Characterization of serum-derived and recombinant rat IGF-I and their use for measuring true concentrations of IGF-I in rat plasma.
While numerous researchers have used rat models to investigate the in vivo actions of IGF-I, interpretation of the results in terms of true concentrations of rat IGF-I (rIGF-I) in plasma has been hampered by the absence of homologous reference standards. In order to overcome this we have produced recombinant rIGF-I (rrIGF-I) from Escherichia coli using procedures similar to those we have previously described for the production of other recombinant IGFs. The rrIGF-I is indistinguishable from serum-derived rIGF-I when characterized in a number of in vitro assays including ability to stimulate protein synthesis and inhibit protein degradation in cultured rat cells, as well as in interactions with the rat type-1 IGF receptor and with rat IGF-binding proteins. Moreover, both the serum-derived and the recombinant rat proteins are similar to recombinant human IGF-I (rhIGF-I) in these assays. However, differences between the human and rat IGFs are apparent when tested in immunoassays using some antibodies raised against rhIGF-I. Furthermore, the differences between rhIGF-I and rrIGF-I are even greater when rhIGF-I is used as the competing radiolabel in these assays, a situation that can lead to a two- to threefold underestimation of the actual concentration of IGF-I in rat plasma. These results indicate that, while immunoassays employing antibodies raised against rhIGF-I and rhIGF-I reference standards reliably indicate trends in IGF-I concentrations in rat plasma, the true amounts of rIGF-I present can only be assured in an assay using homologous tracer and reference peptides.
Upton
Z
Z
Cooperative Research Centre for Tissue Growth and Repair, CSIRO Division of Human Nutrition, Adelaide, South Australia.
Webb
H
H
Tomas
F M
FM
Ballard
F J
FJ
Francis
G L
GL
D000818:Animals; D001483:Base Sequence; D007118:Immunoassay; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D018971:Insulin-Like Growth Factor Binding Protein 2; D009841:Oligonucleotides; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I