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Clinical Chemistry 50: 2059-2068, 2004. First published September 2, 2004; 10.1373/clinchem.2004.038232
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(Clinical Chemistry. 2004;50:2059-2068.)
© 2004 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

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

Timo Piironen1, Birgitte Laursen2, Jesper Pass, Karin List3, Henrik Gårdsvoll, Michael Ploug, Keld Danø and Gunilla Høyer-Hansena

1 Finsen Laboratory, Copenhagen, Denmark.

aAddress correspondence to this author at: Finsen Laboratory, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark. Fax 45-35385450; e-mail gunilla{at}finsenlab.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The main cause of morbidity and mortality in cancer patients is metastatic disease. It is now well established that a complex array of extracellular proteases plays an essential role in matrix degradation during the process of tissue remodeling associated with cancer invasion and metastasis (1). These proteases and their regulators are potential diagnostic and prognostic tumor markers.

Cell surface plasminogen activation is catalyzed by urokinase plasminogen activator (uPA).4 uPA is secreted as an inactive proenzyme, which localizes on the cell surface by binding to a specific glycolipid-anchored receptor, uPAR (2). Activation of receptor-bound pro-uPA is facilitated by the concomitant binding of plasminogen to the cell surface, which consequently acts as a catalytic template (3). uPAR consists of three homologous domains; the amino-terminal domain is required for binding to uPA, but a structurally intact uPAR is needed for high-affinity binding (4)(5)(6). uPA cleaves uPAR(I-III) between domains I and II, liberating uPAR(I), whereas uPAR(II-III) remains bound to the cell surface (7). Several proteases, including plasmin, can also cleave uPAR(I-III) in vitro (8). Glycolipid-anchored, intact, and cleaved forms of uPAR are present on several neoplastic cell lines in vitro (7), and these are also detected in vivo in transplanted Lewis lung carcinoma, in xenografted human mammary carcinoma (9), and in human ductal breast carcinoma (Høyer-Hansen et al., unpublished results). uPAR may be shed from cells either by phospholipase hydrolysis of the lipid anchor or by proteolytic cleavage (10)(11). A soluble form of uPAR(I-III) has been identified in plasma from healthy individuals and in patients with paroxysmal nocturnal hemoglubinuria (12). Interestingly, the uPAR concentration is increased in blood from patients with non-small-cell lung cancer (13), colon cancer (14), and breast cancer (15). Furthermore, a cleaved soluble form, uPAR(II-III), has been identified in cystic fluid from ovarian cancer patients (16), and immunoprecipitation and Western blotting have revealed that urine samples from patients with acute myeloid leukemia contain uPAR(I) and uPAR(II-III) (17).

It has previously been reported that increased concentrations of uPAR in resected tumor tissue are correlated with poor prognosis for patients with squamous cell lung cancer (18), colon cancer (19), and breast cancer (20). Furthermore, the amount of uPAR in preoperatively collected plasma from patients with colorectal cancer was significantly correlated to patient prognosis (21). Similar results have been obtained recently with serum from patients with primary breast cancer (15).

These results have demonstrated the strong prognostic potential of quantitative uPAR immunoassays and encourage additional studies of assays that not only measure a combination of various forms of uPAR but also differentiate among full-length and cleaved uPAR forms by use of selective monoclonal antibodies (Mabs). The amounts of uPAR fragments in tissues and circulation may reflect the activity of the plasminogen activation system and thus have a stronger prognostic significance than the traditional measurements of only the total uPAR content.

Cancers of the breast, prostate, colon, and lung all have high incidences, and there is a strong need for new methods that enable early detection and stratification of patients at high vs low risk of recurrence to assist in selection of patients for adjuvant therapy. To enable studies of the diagnostic and prognostic potential of the individual uPAR forms, we have designed sensitive and specific time-resolved fluorescence immunoassays (TR-FIAs) that reliably identify and accurately quantify the individual uPAR forms in blood plasma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
biological samples
Pools of donor EDTA and citrate plasma were obtained as described previously (13)(14). Archival serum and EDTA-plasma samples were from a collection described previously (22). Blood from six healthy volunteers at the Department of Biotechnology, University of Turku, Finland, was collected by venipuncture in tubes containing either no additional agents or EDTA. These samples were processed within 1 h after venipuncture and frozen immediately. EDTA plasma from a patient with prostate cancer was from Department of Clinical Chemistry, University Hospital Malmö, Sweden (Prof. Hans Lilja). Urine from a patient with acute myeloid leukemia was from an earlier described study (17). The procedures followed were in accordance with the Helsinki Declaration of 1975.

reagents and instrumentation
Superdex 75 and 200 HR 10/30 FPLC columns, and NAP-5 and NAP-10 gel-filtration columns were from Amersham Pharmacia Biotech. The FluoStar Galaxy fluorometer was from BMG LabTechnologies. The DELFIA® europium-labeling reagents, DELFIA assay buffer (cat. no. 1244-111), wash solution, and enhancement solution (cat. no. 1244-105) were from Perkin-Elmer Life Sciences. White Maxisorp microtitration fluorostrips were obtained from NUNC.

MABS, peptide antagonist, and recombinant proteins
The monoclonal anti-uPAR antibodies R3, R5, and R9 all react with uPAR(I) and were raised against human uPAR purified from phorbol 12-myristate 13-acetate-stimulated U937 cells (23); U937 is a histiocytic lymphoma cell line. R3 is a competitive inhibitor of uPA binding (23)(24), whereas R5 and R9 actively displace receptor-bound uPA (24). The anti-uPAR Mab R2 reacts with an epitope located on uPAR(III) (24) and is from the same fusion as R3, R5, and R9 (23). The fifth anti-uPAR Mab used in this study is denoted R23 and was raised against recombinant uPAR(I-III) (25). R23 is a competitive inhibitor of uPA binding and reacts with uPAR(II-III) but fails to recognize isolated uPAR(II) or uPAR(III) (List et al., unpublished data).

AE120 is a synthetic peptide antagonist of the uPA-uPAR interaction (26) and has the amino acid sequence [D-Cha-F-s-r-Y-L-W-S]2-ßA-K; where "Cha" is ß-cyclohexyl-L-alanine, "s" is D-serine, and "r" is D-arginine. The Kd value of AE120 for immobilized uPAR has been determined by surface plasmon resonance to be 0.6 nmol/L (26).

Recombinant uPAR(I-III), consisting of amino acids 1–277 (calculated mass, 30 741 Da), was produced in Chinese hamster ovary cells and purified by immunoaffinity chromatography as described previously (25). Human uPAR(II-III), consisting of amino acids 88–277 (calculated mass, 21 006 Da), was generated by chymotrypsin cleavage of recombinant uPAR(I-III) (27) and purified by size-exclusion chromatography using a Sephadex HR 75 column (28). uPAR(II-III) purified from U937 cells is a mixture of two forms with either A84 or S90 as the NH2-terminal amino acid (7).

Recombinant uPAR(I), consisting of amino acids 1–92 (calculated mass, 10 400 Da), was produced in a Drosophila Expression System (Invitrogen). The expression vector pMTC-uPAR(I) was constructed by generating a fragment encoding uPAR(I) by standard PCR techniques using Pfu DNA polymerase followed by cloning into the plasmid pMT/V5-His. In brief, with pCI-neo/suPAR as template (29), a SpeI site was introduced in front of the noncoding 5' end, and a TAA stop codon followed by an EcoRI site was introduced in the 3' end at the position immediately after the nucleotides encoding amino acid 92 of uPAR. The native signal sequence of uPAR is retained in this construct, allowing the recombinant uPAR(I) to be produced as a secreted soluble product. Drosophila S2 cells were stably transfected with pMTC-uPAR(I) and propagated for large-scale production as described previously (30). uPAR(I) was purified from the conditioned medium of S2 cells by immunoaffinity chromatography using Mab R3 followed by reversed-phase HPLC as described previously (29).

The purified uPAR(I-III), uPAR(II-III), and uPAR (I) preparations used in this study were all >95% pure as judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Coomassie staining of reduced and alkylated samples (data not shown). The exact protein contents of these samples were determined by amino acid analysis (25).

Active site mutated pro-uPAS356A was produced by Drosophila S2 cells and purified from the medium by immunoaffinity chromatography (Gårdsvoll et al., unpublished results). The protein was converted to two-chain uPA by incubation with immobilized plasmin.

coating and europium labeling of MABS
Antibodies were treated with acid before coating. In brief, one volume of antibody solution (1 g/L) was incubated with six volumes of 0.01 mol/L HCl (pH ~1.8) for 5 min, after which the antibody was diluted to 5 mg/L in 0.1 mol/L NaH2PO4 (pH ~4.2). This solution was used for coating of White Maxisorp microtitration fluorostrips (NUNC) overnight at room temperature (200 µL/well) in a moisturized container. Subsequently, the plates were washed twice with wash solution containing 0.5 mL/L Tween 20 and blocked with a solution containing 50 mmol/L NaH2PO4, 1 g/L diazolidinyl urea, 60 g/L sorbitol, and 1 g/L diethylenetriaminepentaacetic acid-purified bovine serum albumin (300 µL/well). The following day, plates were emptied, dried for 3 h in a fume hood, and then covered with sealing tape. Coated plates could be stored for up to 4 weeks at 4 °C.

Mabs were labeled with Eu3+ chelates (31) to a density of 0.8–2.3 Eu3+/IgG according to the manufacturer’s recommendations (DELFIA Eu-labeling reagents; Perkin-Elmer Life Sciences). To remove components that might interfere with the coupling chemistry, the antibody solution was buffer-exchanged to 0.05 mol/L NaHCO3 (pH 9.8) before addition of a 30- to 125-fold molar excess of Eu3+ chelated to N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1, N2,N3,N3-tetraacetate and incubation overnight at 4 °C. The Eu3+-labeled antibody was separated from uncoupled Eu3+ chelate by size-exclusion chromatography using a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech). The affinities of the Eu3+-labeled anti-uPAR antibodies were determined as described previously (32) and are shown in Table 1 .


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  		Table 1. Affinity constants (L/mol) of four Eu3+-labeled anti-uPAR Mabs against bound uPAR forms.

separation of UPAR forms by chromatography
Samples, including the donor EDTA-plasma pool with or without added proteins and the patient EDTA plasma and urine described above, were analyzed by size-exclusion chromatography using a Superdex 75 HR 10/30 column. The samples were filtered through 0.22 µm filter before use. Tris-saline-azide buffer [500 mmol/L Tris-HCl (pH 7.8), 9 g/L NaCl, 5 g/L NaN3] containing 1 µmol/L EDTA was used for elution. The biological samples were diluted in this buffer (1:1 by volume) before loading on the column. Collected fractions were stored at –80 °C.

assay designs
The designs of the individual assay are outlined in Fig. 1 .



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  		Figure 1. Assay designs for the three TR-FIAs.

TR-FIA 1 for quantification of uPAR.
The Mab R2 was used for coating according to the procedure described. Calibrators [threefold serial dilutions of uPAR(I-III) ranging from 0.455 to 325 pmol/L] and samples were diluted in assay buffer and added to the plates in duplicate (100 µL/well), followed by incubation with shaking for 1 h at room temperature. After six washes, 100 µL of Eu3+-labeled R3 (R3-Eu) was added (1 mg/L) and incubated 2 h at room temperature with shaking. After another six washes, 100 µL of enhancement solution was added to each well and incubated for 5 min at room temperature with shaking; the fluorescence was then measured with a FluoStar Galaxy fluorometer with excitation set at 405 nm and emission read at 615 nm with a 400-µs delay and a 400-µs acquisition window.

TR-FIA 2 for quantification of uPAR(I-III) and uPAR(II-III).
With the exception that the detecting antibody was replaced by the Eu3+-labeled R23, the protocol for TR-FIA 2 was identical to that outlined for TR-FIA 1. uPAR(I-III) was used as calibrator because the antibody detects uPAR(II-III) and uPAR(I-III) with the same affinity (Table 1Up ).

TR-FIA 3 for quantification of uPAR(I).
Overall the protocol was the same as for the other assays except that Mab R5 was used for coating, the sample incubation time was 2 h, and recombinant uPAR(I) was used as calibrator (threefold serial dilution ranging from 1.3 to 961 pmol/L). Because none of the available antibodies could differentiate between uPAR(I-III) and uPAR(I), we made use of the high-affinity peptide antagonist, AE120. This peptide reacts only with uPAR(I-III) and is a competitive inhibitor for uPA binding (26). R3 is also a competitive inhibitor of uPA binding (23)(24), and we tested the possible detection of the uPAR(I-III)–AE120 complex by this Mab. As shown in Fig. 2 , this peptide at 1 µmol/L almost completely prevented the binding of R3-Eu to uPAR(I-III). Accordingly, AE120 (1 µmol/L) was added together with the detecting antibody R3-Eu.



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  		Figure 2. Titration of AE120 to reduce the signal produced by uPAR(I-III) in the TR-FIA.

uPAR(I-III) in different concentrations (•, 975 pmol/L; {square}, 325 pmol/L; {blacktriangleup}, 108 pmol/L) was measured with R5 as the capture and Eu-R3 as the detection antibody in the presence of increasing concentrations of AE120. The cross-reactivity was defined as the measured in the assay divided by the added amount of uPAR(I-III).

immunoassay validation
Sensitivity.
The reproducibility of the calibration curve was determined by analyzing the variation of duplicate measurements of calibrators in assay buffer (n = 12 plates). The detection limit was defined as the concentration at which the signal exceeded 3.3 SD (in cps) of 20 replicates of assay buffer. The limit of quantification was determined by adding 0.016–10 µg/L purified calibrators [0.5–325 pmol/L uPAR(I-III), 0.8–476 pmol/L uPAR(II-III), 1.5–961 pmol/L uPAR(I)] to uPAR-depleted citrate plasma and examining the CV in 10 replicates. The limit of quantification was defined as the concentration at which the CV exceeded 20%. uPAR depletion of citrate plasma, diluted 1:5 in 0.2 mol/L phosphate buffer (pH 7.4) containing 0.1 mol/L NaCl, 0.1 mL/L Tween 20, 0.01 mL/L phenol red, 10 g/L bovine serum albumin, and 50 kIU/L heparin, was achieved by adsorption on protein A-Sepharose CL-4B (Pharmacia Biotech) followed by a second adsorption on a protein A-Sepharose column preloaded with a polyclonal rabbit anti-uPAR antibody.

Specificities of the assays.
The specificities of the assays were tested in three ways. First, 200 fmol of each uPAR form was added to assay buffer and measured in the different TR-FIAs. The amounts recovered in each assay were determined. The specificities were then tested by means of TR-FIA measurements of a time-course profile for the cleavage of uPAR(I-III) induced by incubation with either trypsin [modified porcine trypsin (EC 3.4.21.4), sequence grade; Promega] or chymotrypsin [{alpha}-chymotrypsin (EC 3.4.21.1); Worthington]. In brief; 813 nmol/L uPAR(I-III) was incubated at room temperature with 2.5 µg/L of either trypsin or chymotrypsin. Aliquots were withdrawn after incubation for 15 min, 30 min, 1 h, 2 h, 4 h, and 3 days and stored at –80 °C until analyzed by TR-FIA and Western blotting methods. The primary antibodies used in Western blotting were 5 mg/L R2 for detection of uPAR(II-III) and uPAR(I-III), and a mixture of R3 and R9 (2.5 mg/L each) for the detection of uPAR(I) and uPAR(I-III). The membranes were developed with the WesternBreeze Chemiluminescent Immunodetection System for detection of mouse primary antibodies (Invitrogen). A separate set of these samples was subjected to size-exclusion chromatography, and collected fractions were stored at –80 °C before analysis by TR-FIA.

Finally, 961 pmol/L uPAR(I), 333 pmol/L uPAR(II-III), or 325 pmol/L uPAR(I-III) was added to donor EDTA- plasma samples, which were then subjected to size-exclusion chromatography. The collected fractions were stored at –80 °C before analysis by TR-FIA.

To evaluate the impact of uPA/uPAR complex formation on the three TR-FIAs, we preincubated equal volumes of 23 nmol/L uPAR(I-III) and 5, 10, 20, 30, or 60 nmol/L recombinant two-chain uPAS356A on ice for 1 h. Each sample was then diluted to 325 pmol/L uPAR and serially diluted threefold, as for a calibration curve, and measured in the three TR-FIAs as well as in an ELISA (33).

Precision and recovery.
We determined intraassay precision by assaying 12 replicates of donor citrate plasma in the three TR-FIAs and calculating the CVs. In addition, donor citrate plasma to which 480 pmol/L uPAR(I) or 162 pmol/L uPAR(I-III) had been added were assayed, with more than 70 replicates in each assay. All samples were diluted 1:10 in assay buffer before analysis.

We determined interassay precision by analyzing donor citrate plasma (n = 47) and donor citrate plasma to which the protein had been added (n = 21 for TR-FIA 1; n = 20 for TR-FIA 2; and n = 23 for TR-FIA 3). All samples were diluted 1:10 in assay buffer before analysis.

We determined the recoveries of calibrators {0.041–8 µg/L [3.9–769 pmol/L uPAR(I), 1.9–381 pmol/L uPAR(II-III), or 1.3–260 pmol/L uPAR(I-III)]} in 100, 200, and 400 mL/L citrate plasma in assay buffer. The recoveries were in each case calculated from the slopes of the lines representing the uPAR(I-III), uPAR(II-III), or uPAR(I) signals as a function of concentration, and 100% recovery was defined as the slope in assay buffer.

Robustness.
Archival EDTA plasma samples (n = 13) were diluted 1:10 in assay buffer and measured in the three TR-FIAs at 4, 23, and 30 °C.

We addressed assay tolerance toward pH variations in biological samples by adjusting EDTA plasma, citrate plasma, and uPAR-depleted donor citrate plasma to which we had added 961 pmol/L uPAR(I) or 325 pmol/L uPAR(I-III) to pH 6.5, 7.5, 8.5, or 9.5 with negligible amounts of 10 mol/L HCl or 10 mol/L NaOH. These samples were then diluted 1:10 in assay buffer adjusted to the same pH (with 10 mol/L HCl or 10 mol/L NaOH) as the samples before analysis.

stability of UPAR calibrators and endogenousUPAR in biological samples
We diluted assay calibrators containing 325 pmol/L uPAR(I-III) and 961 pmol/L uPAR(I) in assay buffer and froze them; we subsequently thawed the samples quickly at 37 °C and then left them at room temperature for 1 h before refreezing them. The samples passed through one to six such freeze–thaw cycles before they were measured in the three TR-FIAs. Fresh serum and EDTA-plasma samples from six donors were subjected to four to seven freeze–thaw cycles (thawed at room temperature and left thawed for 1 h before refreezing) before they were diluted 1:10 in assay buffer and measured by the three TR-FIAs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
validation of the TR-FIAS
Sensitivity.
The variation between results obtained for calibrators on different plates demonstrated how reproducible the calibration curves were. The means of 12 calibration curves for each of the three assays, with their respective CVs, are shown in Fig. 3 . TR-FIA 2 was the only assay that reliably measured uPAR over the whole concentration range tested, with a CV <20% for the lowest calibrator (0.455 pmol/L) and <15% for the rest (Fig. 3B ). For the other two assays, the CVs were <15% for all but the lowest calibrators measured, for which the CV in both cases was 21% at 0.455 and 1.35 pmol/L for TR-FIA 1 and TR-FIA 3, respectively (Fig. 3 , A and C).



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  		Figure 3. Calibration curves and interassay precision profiles (n = 12).

{square} (right axis) indicate the individual CVs for the measured calibrators; • (left axis) indicate measured concentrations of the calibrators. (A), TR-FIA 1; (B), TR-FIA 2; (C), TR-FIA 3.

The detection limit of TR-FIA 1 and TR-FIA 2 was 0.3 pmol/L, whereas it was 1.9 pmol/L for TR-FIA 3. The limit of quantification was determined in uPAR-depleted citrate plasma to which the analytes were added and was <1.3 pmol/L for uPAR(I-III) in TR-FIA 1. When uPAR(I-III) and uPAR(II-III) were tested in TR-FIA 2, the limits of quantification were 1.3 and 1.6 pmol/L, respectively. The least sensitive assay was TR-FIA 3, for which 2.9 pmol/L uPAR(I) was the lower limit of quantification.

Specificity.
We tested the specificities of the assays by separately adding equimolar amounts of uPAR(I-III), uPAR(II-III), and uPAR(I) to each of the assays (Table 2 ). The amounts of uPAR(I-III) and uPAR(I) were measured directly in TR-FIAs 1 and 3, and the amount of uPAR(II-III) was calculated by subtracting the moles of uPAR(I-III) measured in TR-FIA 1 from those of uPAR(I-III) and uPAR(II-III) measured in TR-FIA 2. In accordance with the specificities of the antibodies used in the assays, TR-FIA 1 was selective for uPAR(I-III), TR-FIA 2 for uPAR(I-III) and uPAR(II-III), and TR-FIA 3 for uPAR(I). The only significant cross-reactivity was in TR-FIA 3, which measured 4% of the uPAR(I-III) added, in good agreement with the effect of the peptide AE120 used in this assay to prevent binding of Eu-R3 to uPAR(I-III) (see Fig. 2Up ).


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  		Table 2. Specificity of TR-FIAs for different uPAR forms.1

To further verify the specificity of the new TR-FIAs, we used them in combination to quantify the cleavage of purified uPAR(I-III) incubated with chymotrypsin in a time-course experiment (Fig. 4A ). The amount of detected uPAR(I) increased concomitantly with a proportional loss of uPAR(I-III). Concordant with the stated equimolar detection of uPAR(I-III) and uPAR(II-III) by R23 (Table 1Up ), the combined concentrations of uPAR(I-III) and uPAR(II-III) remained constant throughout the cleavage reaction as assessed by TR-FIA 2. The cleavage profile detected by the TR-FIAs (Fig. 4A ) was verified by Western blotting (Fig. 4B ). Trypsin cleavage of uPAR(I-III) showed similar results (data not shown).



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  		Figure 4. Chymotrypsin cleavage of uPAR.

We incubated 25 µg of recombinant uPAR(I-III) with 2.5 ng of chymotrypsin. Samples from a time-course experiment were measured in the TR-FIAs (A) and analyzed by Western blotting (B). The primary antibodies used in the Western blots were R2 (5 mg/L) for detection of uPAR(I-III) and uPAR(II-III), and R3 + R9 (5 mg/L) for detection of uPAR(I) and uPAR(I-III). The cleavage products from the sample incubated for 3 days were separated by size-exclusion chromatography on a Superdex 75 HR column (C), and each of the fractions was measured by the three TR-FIAs ({square}, TR-FIA 1; •, TR-FIA 2; {blacktriangleup}, TR-FIA 3) as well as by an assay using the same Mabs as in TR-FIA 3 but omitting AE120 ({triangleup}).

The sample with the most extensive chymotrypsin cleavage was then subjected to size-exclusion chromatography on a Superdex 75 column, and the collected fractions were subsequently measured by the TR-FIAs (Fig. 4CUp ). Different uPAR forms could be identified and quantitatively measured by the TR-FIAs on these fractions. To directly test the effect of AE120 in the TR-FIA 3, we included an assay without the antagonist added. The peaks at the uPAR(I) position that were obtained in this assay and TR-FIA 3 were completely overlapping, whereas omission of AE120 led to a strong increase in the peak at the uPAR(I-III) position (Fig. 4CUp ). This demonstrated that the presence of AE120 in TR-FIA 3 affects only uPAR(I-III) measurements.

Precision and accuracy.
The CVs were <8% in all cases except for plasma with no added analyte assayed in TR-FIA 3 (Table 3 ). This is most probably because the amount of uPAR(I) in the measured 1:10-diluted sample was below the limit of quantification. The CV was 4.8% for TR-FIA 3 when we assayed the sample with analyte added.


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  		Table 3. Intra- and interassay precision of TR-FIAs as determined with donor citrate plasma with and without added uPAR.

For interassay precision, the mean values were essentially the same as those for the intraassay precision study, and the CVs for the plasmas with added protein were acceptable (<11%). For plasma with no added protein, the CVs were higher, ~15% for TR-FIA 1 and TR-FIA 2 and 30% for TR-FIA 3. The most plausible explanation for the high CV in TR-FIA 3 is the low amount of uPAR(I) in the diluted sample, which was below the limit of quantification.

Table 4 shows assay recoveries, calculated as the ratios of the linear regression coefficients of the plasma samples with added protein and those of the pure analytes in assay buffer. In all cases the recoveries were between 93% and 105%.


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  		Table 4. Recoveries in TR-FIAs of different uPAR forms added1 to citrate plasma.

Identical results were obtained when these assays were performed at 4, 23, or 30 °C (data not shown). Furthermore, the assays were insensitive to pH variations between 6.5–9.5 during sample incubation (data not shown).

Because unintended cleavage of uPAR(I-III) in vitro during sample processing could cause interference in the ratios determined for the different uPAR forms, we examined the stabilities of the different uPAR forms under various processing conditions. We observed no significant differences in absolute concentration or changes in the ratio of the uPAR forms being measured subsequent to freezing and thawing of fresh serum and EDTA-plasma samples or in purified preparations of uPAR(I-III) and uPAR(I) (data not shown).

Effect of uPA on uPAR measurements.
The Mabs R3 and R23 used for detection in the TR-FIAs do not bind uPA/uPAR complexes [Rønne et al. (23) and List et al., unpublished results]. Therefore, it would be expected that complexes formed between uPA and uPAR would not be measured in the TR-FIAs. To investigate whether uPA affected the signals obtained in the three TR-FIAs, active-site-mutated uPA (uPAS356A) was added to a uPAR(I-III) preparation, which subsequently was analyzed by TR-FIA and suPAR ELISA (33). Whereas the addition of uPA only marginally affected the signal in the ELISA, the signals in TR-FIA 1 and TR-FIA 2 were reduced by >50% when samples containing equimolar concentrations of uPAR and uPA were measured. A threefold molar excess of uPA reduced signals in the two TR-FIAs further, to 35% and 26%, respectively, of the signals obtained when no uPA was added. In TR-FIA 3, no signal was generated in the presence of uPAR/uPA complexes.

endogenous UPAR forms in patient blood and urine
To define the specificities of the assays more rigorously, we subjected a donor EDTA-plasma pool to which uPAR(I), uPAR(II-III), or uPAR(I-III) had been added to size-exclusion chromatography (Fig. 5 ). Plasma from the same batch, without added analyte, was included for comparison (Fig. 5A ). The results demonstrated the capability of the TR-FIAs to detect the specific uPAR forms. Added uPAR(I) was thus detected by TR-FIA 3 only and at an elution position that corresponded to its small size (Fig. 5B ). Addition of uPAR(II-III) to samples led to a dramatic increase in the signal at a position consistent with the size of uPAR(II-III) in TR-FIA 2 (Fig. 5C ). Finally, when uPAR(I-III) was added, the signals corresponding to uPAR(I-III) were higher in both assays that measured this protein (Fig. 5D ). The recoveries from the column were 84% (n = 9), 98% (n = 9), and 95% (n = 11) for uPAR(I), uPAR(I-III), and uPAR(II-III), respectively.



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  		Figure 5. Elution profiles of uPAR forms by size-exclusion chromatography of donor EDTA-plasma pool with and without added uPAR forms.

Before size-exclusion chromatography, 961 pmol/L uPAR(I) (B), 333 pmol/L uPAR(II-III) (C), or 325 pmol/L uPAR(I-III) (D) was added to plasma samples (100 µL/sample). Plasma with no added uPAR forms is shown in A. Each of the eluted fractions was measured in the three TR-FIAs. {square}, TR-FIA 1; •, TR-FIA 2; {blacktriangleup}, TR-FIA 3.

To assess the performance of the assays on authentic patient samples, we analyzed EDTA plasma from a patient with prostate cancer after fractionation by size-exclusion chromatography (Fig. 6A ). Close examination of the obtained elution profiles revealed that comparable uPAR forms were eluted at identical positions irrespective of their origin (compare Fig. 4CUp and Fig. 6A ). EDTA plasma from the prostate cancer patient contained detectable amounts of uPAR(I), in contrast to the EDTA-plasma pool from healthy donors (Fig. 5AUp ). In addition, the concentrations of uPAR measured with TR-FIA 2 were considerably higher in the patient sample than in the donor plasma (Fig. 5AUp and Fig. 6A ). A sizable fraction of the total uPAR content appeared to be uPAR(II-III); the area under the peak derived from TR-FIA 2 was considerably larger and covered later-eluting fractions than did the peak obtained with TR-FIA 1. Other patient samples, including citrate plasma and serum, yielded comparable profiles (data not shown). In contrast, urine from a patient with acute myeloid leukemia revealed a completely different profile; the prevailing form in this case was uPAR(I). Some uPAR(II-III) was also present, whereas uPAR(I-III) was barely detectable (Fig. 6B ).



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  		Figure 6. Size fractionation of patient samples.

Size-exclusion chromatography of EDTA plasma (100 µL) from a prostate cancer patient (A) and urine (100 µL) from a patient with acute myeloid leukemia (B). Every fraction was measured in the three TR-FIAs. {square}, TR-FIA 1; •, TR-FIA 2; {blacktriangleup}, TR-FIA 3.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The immunoassays presented here specifically and quantitatively measure uPAR(I-III), uPAR(I-III) plus uPAR(II-III), and uPAR(I). In TR-FIA 3, which measures uPAR(I), the signal from uPAR(I-III) is blocked by addition of the peptide antagonist AE120 (26). The functional epitope for this peptide antagonist resides mainly in domain III, suggesting that a conformational change is the cause of the AE120-induced inhibition of R3 binding to uPAR(I-III).

Although uPAR(II-III) cannot be measured directly by the present assays, uPAR(II-III) can be quantified by subtracting the measured moles of uPAR(I-III) in TR-FIA 1 from the moles of uPAR measured with TR-FIA 2. However, a result obtained by such a calculation will have a greater CV than a direct measurement, particularly if the amount of uPAR(I-III) is higher than that of uPAR(II-III). To design an assay that directly measures uPAR(II-III) would, however, necessitate the development of a new antibody specific for this form. Ideally, such an antibody should require a conformational change of the linker region after cleavage. There have been reports that such a conformational change does occur in the linker region as a result of proteolytic cleavage by at least matrix metalloproteinase-12 (34). Characterization of this putative neoepitope presumably specific for uPAR(II-III) is, however, further complicated by the fact that the linker region between domains I and II exhibits different conformations depending on the presence of the glycolipid anchor on uPAR (35).

To date, it has not been possible to design ELISAs to measure the uPAR forms separately because of changes in the affinities and specificities of Mabs after biotinylation (36). The specificity problem was most evident when plasma samples were measured and was related to the degree of biotinylation of the different uPAR Mabs. In ELISA, more than 100 biotin molecules per molecule of detection Mab are often used. However, the very sensitive detection system in TR-FIA requires only ~1 europium molecule per molecule of Mab, and we have not detected any nonspecific reactions with plasma samples when we used Eu3+-labeled R3 or R23.

The TR-FIAs have been validated for measurements of uPAR variants in citrate plasma and were shown to be robust and well suited for analysis of clinical samples. Validation will be needed on any samples before the assays can be used to quantify uPAR forms in serum, urine, ascites, and other human body fluids, tissue and cell extracts, and cell culture media. However, when we subjected urine to size-exclusion chromatography and subsequently measured the different uPAR forms in the eluted fractions, we could identify and quantify the different uPAR forms in this matrix (Fig. 6BUp ).

Cleaved forms of uPAR have previously been identified in extracts from xenotransplanted tumors by Western blotting (9), in urine from patients with acute myeloid leukemia by immunoprecipitation combined with Western blotting (17), and in cystic fluid from patients with ovarian cancer by immunoaffinity purification and Western blotting (16). However, these methods are neither quantitative nor suited for the high throughput required when analyzing clinical samples for diagnostic or prognostic studies.

Complexes of uPA and uPAR have been detected in blood from breast cancer patients and healthy controls by an ELISA designed for the detection of these complexes (37). In tissue lysates, the concentrations of uPA/uPAR complexes are unknown but may be expected to be significant. However, use of the proposed TR-FIAs to detect the presence of such complexes will influence only the quantification of intact uPAR because uPAR(II-III) cannot bind uPA and liberated domain I will bind uPA poorly, if at all (4)(5). To get an estimate of all forms of uPAR in a sample, one could measure the total amounts of both uPAR(I-III) and uPAR(II-III) with the ELISA described previously (15)(33) and use the TR-FIAs described in this study to measure the specific uPAR forms. The amounts measured with the ELISA should equal those measured with TR-FIA 2 if no complexes are present. If all uPAR is in the cleaved form, there will be no signal in TR-FIA 1. If the sample contains a mixture of uPA/uPAR complexes, uPAR(I-III), and uPAR(II-III), then the amount measured by the ELISA will be higher than that measured by TR-FIA 2.

In conclusion, several ELISAs have been developed to quantify uPAR in various patient samples. These assays measure the sum of intact uPAR and most of its degradation products (25)(33)(38). The use of these ELISAs has led to uPAR being identified as a strong prognostic marker in breast, colorectal, and lung cancer (15)(18)(19)(20)(21). Intact uPAR binds pro-uPA and localizes it to the cell surface, where its activation is strongly enhanced by the proximity of cell-surface-bound plasminogen (3). Active uPA is capable of cleaving cell-bound uPAR, liberating domain I and leaving the cleaved uPAR(II-III) on the cell surface (7). In contrast, uPAR purified from plasma or cell culture medium cannot be cleaved by physiologically relevant concentrations of uPA (35). However, purified glycolipid-anchored uPAR and soluble uPAR can both be cleaved by plasmin (35). The concentrations of the cleaved forms of uPAR might therefore be correlated to the activity of the plasminogen activation system. Thus, it is potentially important to identify and quantify the different uPAR forms separately, both for basic biological studies on the mechanism of cleavage and shedding of uPAR and for clinical studies to investigate the diagnostic and prognostic significance of the different forms of uPAR. The TR-FIAs presented here make such studies possible.


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance of Ruth Petersson, Marjo Westerdahl, and Yvonne Delotto. We thank Prof. Hans Lilja and Dr. Satu Mustjoki for supplying clinical samples. We are grateful to Dr. G. Kellerman for critical reading of the manuscript. We thank John Post for photographic assistance. This work was supported by EU contracts QLK4-CT-1999-51464, QLK3-CT-2002-02136, and LSHC-CT-2003-503297 and by the Danish Cancer Society.


   Footnotes
 
1 Current address: Schering Oy, Turku, Finland.

2 Current address: Zealand Pharma A/S, Glostrup, Denmark.

3 Current address: National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD.

4 Nonstandard abbreviations: uPA, urokinase plasminogen activator; uPAR, uPA receptor; uPAR(I), uPAR(II), and uPAR(III), uPAR domains I, II, and III, respectively; Mab, monoclonal antibody; and TR-FIA, time-resolved fluorescence immunoassay.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Danø K, Behrendt N, Brünner N, Ellis V, Ploug M, Pyke C. The urokinase receptor. Protein structure and role in plasminogen activation and cancer invasion. Fibrinolysis 1994;8:189-203.[CrossRef]
  2. Ploug M. Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor. Curr Pharm Des 2003;9:1499-1528.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  3. Stephens RW, Pollanen J, Tapiovaara H, Leung KC, Sim PS, Salonen EM, et al. Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants. J Cell Biol 1989;108:1987-1995.[Abstract/Free Full Text]
  4. Ploug M, Ellis V, Danø K. Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor. Biochemistry 1994;33:8991-8997.[CrossRef][Medline] [Order article via Infotrieve]
  5. Behrendt N, Rønne E, Danø K. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains. J Biol Chem 1996;271:22885-22894.[Abstract/Free Full Text]
  6. Høyer-Hansen G, Behrendt N, Ploug M, Danø K, Preissner KT. The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction. FEBS Lett 1997;420:79-85.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  7. Høyer-Hansen G, Ploug M, Behrendt N, Rønne E, Danø K. Cell-surface acceleration of urokinase-catalyzed receptor cleavage. Eur J Biochem 1997;243:21-26.[ISI][Medline] [Order article via Infotrieve]
  8. Høyer-Hansen G, Rønne E, Solberg H, Behrendt N, Ploug M, Lund LR, et al. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem 1992;267:18224-18229.[Abstract/Free Full Text]
  9. Solberg H, Rømer J, Brünner N, Holm A, Sidenius N, Danø K, et al. A cleaved form of the receptor for urokinase-type plasminogen activator in invasive transplanted human and murine tumours. Int J Cancer 1994;58:877-881.[ISI][Medline] [Order article via Infotrieve]
  10. Wilhelm OG, Wilhelm S, Escott GM, Lutz V, Magdolen V, Schmitt M, et al. Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression. J Cell Physiol 1999;180:225-235.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  11. Beaufort N, Leduc D, Rousselle J-C, Magdolen V, Luther T, Namane A, et al. Proteolytic regulation of the urokinase receptor/CD87 on monocytic cells by neutrophil elastase and cathepsin. G J Immunol 2004;172:540-549.[Abstract/Free Full Text]
  12. Rønne E, Pappot H, Grøndahl-Hansen J, Høyer-Hansen G, Plesner T, Hansen NE, et al. The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol 1995;89:576-581.[ISI][Medline] [Order article via Infotrieve]
  13. Pappot H, Høyer-Hansen G, Rønne E, Hansen HH, Brünner N, Danø K, et al. Elevated plasma levels of urokinase plasminogen activator in non-small cell lung cancer. Eur J Cancer 1997;33:867-872.
  14. Stephens RW, Pedersen AN, Nielsen HJ, Hamers MJ, Høyer-Hansen G, Rønne E, et al. ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer patients. Clin Chem 1997;43:1868-1876.[Abstract/Free Full Text]
  15. Riisbro R, Christensen IJ, Piironen T, Greenall M, Larsen B, Stephens RW, et al. Prognostic significance of soluble urokinase plasminogen activator receptor in serum and cytosol of tumor tissue from patients with primary breast cancer. Clin Cancer Res 2002;8:1132-1141.[Abstract/Free Full Text]
  16. Wahlberg K, Høyer-Hansen G, Casslén B. Soluble receptor for urokinase plasminogen activator in both full-length and a cleaved form is present in high concentrations in cystic fluid from ovarian cancer. Cancer Res 1998;58:3294-3298.[Abstract/Free Full Text]
  17. Mustjoki S, Sidenius N, Sier CF, Blasi F, Elonen E, Alitalo R, et al. Soluble urokinase receptor levels correlate with number of circulating tumor cells in acute myeloid leukemia and decrease rapidly during chemotherapy. Cancer Res 2000;60:7126-7132.[Abstract/Free Full Text]
  18. Pedersen H, Brünner N, Francis D, Østerlind K, Rønne E, Hansen HH, et al. Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue. Cancer Res 1994;54:4671-4675.[Abstract/Free Full Text]
  19. Ganesh S, Sier CF, Heerding MM, Griffioen G, Lamers CB, Verspaget HW. Urokinase receptor and colorectal cancer survival. Lancet 1994;344:401-402.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  20. Grøndahl-Hansen J, Peters HA, van Putten WL, Look MP, Pappot H, Rønne E, et al. Prognostic significance of the receptor for urokinase plasminogen activator in breast cancer. Clin Cancer Res 1995;1:1079-1087.[Abstract]
  21. Stephens RW, Nielsen HJ, Christensen IJ, Thorlacius-Ussing O, Sørensen S, Danø K, et al. Plasma urokinase receptor levels in patients with colorectal cancer: relationship to prognosis. J Natl Cancer Inst 1999;91:869-874.[Abstract/Free Full Text]
  22. Törnblom M, Norming U, Adolfsson J, Becker C, Abrahamsson PA, Lilja H, et al. Diagnostic value of percent free prostate-specific antigen: retrospective analysis of a population-based screening study with emphasis on men with PSA levels less than 3.0 ng/mL. Urology 1999;53:945-950.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  23. Rønne E, Behrendt N, Ellis V, Ploug M, Danø K, Høyer-Hansen G. Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor. FEBS Lett 1991;288:233-236.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  24. List K, Høyer-Hansen G, Rønne E, Danø K, Behrendt N. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology. J Immunol Methods 1999;222:125-133.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  25. Rønne E, Behrendt N, Ploug M, Nielsen HJ, Wöllisch E, Weidle U, et al. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay. J Immunol Methods 1994;167:91-101.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  26. Ploug M, Østergaard S, Gårdsvoll H, Kovalski K, Holst-Hansen C, Holm A, et al. Peptide-derived antagonists of the urokinase receptor. Affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation. Biochemistry 2001;40:12157-12168.[CrossRef][Medline] [Order article via Infotrieve]
  27. Behrendt N, Ploug M, Patthy L, Houen G, Blasi F, Danø K. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem 1991;266:7842-7847.[Abstract/Free Full Text]
  28. Ploug M, Kjalke M, Rønne E, Weidle U, Høyer-Hansen G, Danø K. Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. J Biol Chem 1993;268:17539-17546.[Abstract/Free Full Text]
  29. Gårdsvoll H, Danø K, Ploug M. Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis. J Biol Chem 1999;274:37995-38003.[Abstract/Free Full Text]
  30. Gårdsvoll H, Werner F, Søndergaard L, Danø K, Ploug M. Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites. Protein Expr Purif 2004;34:284-295.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  31. Mitrunen K, Pettersson K, Piironen T, Björk T, Lilja H, Lövgren T. Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum. Clin Chem 1995;41:1115-1120.[Abstract/Free Full Text]
  32. Pettersson K, Piironen T, Seppälä M, Liukkonen L, Christensson A, Matikainen MT, et al. Free and complexed prostate-specific antigen (PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-{alpha}1-antichymotrypsin complex. Clin Chem 1995;41:1480-1488.[Abstract/Free Full Text]
  33. Riisbro R, Piironen T, Brünner N, Larsen B, Nielsen HJ, Stephens RW, et al. Measurement of soluble urokinase plasminogen activator receptor in serum. J Clin Ligand Assay 2002;25:53-56.
  34. Andolfo A, English WR, Resnati M, Murphy G, Blasi F, Sidenius N. Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1–D2 linker region and expose epitopes not present in the intact soluble receptor. Thromb Haemost 2002;88:298-306.[ISI][Medline] [Order article via Infotrieve]
  35. Høyer-Hansen G, Pessara U, Holm A, Pass J, Weidle U, Danø K, et al. Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor. Biochem J 2001;358:673-679.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  36. Høyer-Hansen G, Hamers MJ, Pedersen AN, Nielsen HJ, Brünner N, Danø K, et al. Loss of ELISA specificity due to biotinylation of monoclonal antibodies. J Immunol Methods 2000;235:91-99.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  37. De Witte H, Sweep F, Brünner N, Heuvel J, Beex L, Grebenschikov N, et al. Complexes between urokinase-type plasminogen activator and its receptor in blood as determined by enzyme-linked immunosorbent assay. Int J Cancer 1998;77:236-242.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  38. Rønne E, Høyer-Hansen G, Brünner N, Pedersen H, Rank F, Osborne CK, et al. Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies. Breast Cancer Res Treat 1995;33:199-207.[CrossRef][ISI][Medline] [Order article via Infotrieve]



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