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 insulin-like growth factor I (IGF-I) and IGF-II and modulates their biological 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 pigs; tracer and standard were provided by the rhIGFBP-4 moiety that had been cleaved from the rhIGFBP-4 fusion protein and repurified by reverse phase high pressure liquid chromatography. We report that both IGFBP-4 purified from PC3 human prostate cell-conditioned medium and rhIGFBP-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 immunoreactive with the IGFBP-4 antiserum. Employing this IGFBP-4 RIA, we determined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 antiserum, and that recovery of IGFBP-4 from serum samples exceeded 90% when exogenous IGFBP-4 was added and was unaffected by the addition of IGFs or by repeated freezing and thawing of the sample. We employed this IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human osteosarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D3, agents known to increase the IGFBP-4 messenger ribonucleic acid level. Application of this RIA to the measurement of IGFBP-4 in human serum revealed that the circulating level of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 microgram/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 microgram/L). The mean circulating level of PTH was also 20% higher in the 61-87 yr group compared to that in the 23-40 yr group (P < 0.01). In addition, serum IGFBP-4 amounts showed a significant 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 illustrate its utility in illuminating the physiological mechanisms that regulate IGFBP-4 in vivo and influence its effects on the IGFs in both normal and abnormal pathology and in aging.
Honda
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Y
Department of Medicine, Loma Linda University, California 92357, USA.
Landale
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EC
Strong
D D
DD
Baylink
D J
DJ
Mohan
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S
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000906:Antibodies; D001681:Biological Assay; D015153:Blotting, Western; D001826:Body Fluids / Q000737:chemistry; D001859:Bone Neoplasms; D001842:Bone and Bones / Q000166:cytology; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004926:Escherichia coli; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000032:analysis / Q000096:biosynthesis* / Q000097:blood*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D012516:Osteosarcoma; D011863:Radioimmunoassay / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012016:Reference Values; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000906:Antibodies; D018974:Insulin-Like Growth Factor Binding Protein 4; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II