ANTIBODY AGAINST ACID-STABLE INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN DETECTS 150,000 MOL WT GROWTH HORMONE-DEPENDENT COMPLEX IN HUMAN PLASMA

Abstract

In order to measure the 150,000 mol wt binding complex for insulin–like growth factors in human plasma without the need for acidification to dissociate endogenous ligands, we have purified an acidstable plasma binding protein (BP) and raised a specific rabbit antiserum against it. When used in RIA, with a covalent complex of binding protein and radioiodinated insulin–like growth factor– as tracer, pure BP and human plasma samples gave parallel displacement curves. Incubation of plasma with pure insulin–like growth factor–I or –II (5 μg/ml) had no effect on the immunoreactivity of the samples. Compared to purified BP standard, the immunoreactive BP content of plasma from normal adults was 9.20±1.59 ug/ml (meaniSD, n=10), from acromegalic subjects, 22.5±3.63 μg/ml (n=10), and from GH–deficient subjects, 4.10±1.59 ug/ml (n=7). Although this antibody reacted with a protein of approximately 60,000 mol wt in acidified plasma, gel chromatography of plasma at pH 7 indicated that a GH–dependent protein of mol wt 150,000 was measured almost exclusively in unacddified samples. Direct binding of insulin–like growth factor–II, however, showed the major peak of binding activity in normal plasma in the 40–45,000 mol wt region, whereas in acromegalic plasma most ligand binding was in the 150,000 mol wt region. It is concluded that there is essentially no free immunoreactive acid–stable BP in native plasma, that the unoccupied binding sites of 40–45,000 mol wt, detectable by ligand binding, are present on an immunologically distinct protein, and that the immunoreactive 150,000 mol wt binding complex in native plasma is strongly GH–dependent.