| Research Abstract |
It has been known that various biomolecules displayd on cell surfaces characterize the eukaryote cells. Human monoclonal antibody specific for these biomolecules on human cell surfaces have immense potentials for medical supplies, e.g.regulation of cell function and examination of diseases. However, the difficulty in preparation of hybridoma producing a specific antibody have restricted the supply of the antibody molecules. In this research, human red cell is taken up as a model cell, and two goals have been focused ; (1) preparation of human antibody molecules specific for human cell surface antigen by further improvement of phage display system developed recently, and construction of systematical preparation system of human antibody fragments ; (2) grafting other functions and/or multivalency into an antibody fragment prepared by genetic engineering, and its application of various inspection of cell diseases. The results can be summarized as follows ; (1) An expression system for sin
… More
gle-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli. 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. (2) In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, becterial 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. Less
|
