Vladimir L. Filatov

Biochemical Factors Influencing Measurement of Cardiac Troponin I in Serum

Abstract Troponin I (cTnI), a sensitive and reliable marker of damaged cardiac tissue, is now widely used in clinics. But the existence of different cTnI assays with a wide variety of cut-off values and discrepancies between the results of measurements of one and the same sample by different assays is puzzling for clinicians. The most urgent issue at the moment is the development of the international standard, which can be used for the calibration of different assays, thus decreasing between assay biases. But another important item, which should be considered by manufacturers, is the standardisation of the epitopes of the antibodies used for the assay development. The importance of such standardisation originates from the complicated biochemical nature of cTnI. Here we briefly try to analyse the main factors that can influence antigen recognition by different antibodies and formulate principles of antibody selection for assay development.

Epitope mapping of anti‐troponin I monoclonal antibodies

Two groups of monoclonal antibodies (MAbs) specific to human cardiac troponin I (cTnI) were generated by immunization of mice by isolated cTnI (group I, 16 MAbs) or by the whole troponin complex (group II, 15 MAbs). Two sets of overlapping decapeptides covering the complete sequence of cTnI were prepared and used for epitope mapping by SPOT technique. Majority of MAbs (28 out of 31) interacts with synthetic peptides thus indicating that they recognize liner epitopes. MAbs raised against isolated cTnI preferentially recognize epitopes located at the N‐ or C‐terminal ends of cTnI. Nine out of fifteen MAbs raised against whole troponin complex interact with epitopes located in the N‐terminal part of cTnI. Generation of MAbs recognizing both isolated cTnI and cTnI inside of troponin complex and mapping their epitopes provides reliable detection of TnI in serum of patients with acute myocardial infarction.

Comparison of soluble and placental transferrin receptors as standards for the determination of soluble transferrin receptor in humans

Transferrin receptor is a transmembrane protein that mediates iron transport from blood into cells. The extracellular part of this receptor circulates in blood as soluble transferrin receptor (sTfR) and the immunological determination of this parameter is widely used in clinical practice. This study aimed at comparing the properties of sTfR and placental TfR (pTfR) and to evaluate the validity of pTfR as a standard for the determination of sTfR in human serum. sTfR and pTfR were studied by immunofluorescent assay and fast protein liquid chromatography (FPLC) gel filtration. Serum sTfR levels were calculated using sTfR or pTfR as a standard. The immunological activity of pTfR was lower than that of sTfR in all anti‐TfR monoclonal antibody pairs. Upon FPLC gel filtration, pTfR eluted in a void volume of the column as a protein with a molecular weight (MW) of >1500 kDa, whereas the MW of sTfR corresponded to 237 kDa. This could be a result of micelle formation by pTfR because of its hydrophobic intracellular part. The serum sTfR levels calculated against sTfR were 2.5 times lower than those calculated against pTfR. Serum sTfR levels are overestimated when pTfR is used as the standard.

Monoclonal antibodies with equal specificity to D-dimer and high-molecular-weight fibrin degradation products.

Fibrin degradation results in the formation of fibrin degradation products (FDPs) of different molecular weights, which include D-dimer. Commercial D-dimer assays recognize multiple forms of FDP with different specificity. As a result, the absence of an international D-dimer standard and the marked discrepancy in the D-dimer values in the same samples measured by assays from different manufacturers have become the primary problems that clinicians face in the D-dimer determination. We consider that an assay with equal specificity to all FDP forms regardless of their molecular weights could help to solve these problems. We aimed to produce mAbs that could equally recognize high-molecular-weight FDP (HMW FDP) and D-dimer. mAbs against D-dimer were produced. The HMW FDP/D-dimer ratios in plasma samples were analyzed following protein separation by gel filtration using the developed fluoroimmunoassay. A sandwich immunoassay with equal specificity to HMW FDP and D-dimer was developed and applied to determine HMW FDP/D-dimer ratios in patients with different diseases. Although the HMW FDP levels prevailed in thrombotic patients, the FDP and D-dimer levels were comparable in septic patients. Meanwhile, the D-dimer levels often exceeded the HMW FDP levels in patients who had undergone surgery. The ‘D-dimer’ levels that were detected by different assays also varied greatly depending on the assay specificities to FDP and D-dimer. Our findings show that the introduction of assays with equal specificities to FDP and D-dimer in clinical practice is a possible way of standardizing D-dimer measurements.

Glycosylated and non-glycosylated NT-IGFBP-4 in circulation of acute coronary syndrome patients

BACKGROUND N-terminal and C-terminal proteolytic fragments of IGF binding protein 4 (NT-IGFBP-4 and CT-IGFBP-4) were recently shown to predict adverse cardiac events in acute coronary syndrome (ACS) patients. NT-IGFBP-4 and CT-IGFBP-4 are products of the pregnancy-associated plasma protein-A (PAPP-A)-mediated cleavage of IGFBP-4. It has been demonstrated that circulating IGFBP-4 is partially glycosylated in its N-terminal region, although the influence of this glycosylation on PAPP-A-mediated proteolysis and the ratio of glycosylated/non-glycosylated IGFBP-4 fragments in human blood remain unrevealed. The aims of this study were to investigate i) the presence of glycosylated NT-IGFBP-4 in the circulation, ii) the influence of the glycosylation of IGFBP-4 on its susceptibility to PAPP-A-mediated cleavage, and iii) the influence of glycosylation on NT-IGFBP-4 immunodetection. METHODS Affinity purification was used for the extraction of IGFBP-4 and NT-IGFBP-4 from plasma samples. Purified proteins were quantified by Western blotting and specific sandwich immunoassays, while molecular masses were determined using mass spectrometry. RESULTS Glycosylated NT-IGFBP-4 was identified in the blood of ACS patients. The fraction of glycosylated NT-IGFBP-4 in individual plasma samples was 9.8%-23.5% of the total levels of NT-IGFBP-4. PAPP-A-mediated proteolysis of glycosylated IGFBP-4 was 3-4 times less efficient (p < 0.001) than proteolysis of non-glycosylated protein. A sandwich fluoroimmunoassay that was designed for quantitative NT-IGFBP-4 measurements recognized both protein forms with the same efficiency. CONCLUSIONS Although glycosylation suppresses PAPP-A-mediated IGFBP-4 cleavage, a considerable amount of glycosylated NT-IGFBP-4 is present in blood. Glycosylation does not influence NT-IGFBP-4 measurements using a specific sandwich immunoassay.

OLIGOMERIC ADIPONECTIN FORMS AND THEIR COMPLEXES IN THE BLOOD OF HEALTHY DONORS AND PATIENTS WITH TYPE 2 DIABETES MELLITUS

Adiponectin (Adn) is a protein that circulates in the blood in several oligomeric forms, namely low-, medium-, and high-molecular-weight forms. Adn may serve as a risk factor for type 2 diabetes mellitus (T2DM). The aims of this work were (1) to produce monoclonal antibodies (MAbs) specific to different Adn oligomeric forms, (2) to design immunoassays suitable for measuring the Adn forms present in human blood, and (3) to investigate the changes in Adn forms that occur in patients with T2DM. Gel filtration, fluoroimmunoassays, and Western blotting were utilized as major techniques in this study. MAbs recognizing various oligomeric forms of Adn were obtained. Complexes between Adn and complement component C1q and between the low molecular weight form of Adn and albumin were described in human blood. A decrease in the total Adn and Adn-albumin complex levels in the blood of patients with T2DM and no difference in the levels of the Adn-C1q complex in comparison with healthy volunteers were demonstrated. An Adn94-Adn63 fluoroimmunoassay was selected as the technique that most accurately measured the mass ratio of Adn oligomers in blood samples, and an Adn214-Adn27 assay that measured the low-molecular-weight form of Adn only.