Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies.
Urokinase plasminogen activator (uPA) is a proteolytic enzyme involved in degradation of the extracellular matrix during cancer invasion. The levels of uPA and its inhibitor PAI-1 in tumor extracts have previously been demonstrated to be of prognostic value in breast cancer as well as other types of cancer. We have previously characterized a specific cell surface receptor for uPA (uPAR) which strongly enhances the catalytic activity of uPA and is expressed during mammary cancer invasion. In order to quantitate uPAR in breast cancer tissue, we have now developed a sensitive enzyme-linked immunosorbent assay (ELISA), with polyclonal catching antibodies and three monoclonal detecting antibodies. The detection limit of the assay is approximately 0.16 fmol of uPAR in a volume of 100 microliters (1.6 pM). There is a linear relationship between signal and uPAR concentration up to at least 6.6 fmol per 100 microliters (66 pM). Both free uPAR and uPAR in complex with uPA is detected. The recovery of an internal uPAR standard in breast cancer tissue extracts is above 87%. The intra-assay and inter-assay variation coefficients are 7% and 13%. In order to find a suitable buffer for extraction of various components of the uPA-system from breast cancer tissue, we tested buffers which previously have been used for optimal extraction of estrogen receptor (A), uPA (B), and uPAR (C). Buffer A and B extracted approximately 30% and 50%, respectively, of the amount of uPAR extracted with buffer C.(ABSTRACT TRUNCATED AT 250 WORDS)
Rønne
E
E
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
Høyer-Hansen
G
G
Brünner
N
N
Pedersen
H
H
Rank
F
F
Osborne
C K
CK
Clark
G M
GM
Danø
K
K
Grøndahl-Hansen
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000201:enzymology*; D002021:Buffers; D016466:CHO Cells; D018270:Carcinoma, Ductal, Breast / Q000201:enzymology*; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis*; D055293:Receptors, Urokinase Plasminogen Activator; D012680:Sensitivity and Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis
D000911:Antibodies, Monoclonal; D002021:Buffers; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator