Μελέτη και ανάπτυξη αντιδραστηρίων ανίχνευσης δεικτών διάγνωσης καρδιαγγειακών παθήσεων

Abstract

In the past 15 years, there has been an explosion of new information regarding the presentation, outcomes, and management of patients presenting with suspected acute myocardial infarction. Although today, one of the main problems of the clinic institutions which still remains, is the valid, effective, and “in time” valuation of the patients, who are getting in the emergency departments with chest pain and suspected acute coronary syndromes. Thereby, the scientific community has lately focused their main concern on the development and application of new improved diagnostic techniques, aiming at the optimal and “in time” diagnosis, the significant decrease of the diagnostic time, and finally the better nursing management and treatment of the patients who are dealing with these very serious and risky pathologic conditions. The diagnosis of acute myocardial infarction depends in general upon the patient’s clinical history, interpretation of the electrocardiogram (EGC), and the measurement of serum levels of cardiac enzymes, which are commonly used to reduce any diagnostic uncertainty in the confrontation of myocardial infarction. Serologic biochemical markers such as Creatine Kinase MB Isoenzyme (CK-MB), myoglobin, Glycogen Phosphorylase Isoenzyme BB (GPBB), and C-reactive Protein (CRP), are routinely used to assist cardiologists and other physicians for the diagnosis and management of patients with acute coronary syndromes. In the last decades, the identification and development of assays for the determination of cardiac troponins, has been the most important innovation in the field of cardiovascular laboratory diagnostics. The troponins are proteins integral to the function of striated muscles. They exist as a complex with actin and tropomyosin on the thin filament of the contractile apparatus, playing an important role in regulating skeletal and cardiac muscle contraction. In particular, the troponin complex consists of three protein subunits. Troponin C (18-kDa) binds calcium and regulates the activation of thin filaments during contraction. Troponin T (37-kDa) binds the troponin complex to tropomyosin and finally, troponin I (24-kDa) as an inhibitory subunit, prevents contraction in the absence of calcium and troponin C. Troponins have been found to exist as isoforms specific to the cardiac and skeletal muscle cells, respectively. Three isoforms of troponin I have been identified: one of each in fast and slow skeletal muscles, and one isoform in cardiac muscles. Troponin T is also found in skeletal (fast and slow) and cardiac isoforms, but in skeletal muscle exists as many slightly different subforms. Troponin C exists in 2 isoforms, a fast and a slow one. The fast isoform is found only in skeletal muscle, but the slow isoform is found in both skeletal and cardiac muscle tissues. Because of this lack of cardiac specificity, it has been the least studied troponin. One of the main characteristics of troponins is the high homology between the skeletal and cardiac muscle isoforms, which is estimated to be ~70% for troponin I and T isoforms, and more than 85% for troponin C isoforms (the slow isoform is completely identical in both kind of cells). Cardiac troponins are released in the peripheral blood during irreversible cardiac muscle damage in a time-specific manner. They are detectable in blood 4-6 hours after the onset of the cardiovascular incident, peak at 16-18 hours, and remain detectable for at least 7 days. Cardiac troponin I is released in complexes of troponin l-C-T and troponin l-C, while cardiac troponin T is released in a troponin l-C-T form, as well as free troponin T. Analysis of cardiac troponin I and T have been shown to be effective in detecting minor myocardial injury in patients with acute coronary syndromes. Determination of minor myocardial injury is absolutely significant, as these patients have a higher short-term morbidity and mortality than other unstable angina patients with normal concentrations for these markers. The development of biological assays for the detection of cardiac troponins is based on the application of specific antibodies against the whole troponin complex or some of its regions. The high homology between cardiac and skeletal muscle troponins, establishes the problem of cross reactions, reflecting negatively on the specificity of the diagnostic method. This fact combined with the necessity of a signifying improvement of the method’s sensitivity, enforces the search of more sensitive and specific reagents. Aiming to contribute in the field of developing highly specific and sensitive reagents for the detection and isolation of troponin complex we synthesized five epitope-carrier conjugates. The selection of the appropriate sequences of cardiac troponin isoform was mainly made on the basis of their predicted immunogenicity, minimum homology compared to the skeletal isoforms and the absence of involvement with interfering factors in the sera of healthy and patient population. The sequential oligopeptide carrier SOC₄, formed by the repetitive motif -Lys-Aib-Gly-, was used for conjugation of multiple copies of the selected peptide sequences leading to protein-like molecules of branched architecture. Three synthetic strategies were used for the synthesis of the high molecular weight (~6-8,5kDa) macromolecules of this work. The selected epitopes were conjugated on the sequential oligopeptide carrier (SOC), by using: i) the step-by-step Fmoc/fBu solid phase peptide synthetic methodology, ii) the corresponding Boc/Bzl methodology and iii) chemoselective ligation methods through thioether bonds formation. From the examination and comparison of the synthetic methodologies used, we concluded that the chemoselective ligation methodology is the most effective technique for the synthesis of high molecular weight immunological complexes of branched architecture. The resulted conjugates were used as immunogens for releasing anti-troponin specific antibodies. The performed ELISA experiments confirmed our initial considerations for producing anti-troponin antibodies with high affinity and specificity against the native protein (human cardiac troponin complex). The produced antibodies could be used for the development of improved troponin immunodiagnostic assays. Finally, this work also contributed to the development of a label-free faradaic impedimetric immunosensor for the direct probing of the interaction between cysteine-modified synthetic peptides and their specific antibodies. The prospective contribution of such label-free affinity-based probing concepts in the field of development of new immunodiagnostic techniques is of main importance.

Στο πλαίσιο της παρούσας εργασίας έγινε μια προσπάθεια ανάπτυξης εξειδικευμένων αντιδραστηρίων ανίχνευσης των καρδιακών ισομορφών του συμπλόκου της ανθρώπινης τροπονίνης. Για το σκοπό αυτό: Επιλέχθηκαν κατάλληλες αμινοξικές αλληλουχίες των καρδιακών ισομορφών της ανθρώπινης τροπονίνης. Η λογική που χρησιμοποιήθηκε για την επιλογή των εν λόγω αλληλουχιών περιελάμβανε τον συνυπολογισμό τριών βασικών κριτηρίων: τις αντιγονικές ιδιότητες των επιτόπων, την ελάχιστη ομολογία τους έναντι των σκελετικών ισομορφών και την μη εμπλοκή τους σε αλληλεπιδράσεις με παρεμβατικούς παράγοντες των ορών, υγιών και ασθενικών πληθυσμών. Χρησιμοποιώντας διάφορες τεχνικές σύνθεσης, συντέθηκαν επτά συνολικά πεπτιδικά ανάλογα διακλαδισμένης αρχιτεκτονικής, με μοριακά βάρη που κυμαίνονται από ~6kDa μέχρι ~8,5kDa. Οι συνολικές αποδόσεις σύνθεσης των μακρομορίων κυμάνθηκαν στην περιοχή από 4,5% έως και 36,5%. Μελετήθηκαν και συσχετίστηκαν οι συνθετικές μεθοδολογίες που χρησιμοποιήθηκαν, για την σύνθεση των μακρομοριακών συμπλεγμάτων διακλαδισμένης αρχιτεκτονικής της παρούσας διατριβής. Το μικρό ποσοστό παραπροϊόντων, η ταχύτητα, η αποτελεσματικότητα και η πρακτική διευκόλυνση κατά την διεξαγωγή των αντιδράσεων, ανέδειξαν την συνθετική στρατηγική των χημειοεκλεκτικών αντιδράσεων σχηματισμού θειοαιθερικού δεσμού, ως την πλέον λειτουργική μεθοδολογία. Τα συνθετικά μακρομόρια χρησιμοποιήθηκαν με επιτυχία ως ανοσογόνα για την παρασκευή εξειδικευμένων αντισωμάτων έναντι της ανθρώπινης καρδιακής τροπονίνης. Η συνδυαστική χρήση των αντισωμάτων αυτών προσφέρει τη δυνατότητα ανάπτυξης εναλλακτικών ELISA τεχνικών, για τη βελτίωση των δοκιμασιών ανίχνευσης των ανθρώπινων καρδιακών τροπονινών. Τα αντισώματα αυτά ύστερα από κατάλληλο καθαρισμό, προσφέρονται επίσης για την απομόνωση αλλά και τον καθαρισμό καρδιακών τροπονινών από ορούς ασθενικών πληθυσμών. Τέλος, αναπτύχθηκε μια νέα αναλυτική τεχνική βασιζόμενη στους βιοαισθητήρες εμπέδησης, η οποία επιτρέπει την απ’ ευθείας ανίχνευση της αλληλεπίδρασης συζευγμένου σε ηλεκτρόδια χρυσού γραμμικού ανοσογονικού επιτόπου της καρδιακής ισομορφής της τροπονίνης C, έναντι των ειδικών για τον επίτοπο αντισωμάτων αντιορού. Η συγκεκριμένη τεχνική μπορεί να συμβάλλει ουσιαστικά στην ανάπτυξη νέων ανοσοδιαγνωστικών τεχνικών.