DEVELOPMENT, VALIDATION, AND APPLICATION OF A RADIOIMMUNOASSAY FOR INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5 IN HUMAN SERUM AND OTHER BIOLOGICAL-FLUIDS

Insulin-Like growth factor binding proteins (IGFBPs) serve as transpor t proteins for the IGFs and modulate their biological effects. In orde r to evaluate the relative contribution of IGFBP-5 to the IGF binding capacity of serum and other biological fluids and to study more accura tely 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, a nd standard. The validity of the IGFBP-5 RIA. was established from the follwing data: 1) recombinant human IGFBP-5 and purified human bone-d erived IGFBP-5 inhibited the binding of tracer to the antisera in an i dentical manlier; 2) the immunoreactive IGFBP-5 in serum co-migrated w ith recombinant human IGFBP-5 in both sodium dodecyl sulfate polyacryl amide gel electrophoresis and high performance gel filtration chromato graphy; 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 tha n 90%. In order to further validate our IGFBP-5 RIA, measurements of I GFBP-5 were carried out in the conditioned media samples collected fro m human bone cells treated with effecters that are known to influence IGFBP-5; production. Treatment of human bone cells with bone morphogen etic protein-7, which increases IGFBP-5 production, caused an increase in IGFBP-5 protein levels; dexamethasone, which decreases IGFBP-5 pro duction, caused a decrease in IGFBP-5 protein levels measured in the c onditioned medium by this RIA. Application of this RIA to the measurem ent of the IGFBP-5 level in human serum revealed that the circulating level of IGFBP-5 in healthy women decreased from 664 +/- 108 mu g/l in prepubertal girls to 222 +/- 42 mu g/L in women ages 61-85 yr (P < 0. 001). There was also a small but significant decrease in the mean seru m level of IGFBP-5 in women ages 23-39 yr compared with the level in p repubertal girls (589 +/- 66 us. 664 +/- 108 mu g/L, P < 0.05). Additi onal studies with this assay may provide an insight into the physiolog ical mechanism that regulates IGFBP-5 production in vivo and the poten tial role of this protein as an IGF transport protein in serum and oth er biological fluids.