Specific immunoassays for detection of intact and cleaved forms of the urokinase receptor.
BACKGROUND
The cell surface receptor (uPAR) for urokinase plasminogen activator (uPA) is a strong prognostic marker in several types of cancer. uPA cleaves the three-domain protein uPAR(I-III) into two fragments: uPAR(I), which contains domain I; and uPAR(II-III), which contains domains II and III. Established immunoassays measure a combination of uPAR forms. Our aim was to design immunoassays for specific quantification of the individual forms of uPAR.


METHODS
Using appropriate combinations of epitope-mapped monoclonal antibodies (Mabs) for capture and europium-labeled detection Mabs, we designed two-site sandwich time-resolved fluorescence immunoassays (TR-FIAs): TR-FIA 1 to measure uPAR(I-III) alone; TR-FIA 2 to measure both uPAR(I-III) and uPAR(II-III); and TR-FIA 3 to measure uPAR(I). To avoid detection of uPAR(I-III) in TR-FIA 3, we used a combination of the peptide uPAR antagonist AE120 and a domain I antibody, R3. AE120 blocks the binding of R3 to uPAR(I-III). In contrast, AE120 does not interact with liberated domain I and therefore does not interfere with the binding of R3 to uPAR(I).


RESULTS
The limits of quantification (CV <20%) determined by adding the proteins to uPAR-depleted plasma were <3 pmol/L in all three assays. The interassay CVs in plasma with added analytes were <11%, and recoveries were between 93% and 105%. Cross-reactivities of purified proteins in the three TR-FIAs were no more than 4%. Studies on chymotrypsin cleavage of uPAR and size-exclusion chromatography of plasma with and without added protein further supported the specificity of the assays.


CONCLUSIONS
The three novel TR-FIAs accurately quantify uPAR(I-III) alone, uPAR(I-III) together with uPAR(II-III), and uPAR(I), respectively, in biological samples, including plasma, and thus are well suited for studies of the diagnostic and prognostic value of individual uPAR forms in cancer patients.
Piironen
Timo
T
Finsen Laboratory, Copenhagen, Denmark.
Laursen
Birgitte
B
Pass
Jesper
J
List
Karin
K
Gårdsvoll
Henrik
H
Ploug
Michael
M
Danø
Keld
K
Høyer-Hansen
Gunilla
G
D014408:Biomarkers, Tumor / Q000097:blood; D002918:Chymotrypsin / Q000737:chemistry; D003429:Cross Reactions; D015200:Fluoroimmunoassay; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000652:urine; D008297:Male; D011471:Prostatic Neoplasms / Q000097:blood / Q000175:diagnosis; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D055293:Receptors, Urokinase Plasminogen Activator; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D014408:Biomarkers, Tumor; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D002918:Chymotrypsin; D014568:Urokinase-Type Plasminogen Activator