly efficient cell agglutination. By grafting BAP enzymatic activity into the scFv fragment (enzyme-linked scFv), blood typing could conveniently be performed. These results indicate that bacterially expressed scFv and scFv-BAP would be of practical use in blood typing. The system reported here could also be applied to the examination of other cell surface antigens and cell agglutination.
authors.affiliation: Japanese Red Cross Central Blood Center, Hiroo, Tokyo.
authors.forename: M
authors.initials: M
authors.lastname: Furuta
authors.forename: M
authors.initials: M
authors.lastname: Uchikawa
authors.forename: Y
authors.initials: Y
authors.lastname: Ueda
authors.forename: T
authors.initials: T
authors.lastname: Yabe
authors.forename: T
authors.initials: T
authors.lastname: Taima
authors.forename: K
authors.initials: K
authors.lastname: Tsumoto
authors.forename: S
authors.initials: S
authors.lastname: Kojima
authors.forename: T
authors.initials: T
authors.lastname: Juji
authors.forename: I
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000235:genetics; D001788:Blood Grouping and Crossmatching; D003001:Cloning, Molecular; D019281:Dimerization; D004903:Erythrocyte Aggregation / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015870:Gene Expression / Q000235:genetics; D005803:Genes, Immunoglobulin / Q000235:genetics; D006384:Hemagglutination / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D012204:Rh-Hr Blood-Group System / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D017386:Sequence Homology, Amino Acid
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; C055966:immunoglobulin Fv; D000469:Alkaline Phosphatase