Definition of epitopes within plasminogen activator inhibitor type-1 (PAI-1) using multiple peptide synthesis

Three regions in PAI-1 were selected for epitope analysis on the basis of their proposed significance for the functional activity of PAI-1 and their surface-location. Antisera were raised against 3 peptides synthesized for the regions ¹¹¹P-V^{121 125}D-N¹³⁷ and ³³⁵A-E³⁵⁰ in PAI-1 and were evaluated by dot immunobinding assay. Confirmation of the antigenicity of the peptides was followed by synthesis of solid-phase sequential overlapping octapeptides for each region. Binding of the octapeptides to antisera against the 3 peptides and to a panel of monoclonal antibodies against PAI-1 was evaluated by ELISA.Antipeptide serum ¹²⁵D-N¹³⁷ was most reactive with the amino acid sequence ¹²⁶FSEVERAR¹³³ and antipeptide serum ³³⁵A-E³⁵⁰ reacted strongly with the 2 octapeptides in the sequence ³⁴²IVSARMAPE³⁵⁰. Two of 10 monoclonal antibodies recognized continuous epitopes within the synthesized peptides. ESPI-10 bound to ¹²⁵DFSEVERA¹³² and ESPI-12 bound to ³⁴²IVSARMAP³⁴⁹, a region spanning the reactive centre of PAI-1.