Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection

Abstract

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

Aptamers against Immunoglobulins: Design, Selection and Bioanalytical Applications

“…This is in agreement with previously published papers that the GQ assembly is induced most effectively in K + -rich environment (>50 mM) [63][64][65]. 4 The underline letters indicate the locked nucleic acid bases.…”

“…This is extremely important when generating aptamers for diagnostic applications as their cross-reactivity can be effectively minimized. Of note, the counter-selection can be made against the sample matrix itself, e.g., by using magnetic nanoparticles with autoreactive cyanuric-groups on their surface that by incubation in target-free serum are modified with typical serum proteins [4]. Aptamers, which can bind universally on Fc region of antibodies can be selected with target replacement strategy coupled to conventional SELEX method, i.e., to achieve universal binding capability, multiple immunoglobulin subclasses have to be consecutively substituted in a single SELEX process [5].…”

Establishment of a microspheres-based homogeneous fluorescence immunoassay for the rapid detection of cardiac troponin I

“…However, the experimental conditions were not optimized, and the entire detection process took 15 min to complete. Zoltán et al 34 used aptamers to replace antibodies. However, aptamers are difficult to screen and are easily inactivated.…”