Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification.
Hybridomas producing a monoclonal IgG1 antibody to a human plasminogen-activating enzyme with an apparent mol. wt. of 66,000 (66 K, HPA66) from human melanoma cells were obtained by fusion of NSI-Ag 4/1 mouse myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of the enzyme. Screening for clones of hybridomas producing antibodies to HPA66 was performed with the impure enzyme preparation. A preliminary screening included enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting; the final identification was based on inhibition of the enzymatic activity of HPA66 which was complete at high antibody concentrations. No inhibition of three other human and murine plasminogen activators or of plasmin was observed. Employing a one-step affinity procedure with the antibody coupled to Sepharose, HPA66 was purified approximately 200-fold from conditioned medium from the melanoma cells with a yield of 79%. The purified HPA66 was homogeneous as evaluated by SDS-PAGE. Electrophoresis under reducing conditions indicated that it consisted of one polypeptide chain. The binding constant between the antibody and 125I-labelled HPA66 was approximately 2.5 x 10(9) l/mol. The antibody did not bind to a variety of other plasminogen activators, including 52-K and 36-K human enzymes and 48-K and 75-K murine enzymes. Previously, a monoclonal antibody against another enzyme was derived by the sole use of enzyme inhibition for screening. The present study represents a modification of this procedure that can be used when antibody-unrelated inhibitors of the enzyme are present in hybridoma culture fluid.
Nielsen
L S
LS
Laboratory of Tumor Biology, Institute of Pathology, University of Copenhagen, Denmark.
Hansen
J G
JG
Andreasen
P A
PA
Skriver
L
L
Danø
K
K
Zeuthen
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D006801:Humans; D008545:Melanoma; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors* / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D010960:Plasminogen Activators