RELIABILITY OF THE WESTERN LIGAND BLOT METHOD FOR THE SIMULTANEOUS RELATIVE ESTIMATIONS OF SERUM INSULIN-LIKE-GROWTH-FACTOR BINDING-PROTEINS (IGFBPS)

It is well established that nutrition is an important regulator of bot h serum insulin-like growth factor-I (IGF-1) and its binding proteins (IGFBPs). The Western ligand blot method (WLB) for simultaneous determ inations of IGFBPs in serum or plasma samples was evaluated and valida ted with emphasis on its reproducible capabilities. After electrophore tic separation and transfer, the membranes were incubated with a mixtu re of recombinant labeled human (GF-I/IGF-II (rhIGF-I/rhlGF-II) and ba nd intensities measured by autoradiography. The typical electrophoreti c profile for pig serum, as determined with molecular weight markers, showed four mainbands of approximately 42-39, 32, 30-28 and 24 kDa whi ch seemed to correpond to IGFBP-3,IGFBP-2, IGFBP-1 and IGFBP-4 respect ively. Likewise, a triplet of approximately 42-39 kDa (IGFBP-3), a bro ad area called IGFBP-30 region (most probably IGFBP-1, -2 and -3 varia nts) and a third band of similar to 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% gels was difficult due to their close electropho retic migration and possibly to the reported lower levels of IGFBP-2 i n adult rat serum. Dilutions tested on 0.2 mu m nitrocellulose membran es with samples volumes between 0.25 to 1.5 mu l (1:10-1:60 dilutions) , showed IGFBPs curves with good linearity which suggest first, that t here exist a quantitative relation between the amount of each protein and the densitometric response and second, that the transfer of the pr oteins was linear across the range of 0.25 to 1.5 mu l (1:10-1:60 dilu tions). Moreover, the results also suggest that losses were equally sp read and that the proteins retained their bind:ng properties after the transfer process. Reproducibility showed intraassay coefficients of v ariation (CVs) of 15% or lower using either a transfer device without cooling system or a combination of a transfer device with cooling syst em and manually defined band boundaries. In summary, it was shown that the optimized experimental conditions here described for the WLB meth od, allow realiable simultaneous measurements of the main pig and rat serum IGFBPs and therefore, could be utilised to detect changes in the serum profile after dietary manipulations.