Domain specific monoclonal anti-factor VIII antibodies generated by inclusion body-renatured factor VIII peptides

Production of monoclonal anti-factor VIII (FVIII) antibodies was hampered b y the availability of FVIII proteins devoid of albumin and the von Willebra nd factor (VWF). We showed a successful way to generate domain specific ant i-FVIII antibodies by using a series of Escherichia coli expressed FVIII. f usion peptides. A total of eight fusion peptides were synthesized to cover almost the entire coding region of FVIII. All except one of the fusion pept ides were insoluble and became aggregated as Inclusion bodies. Purification and refolding of the peptides were accomplished by solublizing them with d enaturants and dialyzing them in appropriate buffers, this being followed b y chromatography of the refolded fractions on a metal-ion chelating column. These purified FVIII fusion peptides were used individually or as a pool t o immunize mice and generate antibodies. Three monoclonal antibodies, D2, E 6 and B12, were obtained. D2 recognizes a region (residues 1680-1703) of th e light chain of FVIII, E6 recognizes a fragment (residues 744-1021) in the heavy chain, and B12, the Al domain (residues 89-326). Both D2 and B12 inh ibited >80% FVIII function. The affinities (k(A)) Of the antibodies for FVI II were 1.62 x 10(7) M-1 for D2 and 2.2 x 10(8) M-1 for E6. Although B12 is inhibitory, it did not show a strong binding affinity with FVIII. The spec ificity of D2 and E6 for FVIII was demonstrated by immunoprecipitation of t he FVIII protein in full-length recombinant FVIII (rFVIII) supplemented FVI II-deficient plasma, but not in FVIII-deficient plasma alone. An enzyme-lin ked immunosorbant assay (ELISA) using D2 or E6 was designed to detect plasm a FVIII. The system may be useful in monitoring FVIII in cultured supernata nts and in mouse models for gene therapy experiments. (C) 2001 Elsevier Sci ence Ltd. All rights reserved.