BACKGROUND: Detection of incompletely processed precursor forms of insulin-like growth factor-II ("big" IGF-II) in plasma is essential for both the diagnosis and follow-up of non-islet cell tumor-induced hypoglycemia (NICTH) and may be relevant to other diseases as well. RIA using an antibody raised against a synthetic peptide consisting of the first 21 amino acids of the E domain [E(68-88)] of human pro-IGF-II cannot distinguish between E-peptide-containing big IGF-II and cleaved E domain or fragments. We therefore developed and validated an ELISA that specifically detects big IGF-II in plasma. METHODS: The ELISA used a solid-phase antibody to E(68-88) and a liquid-phase monoclonal hIGF-II antibody. Pro-IGF-II purified from normal human plasma was used as a calibrator. Acid Sep-Pak C(18) extracts of plasma from NICTH patients were analyzed, and the results were compared with those obtained for plasma samples from healthy individuals. In addition, blood specimens derived from dialyzed patients with chronic renal failure, which contained relatively high concentrations of cleaved E domain or fragments, were studied. The results were validated by acid Sephadex G-50 gel filtration. RESULTS: Results from this ELISA indicated that the concentration of big IGF-II in NICTH plasma was higher (mean +/- SD, 22.6 +/- 9.4 nmol/L) than in normal plasma (3.8 nmol/L). Conversely, the concentrations in pooled CRF plasma (2.0 +/- 0.8 nmol/L) were low. Antibodies directed against either E(68-88) or E(13-134) of pro-IGF-II could be used to detect these peptides in tumor tissue by immunohistochemistry. CONCLUSIONS: The possibility of quantifying pro-IGF-II by ELISA in plasma represents a potentially useful tool for the diagnosis and follow-up of NICTH and should facilitate further in vitro and in vivo studies on its regulation and function in humans.
BACKGROUND: Detection of incompletely processed precursor forms of insulin-like growth factor-II ("big" IGF-II) in plasma is essential for both the diagnosis and follow-up of non-islet cell tumor-induced hypoglycemia (NICTH) and may be relevant to other diseases as well. RIA using an antibody raised against a synthetic peptide consisting of the first 21 amino acids of the E domain [E(68-88)] of human pro-IGF-II cannot distinguish between E-peptide-containing big IGF-II and cleaved E domain or fragments. We therefore developed and validated an ELISA that specifically detects big IGF-II in plasma. METHODS: The ELISA used a solid-phase antibody to E(68-88) and a liquid-phase monoclonal hIGF-II antibody. Pro-IGF-II purified from normal human plasma was used as a calibrator. Acid Sep-Pak C(18) extracts of plasma from NICTH patients were analyzed, and the results were compared with those obtained for plasma samples from healthy individuals. In addition, blood specimens derived from dialyzed patients with chronic renal failure, which contained relatively high concentrations of cleaved E domain or fragments, were studied. The results were validated by acid Sephadex G-50 gel filtration. RESULTS: Results from this ELISA indicated that the concentration of big IGF-II in NICTH plasma was higher (mean +/- SD, 22.6 +/- 9.4 nmol/L) than in normal plasma (3.8 nmol/L). Conversely, the concentrations in pooled CRF plasma (2.0 +/- 0.8 nmol/L) were low. Antibodies directed against either E(68-88) or E(13-134) of pro-IGF-II could be used to detect these peptides in tumor tissue by immunohistochemistry. CONCLUSIONS: The possibility of quantifying pro-IGF-II by ELISA in plasma represents a potentially useful tool for the diagnosis and follow-up of NICTH and should facilitate further in vitro and in vivo studies on its regulation and function in humans.
Authors: Sameer A Greenall; John D Bentley; Lesley A Pearce; Judith A Scoble; Lindsay G Sparrow; Nicola A Bartone; Xiaowen Xiao; Robert C Baxter; Leah J Cosgrove; Timothy E Adams Journal: J Biol Chem Date: 2012-11-19 Impact factor: 5.157