An antiserum (R8) raised against a purified specific low-Mr (16,000-18,000) insulin-like-growth-factor (IGF)-inhibitor/binding protein, which is immunologically related to the native growth hormone (GH)-dependent Mr-150,000 IGF-binding protein in serum, has been used to probe a possible additional relationship to the predominant non-GH-dependent IGF-binding protein (Mr approximately 30,000) of human amniotic fluid. Amniotic-fluid fractions and an IGF-inhibitory fraction of serum were analysed by covalent cross-linking, ligand-blotting and immunoblotting techniques. Western blotting of the serum fraction using the R8 antiserum gave five immunoreactive (ir) bands, of which at least three (Mr 38,000, 34,000 and 23,000) possessed IGF-binding activity, as indicated by ligand (125I-IGF-I) blotting. Western blotting of two differently prepared amniotic-fluid fractions, which both showed potent IGF-inhibitory bioactivity, gave several (Mr range 14,500-73,000) ir bands, of which two (Mr 28,000 and 17,000) were most prominent. Ligand-blotting analysis gave a single intensely labelled band at Mr 28,000, consistent with the major presence of the Mr-28,000-30,000 amniotic-fluid IGF-binding protein. Covalent cross-linking of the amniotic-fluid fractions to 125I-IGF-I gave three specifically cross-linked complexes (Mr 36,000, 32,000 and 23,000), which, assuming a 1:1 binding stoichiometry with IGF (Mr 7500), represent binding proteins of Mr 28,500, 24,500 and 15,500 respectively. The Mr-15,500 binding protein, very similar to the Mr-17,000 ir band, in all likelihood represents the IGF-inhibitor protein. Our results indicate that inhibitor-sized binding proteins and IGF-inhibitor bioactivity are present in human amniotic fluid, and that the IGF-inhibitor protein (Mr 16,000) and amniotic-fluid IGF-binding protein (Mr 28,000) are immunologically related. Since the IGF-inhibitor protein is also immunologically and structurally related to the native, GH-dependent, Mr-150,000 binding protein in serum, our data suggest a heretofore-unrecognized immunological similarity between the Mr-150,000 binding protein and the amniotic-fluid binding protein and its serum analogue, the Mr approximately 30,000, non-GH-dependent, binding protein.

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