Preparation, characterization and application of monoclonal antibodies against PAI-1.
Six hybridoma cell lines (AP1-AP6) secreting monoclonal antibodies (McAb) against PAI-1 were obtained by fusing the murine myeloma cell line SP2/0 with the spleen cells from Balb/c mouse immunized with recombinant PAI-1 expressed in E. coli. These antibodies were purified by SPA affinity chromatography. All McAbs recognized rPAI-1 and PAI-1 from the human hepatoma cell line HepG2. The titers of ascites were more than 10(6). The antibody-antigen affinity constants (Kaff) for anti-PAI-1 McAb measured by EIA were between 3.45 x 10(7)-1.05 x 10(10) M. AP2 and AP3 McAbs were effective in quenching the activity of PAI-1. Partial quenching of PAI-1 activity was achieved with AP4, AP5 and AP6 McAbs respectively. AP1 McAb had no effect upon PAI-1 activity. Three of the six McAbs (AP1, AP4 and AP5) bound to the PAI-1/t-PA complex, while the others did not. The PAI-1 was purified 51 folds to homogeneity from serum free medium of HepG2 with the recovery rate of 92% by one-step procedure using Sepharose 4B conjugated with anti-PAI-1 McAb (AP1, AP3 and AP4). A sandwich ELISA for the measurement of PAI-1 antigen in human plasma was developed, based on anti-PAI-1 McAb against non-overlapping epitopes. The mean value of plasma PAI-1 for the healthy donors was 24.7 +/- 7.75 ng/ml measured by ELISA.
Ding
H
H
Laboratory of Molecular Genetics, Shanghai Medical University, China.
Wu
X
X
Zhou
X
X
Zhu
Y
Y
Song
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D017395:Plasminogen Activator Inhibitor 1 / Q000097:blood* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator