EPITOPE MAPPING OF HUMAN COAGULATION FACTOR VIII WITH IN VITRO SYNTHESIZED FRAGMENTS OF THE PROTEIN

We have used recombinant DNA techniques to map the epitopes of the coagulation Factor VIII for several monoclonal antibodies, raised against this protein. For this purpose, we cloned full- length- and partial Factor VIII cDNA sequences in the vector pSP64. Corresponding RNA fragments were synthesized in vitro with SP6 RNA polymerase and translated in a rabbit reticulocyte lysate system. The resulting Factor VII. I polypeptide fragments were immunoprecipitated. We have located the binding sites of a panel of monoclonal anti-Factor VIII antibodies. Two examples are shown in the figure below. The epitopes for anti-Factor VIII CLB-9 and CLB-65 have been confined to areas of 29 (711-740) and 142 (1635-1777) amino acids, respectively. The results of these studies will be useful in determining structure-function relationships of Factor VIII