A new radioimmunoassay to measure rat insulin-like growth factor binding protein-4 (IGFBP-4) in serum, wound fluid and conditioned media.

Abstract

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor-I (IGF-I) and IGF-II. Because of the possible role of IGFBP-4 in multiple systems including reproduction, pregnancy, bone formation and wound healing, we developed a radioimmunoassay (RIA) to measure the concentration of IGFBP-4 in the serum, extracellular fluid and conditioned media of cells of rodents so that these animal models could be better exploited. Rat IGFBP-4 was expressed in Escherichia coli and purified to homogeneity. The recombinant material was used to raise a rabbit polyclonal antibody against rat IGFBP-4 and for iodination and standards. The RIA developed was sensitive to less than 1 ng/mL of rat IGFBP-4 and IGF-I did not interfere. There was no cross-reactivity with other rat IGFBPs on immuno-Western analysis of serum and wound fluid. IGFBP-4 from mouse serum did cross-react in our assay; however, serum from horse, pig or human showed no cross-reaction. Human IGFBP-1 and IGFBP-3 showed a very weak cross-reactivity. Serum IGFBP-4 levels showed no gender difference but did reveal a significant 66% increase in older rats. During the course of wound healing, which is IGF-I dependent, IGFBP-4 showed no changes. In conclusion an RIA for rat IGFBP-4 has been developed that specifically measures the concentration of IGFBP-4 in the sera, extracellular fluid and conditioned media of rodents. With this assay, the role of IGFBP-4 and IGFs in growth, development and the function of multiple systems can be further investigated using rodent models.