Analysis of molecular forms of urine Retinol-Binding Protein in Fanconi Syndrome and design of an accurate immunoassay

Retinol-Binding Protein in urine (uRBP), a biomarker for the proximal renal tubular disease of congenital and acquired Fanconi Syndrome (FS) occurs in multiple forms. However these have not had quantitative mass spectrometric (MS) analysis, nor is there a validated assay for defined molecular species of uRBP with linearity on sample dilution. A ‘Top-down’ MS approach identified distinct forms of uRBP differing by only one amino acid. Based on this, we designed a dual-monoclonal antibody-based fluorescence immunoassay calibrated with intact plasma RBP4. LC–MS showed that uRBP in FS (one Dent disease urine) comprised intact plasma RBP4 and C-terminal-truncated RBP4, desL-RBP4 and desLL-RBP4 in molar ratio 2:2:1. DELFIA® assay calibrated with plasma RBP4, formulated with two monoclonal antibodies (HyTest, Finland), mAb48 for capture and biotinylated-mAb42 for detection, provided good sensitivity (1 μg/L), working range > 500 μg/L and good linearity on sample dilution. The three predominant forms of uRBP were equipotent over the assay working range. uRBP reference range was < 3 μg/mmol creatinine and FS patients had concentrations of 1000–5000 μg/mmol creatinine. Using ‘Top-down’ MS analysis of uRBP we devised an accurate, linear, fluorescence immunoassay with defined RBP molecular targets optimal for uRBP measurement. Discrimination of elevated uRBP from the upper limit of normal was some 10-fold greater than previous assays. ► ‘Top-down’ mass spectrometry quantitated forms of RBP in Fanconi urine. ► RBP4, desL-RBP4 and desLL-RBP4 were found in ratio 2:2:1. ► A validated dual mAb fluorescence immunoassay with RBP4 calibrant was devised. ► Assay overcame severe non-linearity seen on dilution of Fanconi urine in previous assays. ► Discrimination of elevated uRBP levels from normal was improved about 10-fold.