Neutral Endopeptidase (Neprilysin) in Therapy and Diagnostics: Yin and Yang.

Neprilysin (NEP) is a zinc-dependent metalloproteinase that exists in organisms in both transmembrane and soluble forms. NEP substrates are involved in regulating the cardiovascular and nervous systems. In this review, we discuss some of the biochemical characteristics and physiological functions of this enzyme with special emphasis on the use of NEP as a therapeutic target. The history and various physiological aspects of applying NEP inhibitors for treating heart failure and attempts to increase NEP activity when treating Alzheimer's disease using gene and cell therapies are described. Another important issue discussed is the role of NEP as a potential marker for predicting the risk of cardiovascular disease complications. The diagnostic and prognostic performance of soluble NEP in various types of heart failure is analyzed and presented. We also discuss the methods and approaches for measuring NEP activity for prognosis and diagnosis, as well as a possible new role of natriuretic peptides (NEP substrates) in cardiovascular diagnostics.

N-terminal and C-terminal fragments of IGFBP-4 as novel biomarkers for short-term risk assessment of major adverse cardiac events in patients presenting with ischemia.

OBJECTIVES: Pregnancy Associated Plasma Protein A (PAPP-A)-derived N- and C-terminal fragments of IGF-binding protein-4 (NT- and CT-IGFBP-4) released from vulnerable atherosclerotic plaques are proposed to be used for cardiovascular risk assessment. DESIGN AND METHODS: NT- and CT-IGFBP-4 were measured by novel immunoassays in EDTA-plasma of 180 patients admitted to the emergency department with symptoms of myocardial ischemia but without ST-segment elevation. Six-month incidence of major adverse cardiac events (MACE), including myocardial infarction, cardiac death, percutaneous coronary interventions, and coronary artery bypass grafting was recorded. RESULTS: Sixteen patients met the endpoint. NT- and CT-IGFBP-4 were strong predictors of MACE: area under ROC curve (AUC) 0.856 and 0.809, respectively. NT-IGFBP-4 concentrations≥214µg/L and CT-IGFBP-4 concentrations≥124µg/L were associated with increased risk of future MACE: adjusted hazard ratio 13.79 and 7.93, respectively. CONCLUSIONS: IGFBP-4 fragments can be utilized as biomarkers for MACE prediction in patients with suspected myocardial ischemia.

Generation of recombinant antibodies and means for increasing their affinity.

Highly specific interaction with foreign molecules is a unique feature of antibodies. Since 1975, when Keller and Milstein proposed the method of hybridoma technology and prepared mouse monoclonal antibodies, many antibodies specific to various antigens have been obtained. Recent development of methods for preparation of recombinant DNA libraries and in silico bioinformatics approaches for protein structure analysis makes possible antibody preparation using gene engineering approaches. The development of gene engineering methods allowed creating recombinant antibodies and improving characteristics of existing antibodies; this significantly extends the applicability of antibodies. By modifying biochemical and immunochemical properties of antibodies by changing their amino acid sequences it is possible to create antibodies with properties optimal for certain tasks. For example, application of recombinant technologies resulted in antibody preparation of high affinity significantly exceeding the initial affinity of natural antibodies. In this review we summarize information about the structure, modes of preparation, and application of recombinant antibodies and their fragments and also consider the main approaches used to increase antibody affinity.

[Admission and predischarge levels of NT pre brain natriuretic peptide in patients hospitalized because of worsening heart failure. relation to risk of death in the next 6-12 months].

UNLABELLED: Inhospital treatment of patients with heart failure (HF) can cause changes of N-terminal pro-brain natriuretic peptide (NT proBNP) levels. It has not been established yet which NT proBNP value (before or at height of treatment activation) is closer related to prognosis of unfavorable outcome after discharge. AIM: To compare relation to risk of post discharge unfavorable outcome of patients with HF of NT proBNP levels measured close to hospital admission and discharge. MATERIAL AND METHODS: We studied 69 patients (64% men) aged 66.6 +/- 11.0 years with coronary heart disease or hypertension hospitalized because of worsening HF. Median left ventricular ejection fraction was 28%. NT-proBNP was measured during first 3 days of hospitalization (admission level) and in 2 weeks after first measurement (predischarge level). Duration of follow-up was 6-12 (mean 11.6 +/- 1.3) months. RESULTS: Admission NT-proBNP level (median 13.23, interquartile range 5.95-25.89 ng/ml) exceeded upper limit of normal (ULN) in 67 patients (97.1%). Predischarge NT-proBNP became significantly lower (median 6.02 ng/ml, interquartile range 2.52-12.23 ng/ml; p=0.012), but remained above ULN in 62 patients (89.8%). During follow-up 27 patients (39.1%) died. Median NT-proBNP in the group of patients who later died compared with those who survived was insignificantly higher at admission (15.03 vs. 9.9 ng/ml, respectively, p=0.09) and significantly higher at predischarge (8.65 vs. 3.60 ng/ml, respectively, p=0.012). Analysis of receiver operating characteristic curves identified predischarge NT-proBNP level 3.5 ng/ml as cut - off value for increased risk of death. Multivariate regression analysis selected predischarge NT-proBNP more or equal 3.5 ng/ml as independent predictor of death during follow-up. CONCLUSION: In this group of patients hospitalized because of worsening HF predischarge but not admission NT-proBNP level was independently related to risk of death during next 6-12 months.

[Diagnostic value of cardiac troponin T increase in critically ill patients].

There are presently reports on elevated levels of cardiac troponins in patients without acute myocardial infarction (AMI). The objective of this investigation was to study the diagnostic value of increased blood cardiac troponin T levels in patients without its clinical picture and ECG changes characteristic of AMI. The study covered 72 patients (48 males and 24 females) aged 54 to 87 years (mean 69.8 +/- 11.2 years). The inclusion criteria were increased cardiac troponin T; the main exclusion criteria were AMI-typical anginal pain and characteristic ECG changes (ST-segment elevation, the appearance of pathological Q waves). The final diagnosis of AMI was established in only 29 (40.3%) patients; the other 43 patients were diagnosed as having the following diseases: septic state in 21; oncopathology in 10; diabetic nephropathy with chronic renal failure in 6; brain infarct in 4; and B12 deficiency anemia in 2. In dead patients, the level of troponin T was significantly higher than that in discharged patients, respective of the underlying disease. There was a direct correlation between the cardiac troponin T levels and the SAPS II index that reflected the severity of a patient's general condition (r = 0.44; p = 0.0001) and an inverse correlation between the cardiac troponin level and the left ventricular ejection fraction (r = -0.45; p = 0.003). Thus, despite the cardiospecificity of troponin T, its detection in the blood of critically ill patients without other manifestations of AMI is not a specific symptom of AMI, but it is suggestive of the severity of the disease, probably with the involvement of the myocardium into the pathological process.

[Heart fatty acid binding protein in patients hospitalized because of worsening heart failure. Relation to prognosis of death].

PURPOSE: We tested the hypothesis that serum heart fatty-acid binding protein (FABP), an early marker of myocardial necrosis, is related to prognosis of patients hospitalized because of worsening heart failure (HF). METHODS: Sixty nine patients (64% men, age 66.6 +/- 11.0 years) with NYHA class II, III, IV HF (1, 18, and 50 patients, respectively) at hospital admission were followed for 6-12 (mean 11.6 +/- 1.3) months. Forty seven patients (68.1%) had history of myocardial infarction (MI), 56 (81.2%) - hypertension, 15 (21.7%) -- diabetes, and 17 (24.6%) had echocardiographical signs of aortic stenosis. Median left ventricular ejection fraction was 28%. Serum FABP, cardiac troponin I (Tn I) and N-aminoterminal pro brain natriuretic peptide (NT proBNP) were measured within 3 days after admission ( " admission " levels) and 2 weeks later (minimal hospital stay). Manufacturer recommended upper limits of norm (ULN) were 4.0 ng/ml for FABP, 0.35 ng/ml for Tn I, 0.1 ng/ml for NT proBNP. RESULTS: Median admission FABP was insignificantly higher than level measured 2 weeks later (4.17 vs 4.03 ng/ml, p=0.069). FABP exceeded ULN in 38 (55.1%) patients and in 35 (50.7%) patients at admission and in 2 weeks, respectively (p=0.65). Median admission NT proBNP was significantly higher than 2 weeks level (13.23 vs 6.02 ng/ml, p < 0.0001). Median admission and 2-weeks levels of Tn I were similar and greatly lower than ULN. There were 27 all cause deaths (39.1%) during follow up. Median admission levels of TnI, FABP and NT proBNP were similar in patients who died and survived. Two weeks NT proBNP was significantly higher in patients who died (8.65 vs 3.62 ng/ml, p=0.012). ROC curve derived cut-off levels of FABP and NT proBNP (3.31 ng/ml and 3.5 ng/ml, respectively) were used in univaritate regression analysis. According to this analysis FABP >or= 3.31 ng/ml was related to occurrence of death (OR 3.54; 95% CI 1.03-12.17, p=0.044). FABP and variables with p > 0.1 (age, history of MI and diabetes, regular treatment with nitrates, signs of aortic stenosis, pulmonary rales at admission, and 2 weeks level of NT proBNP >or = cut-off) were included into multivariate logistic regression model. Independent predictors of death were aortic stenosis (OR 31.67; 95% CI 6.11-164.00) and NT proBNP >or= 3.5 ng/ml (OR 5.75; 95%CI 1.69- 19.52). CONCLUSION: In this group of patients hospitalized due to worsening of HF admission values of neither FABP nor other biomarkers studied were predictors of death during about 1 year of follow up. FABP level after 2 weeks of hospital stay was related to occurrence of death but as predictor was inferior to NT-proBNP measured at the same time point.

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.

[Levels of fatty acid binding protein before and after direct current cardioversion of atrial fibrillation or flutter in patients without acute coronary syndrome].

UNLABELLED: It is not known whether FABP suggested as an early marker of myocardial necrosis increases after direct current cardioversion (DCC). Moreover there are no data on FABP levels in patients with atrial fibrillation (AF) and flutter. AIM: To find out whether DCC induces acute changes of FABP levels in patients with AF or flutter without acute coronary syndrome. METHODS: Serum FABP and troponin I (TnI) were measured in 63 pts treated with DCC (monophasic shocks) because of AF (n=45, 71.4%) or atrial flutter (n=18, 28.6%). Initial energy was 50J for atrial flutter and 200J for AF. Median total energy was 200J, max 660J. Blood was sampled before DCC and in 30, 60 min, 3, 6 h after first shock. TnI and FABP were measured with single-step sandwich method ("Hytest" Finland). Upper limit of normal range (ULN) for TnI was 0.4 ng/ml (recommended by manufacturer). Serum FABP was measured in healthy volunteers and 95th percentile was used as ULN (4.67 ng/ml). RESULTS: Most pts (n=40, 63.5%) had baseline FABP > ULN (median for all pts--5.55 ng/ml). In 11 pts (17.4%) baseline FABP was > 2 ULN. No relationship was found between baseline FABP and age, sex, duration of arrhythmia, concomitant ECG changes, and presence of overt coronary heart disease or clinical signs of heart failure. Median FABP level increased after DCC (p for trend 0.00014). FABP levels after DCC correlated with total delivered energy. Delivery of > or = 2 shocks compared with 1 shock was associated with higher FABP (median 60 min--9.65 and 5.24 ng/ml, p=0.009; 3 h--12.41 and 5.84 ng/ml, p=0.01, respectively). Median TnI levels were below ULN at each study point. After DCC TnI did not exceed ULN in 61 pts and remained unchanged in 2 pts with baseline TnI above ULN. CONCLUSION: Unexpectedly most pts with AF and atrial flutter had elevated FABP at baseline. After DCC FABP increased in proportion with total delivered energy. Elevations of FABP levels were not associated with rapid increases of TnI so skeletal muscle damage can be a likely cause of elevated FABP. AF, atrial flutter and DCC for these arrhythmias should be considered as sources of false positive results when FABP is used for diagnosis of acute myocardial infarction.

[Prognostic significance of heart fatty acid binding protein in patients with non-ST elevation acute coronary syndrome: results of follow-up for twelve months].

UNLABELLED: Value of heart fatty acid binding protein (FABP) for medium term prognosis in patients with non-ST elevation acute coronary syndrome (NSTEACS) is not well established. AIM: To compare prognostic value of FABP levels with those of troponin I (TnI) and creatine kinase MB (CK MB) activity in patients with NSTEACS. METHODS: Serum FABP and TnI levels (HyTest), CK MB activity (Biocon) were measured in 203 patients with NSTEACS (mean age 63.9+/-11.5 years, 52.2% male). Blood was sampled at admission within 12 (median 3.83) hours and in 6 and 12 hours after onset of pain. Upper limits of normal range (ULN) for TnI and CK MB were 0.4 ng/ml and 25 U/l, respectively. Serum FABP was measured in 53 healthy volunteers (mean age 44.3+/-13.3) and 95th percentile was used as ULN (4.67 ng/ml). Deaths and nonfatal MIs (events) were registered during one year follow-up. RESULTS: There were 47 events (23%, 23 deaths and 24 nonfatal MIs). Patients with events compared with those without events had significantly higher TnI and CK MB 12 hours after onset of pain and significantly higher FABP at all time points of blood sampling. Multivariate (step-up) analysis selected the following independent predictors of events: elevated FABP 6 hours after pain onset (OR 2.45, 95% CI 1.14-5.24; p=0.021), T-wave inversion on admission ECG, age >65 and regular use of nitrates before hospitalization. Sensitivity of elevated FABP 6 hours after pain onset was 78.4%, specificity -- 45.1%. After exclusion from analysis of all or just admission and 6 hours FABP data elevated TnI 12 hours after onset of pain became an independent predictor of events. CONCLUSION: In this group of patients with NSTEACS among markers of myocardial necrosis (FABP, TnI, MB CK) obtained serially during first 12 hours after pain onset elevated FABP was the best predictor of events during 1 year follow up for subjects in whom blood sample could be done 6 hours after pain onset.

Immunological study of complex formation between soluble transferrin receptor and transferrin.

Transferrin receptor (TfR) is a dimeric transmembrane protein that provides iron transport from plasma to cells by binding and internalization of iron-loaded transferrin (Tf). Soluble transferrin receptor (sTfR) is an extracellular part of the TfR molecule that is truncated from the cell surface and released into the blood stream. Using monoclonal antibodies (HyTest Ltd., Turku, Finland), immunofluorescent methods for sTfR and sTfR-Tf complex determination were developed. Soluble TfR was isolated from human plasma, and complex formation between sTfR and Tf was studied by stepwise complex construction and by FPLC gel filtration. It was found that sTfR could bind two Tf molecules step by step when the sTfR-Tf complex is constructed in the plate wells. FPLC gel filtration of sTfR-Tf mixtures and analysis of sTfR and sTfR-Tf immunological activities in collected fractions showed that sTfR can form different complexes with TF depending upon the ratios between them: a 291-kDa compound is assumed to be a 2:1 sTfR/Tf complex, and a 345-kDa compound is assumed to be a 2:2 sTfR/Tf complex. Isolated sTfR eluted as a 237-kDa protein. FPLC gel filtration of serum revealed that all sTfR in serum is bound to Tf in a 2:2 complex, and no isolated sTfR can be found in serum. This raises the question as to the nature of the bonds that hold two molecules of sTfR together to form a dimer.

[Diagnostic value of heart fatty-acid binding protein in early hospitalized patients with non ST elevation acute coronary syndrome]. / Ostryi koronarnyi sindrom bez pod"emov segmenta ST na EKG: diagnosticheskoe znachenie serdechnogo belka, sviazyvaiushchego zhirnye kisloty.

BACKGROUND: Heart fatty-acid-binding protein (FABP) is supposed to be the most sensitive biomarker of myocardial necrosis in patients with Q-wave myocardial infarction (MI) and non-diagnostic ECG during first hours after onset of symptoms. However, diagnostic value of FABP in patients with non-ST elevation acute coronary syndrome (NSTEACS) is not well established. AIM: To elucidate diagnostic value of FABP in patients with NSTEACS hospitalized within time interval considered to be too early for a majority of biochemical tests. MATERIAL AND METHODS: FABP levels were measured by immunofluorometry (HyTest, Finland) in 44 patients (26 men, mean age 69+/-8.9 years) at admission within 6 hours (median - 2 h) from onset of index attack of angina and in 6, 12, 24 hours after onset of pain. Cut off FABP level was 12 ng/ml. Serum cardiac troponin I was measured for diagnosis of MI on admission and twice during first 24 hours of hospital stay. Cut off TnI level was 0.4 ng/ml. RESULTS: Acute MI was diagnosed by TnI above cut off in 31 patients (70.5%). There were no new-Q-wave MIs. Average ratio of observed serum FABP level to diagnostic cut off value on admission and in 6, 12, 24 hours after onset of pain was higher in patients with MI than in patients with unstable angina (1.01, 1.53, 0.81, 0.66 and 0.78, 0.51, 0.65, 0.56, respectively). The difference was maximally significant in 6 hours after onset of pain (p=0.018). Among patients with MI admission FABP compared with admission TnI more frequently exceeded diagnostic level (in 18 vs 9 patients, respectively, p=0.009). Sensitivity and specificity of admission levels of FABP and TnI for diagnosis of MI were 58 and 85%, 29% and 100%, respectively. CONCLUSION: In patients with NSTEACS during first 6 hours after pain onset FABP compared with TnI has greater sensitivity for detection of MI and sufficient specificity. FABP can be used as additional diagnostic tool for MI detection in early admitted patients with NSTEACS.

[Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells]. / Vliianie reguliatornykh genov virusa immunodefitsita cheloveka tipa 1 na proliferatsiiu i differentsirovku émbrional'nykh stvolovykh kletok myshi.

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.

[Heart fatty acid binding protein in patients with ST-elevation acute coronary syndrome hospitalized within 6 hours after onset of pain]. / Serdechnyi belok, sviazyvaiushchii zhirnye kisloty, u bol'nykh ostrym koronarnym sindromom s pod"emami segmenta ST, gospitalizirovannykh v pervye 6 ch zabolevaniia.

AIM: To compare diagnostic value of a novel marker of myocardial necrosis heart fatty acid binding protein (FABP) with that of troponin I (TnI) and total creatine kinase (CK) in patients admitted early after onset of ST-elevation acute coronary syndrome. MATERIAL: Fifty seven patients with ST-segment elevations justifying thrombolytic therapy admitted within 6 hours (29/57 within 3 and 12/57 - 2 hours) after onset of chest pain. In all patients myocardial infarction (MI) was eventually confirmed by development of Q waves and/or diagnostic increase of CK. METHODS: Samples of blood were taken at admission to coronary care unit. Cut-off values for an elevated level of FABP was 12 ng/ml, TnI - 1.2 and 0.4 ng/ml, CK - 400 IU/l. RESULTS: Overall FABP was elevated in 47 (83%), TnI - in 16 (28.1%), CK in 7 (12.3%) patients. Among patients admitted within first 3 and 2 hours FABP was elevated in 23/29 (79.3%) and 11/12 (91%), TnI - in 9/29 (31%) and 5/12 (41.7%), CK in 3/29 (10.3%) and 1/12 (8.3%) patients, respectively. The use of lower cut-off of abnormality (0.4 ng/ml) increased proportion of patients with elevated TnI up to 56.1% in the group as a whole, to 48.3% and 50% among patients admitted within first 3 and 2 hours, respectively. Nevertheless proportion of patients with elevated FABP remained higher with difference being significant for the whole group and patients admitted within first 3 hours (p=0.004 and 0.016, respectively). CONCLUSION: Most patients with ST-elevation acute coronary syndrome hospitalized within 2-6 hours after onset of pain had elevated levels of heart FABP.

Troponin: structure, properties, and mechanism of functioning.

This review discusses the structure and properties of the isolated components of troponin, their interaction, and the mechanisms of regulation of contractile activity of skeletal and cardiac muscle. Data on the structure of troponin C in crystals and in solution are presented. The Ca2+-induced conformational changes of troponin C structure are described. The structure of troponin I is analyzed and its interaction with other components of actin filaments is discussed. Data on phosphorylation of troponin I by various protein kinases are presented. The role of troponin I phosphorylation in the regulation of contractile activity of the heart is analyzed. The structural properties of troponin T and its interaction with other components of thin filaments are described. Data on the phosphorylation of troponin T are presented and the effect of troponin T phosphorylation on contractile activity of different muscles is discussed. Modern models of the functioning of troponin are presented and analyzed.

Antibodies to the nonnative forms of d-glyceraldehyde-3-phosphate dehydrogenase: identification, purification, and influence on the renaturation of the enzyme.

Monoclonal antibodies of two clones reacting with the nonnative forms of d-glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12 (GAPDH), were obtained. Antibodies of clone 6C5 belonged to IgG1 subtype; antibodies of clone 6G7 belonged to IgM type. The interaction of antibodies of both clones with the immobilized and soluble enzyme was studied. The specificity of antibodies to the definite oligomeric forms was demonstrated on immobilized monomers, dimers, and tetramers of GAPDH. The affinity of antibodies to monomeric and dimeric forms of GAPDH, either active or not, was demonstrated. At the same time the antibodies did not react with the tetrameric enzyme. The binding of antibodies had no influence on the enzymatic activity. However, the addition of antibodies to the denatured enzyme blocked the spontaneous renaturation of GAPDH. The immobilized antibodies of both clones were successfully used for the purification of GAPDH solution from the denatured admixtures.

New approach to standardisation of human cardiac troponin I (cTnI).

Six different cTnI assays (5 commercial and one assay under development) were run with either their own calibrators or with standard dilutions of different forms of cTnI (free or complexed). Twenty-one serum samples from 7 AMI patients were analysed using these combinations of calibrators and assay constructs. The range of cTnI concentrations as measured in AMI serum samples by different assays was more than 10-fold using each assays own standards or when using free native or recombinant cTnI. With recombinant or in vitro formed ternary cTnI-cTnT-TnC complexes the differences were smaller (3-fold). The lowest between-manufacturer bias (1.4-fold) was obtained when the heart tissue derived native troponin complex was used for the calibration of the assays. For all assays, calibrated against this troponin I complex, the recalculated cut-off values were within 0.1-0.25 microgram/L. We conclude that to reduce assay-to-assay variation, native troponin complex is presently the preferred alternative for the standardisation of cTnI assays.

Biochemical factors influencing measurement of cardiac troponin I in serum.

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.

Degradation of cardiac troponin I: implication for reliable immunodetection.

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.

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.