Comparative Use of Immunological Methods & Ligand Blotting of Insulin-Like Growth Factor Binding Proteins in Serum and Other Biological Fluids

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

Research output: Contribution to journalArticle

Abstract

There is controversy as to which methodology most accurately detects the Insulin-like growth factor binding proteins (IGFBPs). We have compared immunological and ligand binding techniques to assess the IGFBPs in serum from non-pregnant women and women in late pregnancy, fetal cord serum and amniotic fluid. a specific radioimmunoassay for IGFBP-3 measured mean levels of 3.5 and 3.7μg/ml for non-pregnancy and pregnancy serum, respectively, 0.7μg/ml for fetal cord serum and 5.1μg/ml for amniotic fluid. in contrast, Westem-ligand blot of fluid showed IGFBP-3 as a 40 kDa doublet in non-pregnancy serum and fetal cord serum, unlike pregnancy serum or amniotic fluid, where IGFBP-3 was undetectable by standard blot analysis. However, analysis of fluids at 25-50 times more volume showed that IGFBP-3 was detectable as a 40 kDa doublet in pregnancy serum and amniotic fluid. the molecular mass of immunoreactive IGFBP-3 was examined by Western-immunoblot, probed with antibody ±IGFBP-3gl. As in Westem-ligand blot, intact IGFBP-3 (40 kDa) was easily detected in non-pregnancy serum and fetal cord serum, but required greater volumes of sample for detection in pregnancy serum and amniotic fluid. in addition, smaller immunoreactive fragments were found in all fluids: A faint 27 and 22 kDa band was found in non-pregnancy serum and fetal cord serum, with more dominant bands of 31-27, 26, 25, 24 and 22 kDa in pregnancy serum and 31-26, 25, 22,19-17 kDa in amniotic fluid. To address the obvious discrepancies between ligand and immunological analysis, samples were assessed for IGFBP-3 protease activity. Non-pregnancy serum proteolyzed 14% of the substrate, pregnancy serum 61%, fetal cord serum 33%, and amniotic fluid 50-61%. Thus, fluids with apparently low IGFBP-3 by Westem-ligand blot, but high IGFBP-3 by RIA, showed high levels of protease activity. Previously, IGFBP-proteases have been shown to degrade IGFBP to smaller fragments with a reduced affinity for IGF. We conclude that the discrepancy between ligand binding techniques and immunological techniques can be explained, at least in part, by the presence of IGFBP-protease activity.

Original languageEnglish (US)
Pages (from-to)21-29
Number of pages9
JournalClinical Pediatric Endocrinology
Volume2
Issue number1
DOIs
StatePublished - 1993
Externally publishedYes

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Keywords

  • IGF binding protein (IGFBP)
  • insulin-like growth factor (IGF)
  • protease

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health
  • Endocrinology, Diabetes and Metabolism
  • Endocrinology

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