Quantitative assay of the carbohydrate in urokinase-type plasminogen activator by lectin-enzyme immunoassay.
We developed an enzyme immunoassay (EIA) for the measurement of Asn302-linked carbohydrate in urokinase-type plasminogen activator (u-PA) using peroxidase (HRP)-labelled lectins. u-PA antigen in the sample was immunologically bound to microtitre plate wells by anti-u-PA IgG and the binding of HRP-labelled lectins [Con A (Concanavalin A), WGA (wheat germ agglutinin), PNA (peanut agglutinin), CSA (Scotch broom), GS-I (Groffonia simplicifollia) and SBA (soybean agglutinin)] to the carbohydrate of u-PA was measured. The lectin-EIA was dose-dependent in the range 6-6000 IU/ml of u-PA using Con A and WGA. The assay did not detect the carbohydrate of bovine albumin, ovalbumin, human albumin, plasminogen, tau-globulin and fibrinogen. The binding of HRP-labelled Con A and WGA to the carbohydrate of u-PA was specifically inhibited by alpha-methylmannose and N-acetylglucosamine respectively. Endo F treatment of the carbohydrate of u-PA decreased the binding of Con A and WGA. The carbohydrate of u-PA obtained from chest fluid, ascites and U937 cell culture medium reacted with Con A and WGA by this assay forming a band in the 55 kDa region. These results suggest that the lectin-EIA method is suitable for the assay of the carbohydrate in the B-chain of u-PA.
Hayashi
S
S
Thrombosis Chemical Institute, Tokyo, Japan.
Yamada
K
K
D000117:Acetylglucosamine / Q000494:pharmacology; D001202:Ascitic Fluid / Q000201:enzymology; D002241:Carbohydrates / Q000032:analysis*; D002460:Cell Line; D003208:Concanavalin A / Q000378:metabolism; D005122:Exudates and Transudates / Q000201:enzymology; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D037102:Lectins / Q000378:metabolism*; D008264:Macrophages / Q000201:enzymology; D008297:Male; D008766:Methylmannosides / Q000494:pharmacology; D008875:Middle Aged; D009000:Monocytes / Q000201:enzymology; D013909:Thorax; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D002241:Carbohydrates; D037102:Lectins; D008766:Methylmannosides; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; C008466:methylmannoside; D014568:Urokinase-Type Plasminogen Activator; D000117:Acetylglucosamine