Reliability of the western ligand blot method for the simultaneous relative estimations of serum insulin-like growth factor binding proteins (IGFBPs) / Confiabilidad del método de \"western ligand blot\" para la determinación simultánea de las proteínas de unión de los factores de crecimiento similares a la insulina (IGFBPs) en suero

RESUMEN

It is well established that nutrition is an important regulator of both serum insulin-like growth factor-I (IGF) and its binding proteins (IGFBPs). The Western ligand blot method (WLB) for simultaneous determinations of IGFBPs in serum or plasma sambples was evaluated and validated with emplasis on its reproducible capabilities. After electrophoretic separation and transfer, the membranes were incubated with a mixture of recombinant labeled human (GF-I/IGF-II(rhIGF-II) and band intensities measured by autoradiography. The typical electrophoretic profile for pig serum, as determined with molecular weight markers, showed four mainbands of approximately 42-39,32,30-28 and 24 kDa which seemed to correspond to IGFBP-3, IGFBP-2, IGFBP-1 and IGFBP-4 respectively. Likewise, a trinlet of approximately 42-39 kDa (IGFBP-3), a broad area called OGFBP-30 region (most probably IGFBP-1, -2 and -3 variants) and a third band of 24 kDa IGFBP-4) were seen in rat samples. Determination of IGFBP/2 and 1 in rat serum samples, as two separate bands on 12 por ciento gels was difficult due to their close electrophoretic migration and possibly to the reported lower levels of IGFBP-2 in adult rat serum. Dilutions tested on 0.2 µm nitrocellulose membranes with samples volumes between 0.25 to 1.5 µl(110-160 dilutions), showed IGFBPs curves with good linearity which suggest first, that there exist a quantitative relation betweem the amount of each protein and densitometric response and second, that the transfer of the proteins was linear across the range of 0.25 to 1.5 µl(110-160 dilutions). Moreover, the results also suggest that losses were aqually spread and that the proteins retained their binding pro[erties after the process. Reproducibility showed intra-assay coefficients of variation (CVs) of 15 per cent or lower using either a transfer device without cooling system or a combination of a transferdevice with cooling system and manually defined band boundaries. In summary, it was shown that the optimized experimental conditions here described for the WLB method, allow realiable simultaneous measurements of the main pig and rat serum IGFBPs and therefore, could be utilised to detect changes in the profile after dietary manipulations