Specific Immunoassays for Detection of Intact and Cleaved Forms of the Urokinase Receptor

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 50; no. 11; pp. 2059 - 2068
• Main Authors: Piironen, Timo, Laursen, Birgitte, Pass, Jesper, List, Karin, Gardsvoll, Henrik, Ploug, Michael, Dano, Keld, Hoyer-Hansen, Gunilla
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01-11-2004; American Association for Clinical Chemistry; Oxford University Press
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biological samples; Biomarkers, Tumor - blood; Breast cancer; Chymotrypsin - chemistry; Colorectal cancer; Cross Reactions; Europium; Fluoroimmunoassay; Fundamental and applied biological sciences. Psychology; Humans; Immunoassay; Immunoassays; Investigative techniques, diagnostic techniques (general aspects); Leukemia; Leukemia, Myeloid, Acute - diagnosis; Leukemia, Myeloid, Acute - urine; Lung cancer; Male; Medical sciences; Monoclonal antibodies; Patients; Peptides; Phlebotomy; Plasma; Prostate; Prostatic Neoplasms - blood; Prostatic Neoplasms - diagnosis; Proteins; Receptors, Cell Surface - analysis; Receptors, Cell Surface - blood; Receptors, Cell Surface - chemistry; Receptors, Urokinase Plasminogen Activator; Reproducibility of Results; Sensitivity and Specificity; Urokinase-Type Plasminogen Activator - metabolism; Peptidases; u-Plasminogen activator; Serine endopeptidases; Enzyme; Clinical biology; Hydrolases; Biochemistry; Molecular biology; Immunological method; Biological receptor
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2004.038232

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.
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).
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.
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.