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.

Keywords: sandwich assay; spiegelmer; troponinI.

Figure 1

Schematics of spiegelmer-based sandwich assay development for cardiac troponin I detection. These proof of concept results provide the first example for fully spiegelmer-based sandwich assay of diagnostic potential as supported by their applicability for cTnI in blood plasma.

Figure 2

Screening of isolated oligonucleotides by filter binding assay. (A) Non-modified bead or 10 pmol of bead-bound cTnI D-peptide was incubated with 0.4 µM Cy5 labelled D-oligonucleotide solution. Following the washing steps, the beads were transferred onto nitrocellulose membrane and the fluorescence signal was detected by a fluorescence scanner. (B) Relative fluorescence intensities as result of the quantitative evaluation of the fluorescence images.

Figure 3

Selective detection of cTnI monomer and ternary complex by spiegelmer based sandwich assay. (A) Ten times diluted cTnI depleted human plasma was spiked with 5 nM recombinant cTnI and sTnI and analyzed by AlphaLisa in a dedicated plate reader using the N-terminal B10 and the C-terminal epitope selective A6, C6 spiegelmers. (B) The selection buffer was completed by the addition of 5 nM human I-T-C ternary complex and analyzed as in panel A.

Figure 4

AlphaLisa signal of the sandwich assay as a function of the concentration of cTnI. The selection buffer (A) or diluted serum (B) was formulated with various amounts of recombinant cTnI or sTnI to reach the indicated final concentrations. The signals were measured by a fluorescence plate reader. The adjusted R-squared values were 0.9945 and 0.9843, respectively.