Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: Discrepancies between radioimmunoassay and ligand blotting

Sharron E. Gargosky, Hung M. Pham, Kristin F. Wilson, Frances Liu, Linda C. Giudice, Ronald (Ron) Rosenfeld

Research output: Contribution to journalArticle

151 Citations (Scopus)

Abstract

The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 ± 3 ng/ml (mean ± SEM), 110 ± 26 ng/ml, and 209 ± 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 ± 101 ng/ml; peritoneal fluid, 1124 ± 130 ng/ml; follicular fluid, 2356 ± 211 ng/ml; nonpregnancy serum, 3556 ± 508 ng/ml; pregnancy serum, 3718 ± 842 ng/ml; and amniotic fluid, 5150 ± 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot. These results demonstrate the need to use ligand binding, immunological analysis, and the IGFBP-protease assays to characterize and measure IGFBP-3 in biological fluids.

Original languageEnglish (US)
Pages (from-to)3051-3060
Number of pages10
JournalEndocrinology
Volume131
Issue number6
StatePublished - Dec 1992
Externally publishedYes

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Insulin-Like Growth Factor Binding Protein 3
Radioimmunoassay
Ligands
Serum
Amniotic Fluid
Insulin-Like Growth Factor Binding Proteins
Western Blotting
Cerebrospinal Fluid
Follicular Fluid
Ascitic Fluid
Semen
Pregnancy
Urine
human IGFBP3 protein
Immune Sera
Peptide Hydrolases
Immunologic Techniques

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Gargosky, S. E., Pham, H. M., Wilson, K. F., Liu, F., Giudice, L. C., & Rosenfeld, R. R. (1992). Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: Discrepancies between radioimmunoassay and ligand blotting. Endocrinology, 131(6), 3051-3060.

Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids : Discrepancies between radioimmunoassay and ligand blotting. / Gargosky, Sharron E.; Pham, Hung M.; Wilson, Kristin F.; Liu, Frances; Giudice, Linda C.; Rosenfeld, Ronald (Ron).

In: Endocrinology, Vol. 131, No. 6, 12.1992, p. 3051-3060.

Research output: Contribution to journalArticle

Gargosky, Sharron E. ; Pham, Hung M. ; Wilson, Kristin F. ; Liu, Frances ; Giudice, Linda C. ; Rosenfeld, Ronald (Ron). / Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids : Discrepancies between radioimmunoassay and ligand blotting. In: Endocrinology. 1992 ; Vol. 131, No. 6. pp. 3051-3060.
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N2 - The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 ± 3 ng/ml (mean ± SEM), 110 ± 26 ng/ml, and 209 ± 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 ± 101 ng/ml; peritoneal fluid, 1124 ± 130 ng/ml; follicular fluid, 2356 ± 211 ng/ml; nonpregnancy serum, 3556 ± 508 ng/ml; pregnancy serum, 3718 ± 842 ng/ml; and amniotic fluid, 5150 ± 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot. These results demonstrate the need to use ligand binding, immunological analysis, and the IGFBP-protease assays to characterize and measure IGFBP-3 in biological fluids.

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