Background: Peptides derived from brain natriuretic peptide (BNP) precursor (proBNP), BNP, and the Nterminal fragment of proBNP (NT-proBNP) are used as biomarkers of heart failure. It remains unclear which forms of these peptides circulate in blood and which forms are measured by assays for these natriuretic peptides. Methods: To design assays for immunodetection of proBNP, NT-proBNP, and BNP, we used a panel of BNP-and NT-proBNP-specific monoclonal antibodies (MAbs). All MAbs were tested in 2-site combinations in time-resolved fluoroimmunoassays with recombinant or synthetic antigens and plasma from heart failure (HF) patients. ProBNP and related molecules were assayed in HF plasma samples and plasma extracts by means of gel filtration fast protein liquid chromatography (FPLC) before and after protein fractionation on Sep-Pak C18 cartridges. Results: The limits of detection for BNP, proBNP, and NT-proBNP assays were 0.4, 3, and 10 ng/L, respectively. Gel filtration-FPLC studies revealed 1 peak of NTproBNP (ϳ25 kDa), 1 peak of proBNP (ϳ37 kDa), and 2 peaks of BNP immunoreactivity, a major peak (ϳ37

We have analyzed by different immunological methods the proteolytic degradation of cardiac troponin I (cTnI) in human necrotic tissue and in serum. cTnI is susceptible to proteolysis, and its degradation leads to the appearance of a wide diversity of proteolytic peptides with different stabilities. N- and C-terminal regions were rapidly cleaved by proteases, whereas the fragment located between residues 30 and 110 demonstrated substantially higher stability, possibly because of its protection by TnC. We conclude that antibodies selected for cTnI sandwich immunoassays should preferentially recognize epitopes located in the region resistant to proteolysis. Such an approach can be helpful for a much needed standardization of cTnI immunoassays and can improve the sensitivity and reproducibility of cTnI assays.

Fourteen monoclonal antibodies (mAbs) against human cardiac troponin I (cTnI) were generated by commonly used experimental techniques. All these antibodies, as well as antibody 414 (HyTest), were specific for human cTnI. Fifteen antibodies thus obtained were tested in a sandwich cTnI immunofluorescence assay (altogether 196 combinations). Ten pairs giving the highest sensitivity were selected for further investigation. The effect of TnI–TnC complex formation on antibody interaction with antigen was analyzed. The formation of TnI–TnC complex results in a significant decrease of the interaction of mAbs with TnI for seven of 10 analyzed pairs of antibodies. Using two pairs of cTnI-specific mAbs, one that recognized only free cTnI but not cTnI complexed with cTnC, and another that could be used for measurement of total cTnI (free cTnI and cTnI in complex with cTnC), we demonstrated that the main part of cTnI in serum collected from acute myocardial infarction patients is presented in the complex form. We concluded that effective and reliable immunological detection of TnI is possible only when antibodies used for assay development recognize both free TnI and TnI complexed with other troponin components.

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 cTnl 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 acutc myocardial infarction. BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONALstructure of skeletal and cardiac TnI [4,5]. This makes possible production of cTnl specific antibodies which can be used for detection of many types of myocardial cells damage [6,7]. After cell death caused by infarction cTnI is released into the extracellular space and 4 -6 hours after onset of the chest pain can be detected in the blood stream [8,

Urinary microscopy is a diagnostic tool which is largely used by nephrologists. In the opinion of the authors the best results can be achieved when all the aspects concerning this test are properly taken into account. Thus, from the methodological point of view, proper patient guidance, proper urine collection and handling, adequate microscopic equipment, and knowledge of the factors which can influence the results are all necessary. All the elements of clinical importance have to be known, namely, erythrocytes (with their morphological subtypes), leukocytes, tubular cells, uroepithelial cells (both superficial and deep), lipids, casts, crystals, and microorganisms. Then, the urinary findings have to be interpreted and, whenever possible, also combined into urinary profiles (e.g., the nephritic sediment, the nephrotic sediment). This, combined with other laboratory tests, the pathologic findings, and the clinical data, allows for the definition and management of urinary tract diseases.