The pros and cons of nucleic acid-amplified immunoassays-a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification

Abstract

Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor-acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).

Keywords: Diagnostics; ELISA; Fluorescence; PSA; TR-FRET; Terbium.

Fig. 1

a Schematic representation of the direct and amplified immunoassays investigated. Both assay types used 96-well plates coated with PSA-specific capture antibodies (AB). After target incubation and washing, the PSA (bound to the capture antibodies) was recognized by biotinylated detection AB. For the direct approach, fluorophore-labeled streptavidin (sAv) attached to the biotinylated detection AB. For the amplified approach, sAv attached to the biotinylated detection AB, and biotinylated oligonucleotides attached to sAv. The oligonucleotides served as primers for RCA on the circular DNA template, followed by fluorescent DNA-probe labeling of the RCA product. b Excitation (dotted) and emission (solid) spectra of the Tb (excitation/emission wavelengths: 342 nm/490, 550, 588, 624 nm), FAM (excitation/emission wavelengths: 460 nm/524 nm), and Cy5.5 (excitation/emission wavelengths: 690 nm/710 nm)

Fig. 2

FLISA calibration curves for the direct TG PL (a) and CW PL (b) PSA assays. The curves between the blank (0 ng/mL) and the lowest measured concentration (0.001 ng/mL) were estimated and are shown as dotted lines. Insets show a magnified view of the lower concentration range. The LODs were evaluated corresponding to three times the standard deviation of blank, i.e., 3σ₀, above the background, as shown by the dotted lines that cross the calibration curves. Error bars correspond to the standard deviations from three independent measurements (n = 3) for all target concentrations

Fig. 3

FLISA calibration curves for the amplified TG PL (a), CW PL (b), and TG FRET (c) PSA assays. The curves between the blank (0 ng/mL) and the lowest measured concentration (0.0001 ng/mL) were estimated and are shown as dotted lines. Insets show a magnified view of the lower concentration range. The LODs were evaluated corresponding to three times the standard deviation of blanks, i.e., 3σ₀, above the background, as shown by the dotted lines that cross the calibration curves. Error bars correspond to the standard deviations from three independent measurements (n = 3) for all target concentrations

Fig. 4

FLISA calibration curves for the amplified TG PL PSA assays with PSA in PBS (black) and 50% FBS (red). The curves between the blank (0 ng/mL) and the lowest measured concentration (0.001 ng/mL) were estimated and are shown as dotted lines. Insets show a magnified view of the lower concentration range. The LODs were evaluated corresponding to three times the standard deviation of blanks, i.e., 3σ₀, above the background, as shown by the dotted lines that cross the calibration curves. Error bars correspond to the standard deviations from three independent measurements (n = 3) for all target concentrations