Selection of monoclonal antibodies that bind and inhibit tissue-type plasminogen activator.
Three monoclonal antibodies raised against tissue-type plasminogen activator (t-PA) were selected for their ability to inhibit solid-phase bound t-PA. Each monoclonal antibody blocked the release of p-nitroaniline from H-D-Ile-Pro-Arg-pNA (S-2288). The first antibody 1D2 was a gamma 2b, kappa with KD = 8 x 10(-9) M, the second antibody 2B9 was a gamma 1, kappa with KD = 2 x 10(-9) M, and the third antibody 5A9 was a gamma 1,kappa with KD = 4 x 10(-10) M. In solution-phase format each antibody blocked the conversion of plasminogen to plasmin as judged by a plasmin assay and also inhibited t-PA-mediated lysis of plasma fibrin clot in plasma. The binding of each 125I-radiolabeled antibody to t-PA was inhibited by any one of the three antibodies, suggesting that they recognized a common epitope on t-PA which was absent on unfolded t-PA. We concluded these antibodies bind near t-PA active site since PPACK treatment lowered binding of two antibodies. We believe solid-phase chromogenic substrate assay may be a useful way to screen for antibodies directed against the active site of proteases.
Ball
E L
EL
Macromolecular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000.
Dunlop
K
K
Matsueda
G R
GR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D005260:Female; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000031:analogs & derivatives / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism; D010969:Plastics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009842:Oligopeptides; D010969:Plastics; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; C018788:valyl-leucyl-lysine 4-nitroanilide; C027226:(4-amidinophenyl)methanesulfonyl fluoride; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone