RECOMBINANT SYNTHESIS OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-4(IGFBP-4) - DEVELOPMENT, VALIDATION, AND APPLICATION OF A RADIOIMMUNOASSAY FOR IGFBP-4 IN HUMAN SERUM AND OTHER BIOLOGICAL-FLUIDS

Insulin-like growth factor-binding protein-4 (IGFBP-4), like the five other IGFBPs present in human serum, acts as a transport protein for i nsulin-like growth factor I (IGF-I) and IGF-II and modulates their bio logical effects. To investigate the role of IGFBP-4 in the physiology of the IGF system, we developed a sensitive RIA for IGFBP-4 employing, as antigen, tracer, and standard, recombinant human IGFBP-4 (rhIGFBP- 4) expressed in Escherichia coli as a fusion protein with glutathione S-transferase and affinity purified with glutathione-derivatized resin . Antibody against the rhIGFBP-4 fusion protein was raised in guinea p igs; tracer and standard were provided by the rhIGFBP-4 moiety that ha d been cleaved from the rhIGFBP-4 fusion protein and repurified by rev erse phase high pressure liquid chromatography. We report that both IG FBP-4 purified from PC3 human prostate cell-conditioned medium and rhI GFBP-4 bound IGF and migrated in electrophoresis gels in an identical manner; that in gel permeation chromatography, rhIGFBP-4 coeluted with the IGFBP-4 present in human serum; and that both are equally immunor eactive with the IGFBP-4 antiserum. Employing this IGFBP-4 RIA, we det ermined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 anti serum, and that recovery of IGFBP-4 from serum samples exceeded 90% wh en exogenous IGFBP-4 was added and was unaffected by the addition of I GFs or by repeated freezing and thawing of the sample. We employed thi s IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human oste osarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D-3, agents known to increase the IGFBP -4 messenger ribonucleic acid level. Application of this RIA to the me asurement of IGFBP-4 in human serum revealed that the circulating leve l of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 mu g/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 mu g/L). The mean circulating level of PTH was als o 20% higher in the 61-87 yr group compared to that in the 23-40 yr gr oup (P < 0.01). In addition, serum IGFBP-4 amounts showed a significan t positive correlation with age (r = 0.54; P < 0.001) and serum PTH (r = 0.26; P < 0.01). These data validate this IGFBP-4 RIA and illustrat e its utility in illuminating the physiological mechanisms that regula te IGFBP-4 in. vivo and influence its effects on the IGFs in both norm al and abnormal pathology and in aging.