Reactivity of isolated scFv with factor VIII/factor V light-chain hybrids.

(A) Below a sequence alignment of factor VIII region Q1778-D1840 with the corresponding sequence of factor V, a schematic representation of the hybrid factor VIII/factor V light-chain fragments used in this study is depicted. The regions R1803-K1818 (HV1803-1818), Q1778-H1821 (HV1778-1821), and K1804-D1840 (HV1804-1840) in the factor VIII light chain that have been replaced for the corresponding factor V sequence are indicated as dotted boxes. Hatched boxes correspond to the acidic region a3, composed of residues E1649-R1689. (B) Binding of scFv to recombinant factor VIII/factor V light-chain hybrids was assessed by immunoprecipitation. (Lane 1) positive (+) control (antibody CLB-CAg 117); (lanes 2-7) 33, 35, 36, 37, 38, and 41; scFv corresponding to clones KM33, KM35, KM36, KM37, KM38, and KM41; (lane 8) negative (−) control (scFv directed toward the A2 domain of factor VIII); (lane 9)14 positive control (scFv EL-14, directed toward the C2 domain of factor VIII); (lane 10) antibody (A) antibody CLB-CAg A. On the left, the used factor VIII fragments are indicated.