Species: pigs, cattle, mice, sheep, goats, dogs, fishes, horses, rabbits, humans Substrates: plasma, serum, cerebrospinal fluid (CSF), follicular fluid (FF) or seminal plasma Analysis: quantitative Western Ligand Blot (qWLB) developed by Ligandis. Proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. Minimum required dilution for sample loading was determined for each substrate. The blots were blocked and then incubated with biotin labeled IGF-2. The membrane was washed followed by incubation with horseradish peroxidase conjugated streptavidin. The binding proteins were detected by enhanced chemiluminescence. Bands were visualized on KODAK Image Station 4000MM. Signal intensities were corrected for background and quantified by using human recombinant standards as calibrators. Curve fitting was done by a four parametric nonlinear regression of each separate IGFBP. The calculation of the IGFBP concentrations in plasma and CSF was done with the software GraphPad Prism4 and corrected for dilution and volume/lane of each sample. Quality management: analyses are performed according to EMA Guideline on Validation of Bioanalytical Methods (part: Ligand binding assays), July 2011. Intraand inter-assay precision for each IGFBP was determined by using spiked samples as high and low quality controls on each blot.