Document Detail


Immunoassay of insulin-like growth factor-binding protein-3 (IGFBP-3): new means to quantifying IGFBP-3 proteolysis.
MedLine Citation:
PMID:  10852472     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Posttranslational modifications, particularly proteolysis, may play a significant role in the regulation of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) physiology, and thus, measurement of modified variants of IGFBP-3 and/or their combination ratios may have important research and diagnostic relevance. Based on evaluation of a panel of monoclonal and polyclonal IGFBP-3 antibodies, we constructed three new enzyme-linked immunosorbent assays (ELISAs) using a common capture and polyclonal (ELISA-3) or monoclonal (ELISA-1 and -2) detection antibodies and evaluated them in a two-step colorimetric procedure. Evaluation of ELISA-1-3 demonstrated detection limit, dynamic range, overall precision, and recovery of the added IGFBP-3 to be generally less than 0.04 microg/L, 2-100 microg/L, less than 10%, and 91-113%, respectively. IGF-I and -II, and IGFBP-1, -2, -4, -5, and -6 did not interfere. In normal adult sera (n = 26), seminal plasma (n = 14), pregnancy sera (n = 30), and amniotic fluid (n = 30), ELISA-1-3 detected significantly different IGFBP-3 levels (by up to 6-fold, on the average), whereas levels in seminal plasma determined by ELISA-1 were undetectable. Comparison of the values obtained vs. corresponding levels by an established method (Diagnostic Systems Laboratories, Inc., active IGFBP-3 ELISA) were similarly sample dependent and, on the average, varied by up to 19-fold. Only ELISA-3 compared well with the Diagnostic Systems Laboratories, Inc., IGFBP-3 ELISA when samples from normal adults were analyzed. The observed variability could not be totally explained by 50% lower reactivity of ELISA-1-3 for glycosylated IGFBP-3 vs. the nonglycosylated form, and changes in phosphorylation had no effect on immunoreactivity. Evaluation of IGFBP-3 after proteolysis by seminal plasma, plasmin, or thrombin suggested recognition of intact IGFBP-3 by ELISA-1, whereas ELISA-3 appeared to measure intact and proteolyzed IGFBP-3 (total IGFBP-3) with similar potency. In contrast, levels determined by ELISA-2 increased severalfold, indicating preferential recognition of IGFBP-3 fragments. We propose that immunoassay capable of differential determination of IGFBP-3 variants may help better define the physiological importance and potential clinical value of IGFBP-3 measurements.
Authors:
A Diamandi; J Mistry; R G Krishna; J Khosravi
Publication Detail:
Type:  Comparative Study; Journal Article    
Journal Detail:
Title:  The Journal of clinical endocrinology and metabolism     Volume:  85     ISSN:  0021-972X     ISO Abbreviation:  J. Clin. Endocrinol. Metab.     Publication Date:  2000 Jun 
Date Detail:
Created Date:  2000-06-23     Completed Date:  2000-06-23     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0375362     Medline TA:  J Clin Endocrinol Metab     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  2327-33     Citation Subset:  AIM; IM    
Affiliation:
Diagnostics Systems Laboratories, Inc., Toronto, Ontario, Canada.
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MeSH Terms
Descriptor/Qualifier:
Adult
Amniotic Fluid / chemistry*
Antibodies
Antibodies, Monoclonal
Enzyme-Linked Immunosorbent Assay / methods
Female
Fibrinolysin / metabolism
Glycosylation
Humans
Insulin-Like Growth Factor Binding Protein 3 / analysis*,  blood*
Kinetics
Male
Middle Aged
Pregnancy
Reproducibility of Results
Semen / chemistry
Sensitivity and Specificity
Chemical
Reg. No./Substance:
0/Antibodies; 0/Antibodies, Monoclonal; 0/Insulin-Like Growth Factor Binding Protein 3; EC 3.4.21.7/Fibrinolysin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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