Quantitation of human urokinase receptor (u-PAR, CD87): Development of two new ELISAs

V. Magdolen, M. Kotzsch, M. Schmitt, T. Luther

Research output: Contribution to journalArticlepeer-review

Abstract

The serine protease urokinase-type plasminogen activator (u-PA), its inhibitor (PAI-1), and its receptor, (u-PAR) facilitate cancer cell invasion and metastasis. Whereas u-PA and PAI-1 antigen levels determined in tumor tissue extracts of cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of u-PAR is still matter of debate. We devised two new, highly sensitive ELISA formats which are well suited for measuring u-PAR antigen in tumor tissue extracts. A panel of monoclonal (mAb) and polyclonal (pAb) antibodies were tested employing recombinant human u-PAR produced by CHO-cells (CHO-uPAR) as the reference antigen. Highly sensitive combinations regarding signal to background ratio were obtained using pAb HU2771 as the capture antibody and peroxidase-conjugated mAb IIIF102 or mAb HD 13.11 for uPAR detection. mAb IJIF10 recognizes a linear epitope in domain I of uPAR (amiiio acids 52-60), mAb HD 13.1 a conformationdependent epitope located on domain H/IIJ of u-PAR. A linear relationship in the range of 0.16-5 ng/ml between the measured absorbance and the input of u-PAR antigen was observed in both ELISA formats. Intra-assay [inter-assay] coefficients of variation (CV) were 4.3% [11.7%] for the HU/IIIF10-ELISA and 4.0% [10.7%] for the HU/HD13-EUSA, respectively. Recovery values obtained with diluted cell lysates spiked with increasing concentrations of CHO-uPAR (1.25-10 ng/ml) ranged from 82 to 122% and 88 to 113% for the HU/IIIF10- and HU/HD13-ELISA, respectively. No inhibitory activity of u-PA on u-PAR measurements was found in both ELISA formats, even not at a 6-fold excess of uPA over uPAR.

Original languageEnglish
Pages (from-to)16
Number of pages1
JournalFibrinolysis and Proteolysis
Volume14
Issue numberSUPPL. 1
StatePublished - 2000
Externally publishedYes

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