Reactivity of antibodies with modified heparin preparations (inhibition ELISA).

Periplasmic fractions containing antibodies were allowed to bind to chemically modified heparin preparations for 16 hours, and then transferred to heparin-coated wells. Antibodies bound to heparin were detected using anti-VSV mouse monoclonal antibody P5D4, followed by alkaline phosphatase–conjugated rabbit antimouse IgG. Enzymatic activity was measured using p-nitrophenyl phosphate as a substrate. Values represent the reactivity in percent relative to heparin. Chemically modified heparin preparations used were: completely desulfated, N-sulfated heparin (■); N-desulfated and N-acetylated heparin (▴); completely desulfated and N-acetylated heparin (●); unmodified heparin (♦). (A) Antibody EW3F5. (B) Antibody EW4A4. Note reactivity of EW3F5, but not EW4A4, with N-desulfated and N-acetylated heparin.