Epitope mapping of LE2E9.

(A) Structure of circulating fVIII. Each domain of fVIII is identified by a letter in upper case followed by a numeral. The small acidic regions are indicated by a1, a2, and a3, respectively. The copper ion involved in the non-covalent association of the A1 and A3 domains is indicated. (B) Recombinant fVIII fragments labeled with [³⁵S]methionine and expressed in reticulocyte lysates were incubated for 2 hours at 4°C with LE2E9 bound to Protein A Sepharose. After washing, bound material was eluted and analyzed by SDS-PAGE, followed by autoradiography. Recombinant fVIII fragments are indicated by open bars. The amino- and carboxy-terminal extremities of fragments are as indicated. Control experiments were carried out with a human monoclonal antibody, BO2C11, directed toward the fVIII C2 domain19 and a rabbit polyclonal IgG antibody, aA3, directed toward amino acid residues 1797-1815 of the fVIII A3 domain The experiments were repeated at least 3 times with comparable results.