title: Characterization of endogenously circulating IGFBP-4 fragments-Novel biomarkers for cardiac risk assessment.
abstract: BACKGROUND
  Recent findings show that circulating N- and C-terminal fragments of IGF-binding protein-4 (NT-IGFBP-4 and CT-IGFBP-4) can be utilized as biomarkers for cardiac risk assessment in acute coronary syndrome (ACS) patients. The fragments are thought to be the products of pregnancy-associated plasma protein A (PAPP-A)-dependent proteolysis. Two immunoassays for the measurement of IGFBP-4 fragments have been proposed. However, properties of the endogenous IGFBP-4 fragments that could influence the performance of the immunoassays were still not investigated.
  
  
  METHODS
  NT- and CT-IGFBP-4 were extracted from pooled ACS plasma using affinity purification, and their concentrations were measured using sandwich immunoassays utilizing antibodies specific to their proteolytic neo-epitopes or internal epitopes. The extracted fragments were characterized by Western blots (WB) and mass-spectrometry. ACS plasma samples were analyzed by size exclusion chromatography (SEC).
  
  
  RESULTS
  Immunoassays utilizing the neo-epitope-specific and the internal epitope-specific antibodies measured equal concentrations of the analyte in the endogenous IGFBP-4 fragments preparations. Only the 18 kDa NT-IGFBP-4 and 14 kDa CT-IGFBP-4 were detected in the WB analysis. Using mass-spectrometry, peaks corresponding to intact non-truncated and non-modified NT-IGFBP-4 (14626 Da) and CT-IGFBP-4 (11346 Da) were observed. The absence of complexed forms of IGFBP-4 in patients' plasma was demonstrated using SEC.
  
  
  CONCLUSIONS
  Endogenous NT- and CT-IGFBP-4 from ACS patients' plasma correspond to the PAPP-A-derived IGFBP-4 fragments and do not undergo any truncation, modification, or complex formation in the patients' blood. Because of the demonstrated intact state of the circulating IGFBP-4 fragments, the neo-epitope-specific immunoassays perform reliably, allowing further clinical validation of these novel biomarkers.
authors.affiliation: HyTest Ltd, Turku, Finland; School of Biology, Moscow State University, Moscow, Russia. Electronic address: Alexey.Konev@hytest.fi.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Konev
authors.affiliation: HyTest Ltd, Turku, Finland.
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Smolyanova
authors.affiliation: HyTest Ltd, Turku, Finland.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Kharitonov
authors.affiliation: HyTest Ltd, Turku, Finland; School of Biology, Moscow State University, Moscow, Russia.
authors.forename: Daria V
authors.initials: DV
authors.lastname: Serebryanaya
authors.affiliation: HyTest Ltd, Turku, Finland.
authors.forename: Stanislav V
authors.initials: SV
authors.lastname: Kozlovsky
authors.affiliation: School of Biology, Moscow State University, Moscow, Russia.
authors.forename: Andrey N
authors.initials: AN
authors.lastname: Kara
authors.affiliation: HyTest Ltd, Turku, Finland; School of Biology, Moscow State University, Moscow, Russia.
authors.forename: Evgeniya E
authors.initials: EE
authors.lastname: Feygina
authors.affiliation: HyTest Ltd, Turku, Finland; School of Biology, Moscow State University, Moscow, Russia.
authors.forename: Alexey G
authors.initials: AG
authors.lastname: Katrukha
authors.affiliation: HyTest Ltd, Turku, Finland; School of Biology, Moscow State University, Moscow, Russia.
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Postnikov
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000097:blood; D002318:Cardiovascular Diseases / Q000097:blood*; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000097:blood*; D008297:Male; D008875:Middle Aged; D010446:Peptide Fragments / Q000097:blood; D011266:Pregnancy-Associated Plasma Protein-A / Q000378:metabolism; D018570:Risk Assessment; D012307:Risk Factors
chemical_list: D015415:Biomarkers; D018974:Insulin-Like Growth Factor Binding Protein 4; D010446:Peptide Fragments; D011266:Pregnancy-Associated Plasma Protein-A; C579283:PAPPA protein, human
keywords: Acute coronary syndrome (ACS); Biomarkers; Cardiovascular risk; IGF-binding protein-4 (IGFBP-4) fragments; Immunoassay; Ischemia; Major adverse cardiac events (MACE); Myocardial infarction (MI); Pregnancy-associated plasma protein A (PAPP-A)