Endocrine Abstracts

IGF binding proteins (IGFBPs) are important biomarkers for diagnostics and treatment studies. In different physiological conditions IGFBPs are actively degraded by specific proteases. Due to the detection of intact and fragmented IGFBPs, ELISA or RIA techniques may generate biased IGFBP-data. We thus developed quantitative western ligand blotting for comprehensive monitoring of intact IGFBPs and IGFBP-profiles in biological fluids of vertebrates. The dynamic range for each IGFBP was determined and signal intensities were quantified by using recombinant standards as calibrators on each blot. Curve fitting was achieved by a four parametric nonlinear regression of each separate IGFBP. Inter- and intra-assay precision for each IGFBP were determined by using spiked samples as quality controls on each blot. Exploratory measurements were performed in plasma and serum of pigs, cattle, mice, sheep, goats, dogs, fishes and humans. Furthermore, cerebrospinal fluid (CSF), follicular fluid (FF) or seminal plasma (SP) were examined in cattle or horse respectively. For all substrates the minimum required dilution for sample loading was determined. IGFBPs were present in all investigated substrates with a species and substrate specific IGFBP profile. Most abundant plasma and serum IGFBPs were IGFBP3, −2 and −4. In SP we were able to quantify comparably high amounts of IGFBP5. In CSF we further identified an IGFBP2 fragment. In CSF and FF both IGFBP2 and IGFBP3 were detected. The lower limits of detection of IGFBP2, 3, and 4 were 0.062, 0.26 and 0.03 ng respectively. The intra- and inter-assay precision in serum and plasma were <15 and <20% fulfilling the acceptance criteria of recent bioanalytical guidelines. To conclude, the qWLB represents a standardized methodology for measuring IGFBP profile in biological fluids characterized by high reproducibility and sensitivity. QWLB is attractive particularly for translational biomedical research.