ИСТИНА

Background: Recent findings show that N- and C-terminal fragments of IGF-binding protein-4 (NT- and CT-IGFBP-4) can be utilized as biomarkers for the prediction of major adverse cardiac events in patients with suspected acute coronary syndrome (ACS). It was suggested that increased NT- and CT-IGFBP-4 level in patients’ blood is associated with increased expression and proteolytic activity of the dimeric form of the Pregnancy Associated Plasma Protein A (dPAPP-A). dPAPP-A is overexpressed in vulnerable atherosclerotic plaques and is considered to be responsible for the plaque destabilization. Previously we have reported the method of IGFBP-4 fragments measurements. However, immunochemical and biochemical properties of circulating NT- and CT-IGFBP-4 have not been investigated yet. Methods: Monoclonal antibodies (MAb) IBP180 and MAb IBP185 (HyTest,Finland) specific to core region of NT- and CT-IGFBP-4, respectively, were usedfor affinity purification of endogenous fragments from pooled plasma collected from ACS patients. For NT- and CT-IGFBP-4 quantification two immunoassays described by Postnikov et al. (2012) were utilized. Each assay has one antibody that is fragment-specific and recognizes neo-epitope formed after IGFBP-4 cleavage by dPAPP-A. Such antibody has no cross-reaction with full-length IGFBP-4 and recognizes only corresponding fragment. Assays were calibrated using recombinant NT- and CT-IGFBP-4 expressed in HEK293 cells (HyTest, Finland). Size-exclusion chromatography on Superdex 75 column was used for characterization of IGFBP-4 fragments in patients’ plasma. Results: Endogenous IGFBP-4 fragments were purified from patients’ plasma at the quantities corresponding to 80 - 100% of initial immunochemical activity. In SDS-PAGE studies it was shown that purified NT- and CT-IGFBP-4 had apparent molecular masses equal to molecular masses of corresponding recombinant proteins. Identity of IGFBP-4 fragments purified from human plasma and recombinant proteins was confirmed by Western blotting studies with several fragment-specific MAbs as well as by mass spectrometry analysis. Analysis of purified fragments by specific MAbs showed that the most part of both endogenous NT- and CT-IGFBP-4 molecules contained intact, non-truncated neo-epitopes. Thus, the assays utilizing antibodies specific to the neo-epitopes could be used for the NT- and CT-IGFBP-4 quantification in patients’ blood. Size-exclusion chromatography analysis of individual plasma samples of ACS patients revealed the single peak of CT-IGFBP-4 immunochemical activity with the apparent molecular mass very close to that of corresponding recombinant fragment (19.5 and 20.2 kDa, respectively). For NT-IGFBP-4 several peaks of immunochemical activity were identified. The main peak (23.6 - 26.3 kDa, 70 - 80% of immunochemical activity) corresponded to the position of recombinant NT-IGFBP-4 (23.6 kDa). Several additional minor peaks (11, 57, and >300 kDa) of NT-IGFBP-4 immunochemical activity were also identified. Immunochemical activity in these peaks varied significantly (up to 30%) in individual samples. The presence of these NT-IGFBP-4 forms could reflect degradation as well as formation of higher molecular weight complexes with other proteins in human plasma. Conclusions: Here we for the first time describe endogenous NT-IGFBP-4 and CT- IGFBP-4 from ACS patients’ plasma. Both proteins display the biochemical and immunochemical features similar to the proteins expressed in mammalian cell lines.