Development, validation, and application of a radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids.

Abstract

Insulin-like growth factor binding proteins (IGFBPs) serve as transport proteins for the IGFs and modulate their biological effects. In order to evaluate the relative contribution of IGFBP-5 to the IGF binding capacity of serum and other biological fluids and to study more accurately the in vivo regulation of serum IGFBP-5, we have developed an RIA for IGFBP-5 using recombinant human IGFBP-5 as the antigen, tracer, and standard. The validity of the IGFBP-5 RIA was established from the following data: 1) recombinant human IGFBP-5 and purified human bone-derived IGFBP-5 inhibited the binding of tracer to the antisera in an identical manner; 2) the immunoreactive IGFBP-5 in serum co-migrated with recombinant human IGFBP-5 in both sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance gel filtration chromatography; 3) none of the other known IGFBPs (IGFBP-1, -2, -3, -4, and -6) exhibited significant cross-reactivity; 4) the addition of IGF-I or IGF-II to the samples did not affect the recovery of IGFBP-5; and 5) the recovery of the exogenously added IGFBP-5 to serum was greater than 90%. In order to further validate our IGFBP-5 RIA, measurements of IGFBP-5 were carried out in the conditioned media samples collected from human bone cells treated with effectors that are known to influence IGFBP-5 production. Treatment of human bone cells with bone morphogenetic protein-7, which increases IGFBP-5 production, caused an increase in IGFBP-5 protein levels; dexamethasone, which decreases IGFBP-5 production, caused a decrease in IGFBP-5 protein levels measured in the conditioned medium by this RIA. Application of this RIA to the measurement of the IGFBP-5 level in human serum revealed that the circulating level of IGFBP-5 in healthy women decreased from 664 +/- 108 ug/L in prepubertal girls to 222 +/- 42 ug/L in women ages 61-85 yr (P < 0.001). There was also a small but significant decrease in the mean serum level of IGFBP-5 in women ages 23-39 yr compared with the level in prepubertal girls (589 +/- 66 vs. 664 +/- 108 ug/L, P < 0.05). Additional studies with this assay may provide an insight into the physiological mechanism that regulates IGFBP-5 production in vivo and the potential role of this protein as an IGF transport protein in serum and other biological fluids.