Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies

Abstract

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.

Keywords: antibody affinity; immuno-PCR; kinetic analysis; proximity ligation assay; qPCR.

Fig. 1.

Schematic representation of t-PLA and c-PLA. (A) t-PLA detects proteins using pairs of antibody–DNA conjugates (red and blue), which are brought into close proximity on binding to target analyte. The addition of a bridge oligonucleotide and DNA ligase enables ligation of the antibody-tethered oligonucleotides to form a new DNA sequence. Ligation is terminated by selective degradation of the bridge oligonucleotide. The newly formed ligation product is subsequently preamplified followed by quantification using qPCR. (B) In c-PLA, the antibody-tethered oligonucleotides act as bridges for two ligation events between free oligonucleotides, resulting in the formation of a circular ligation product. The addition of an extra oligonucleotide increases stringency compared with t-PLA; it lowers the probability of random background ligation events, since four components must assemble in the absence of the target analyte to generate an independent circular ligation product. Circle formation also allows exonuclease treatment, which terminates ligation and reduces background by degrading all uncircularized DNA. The reduction in background also simplifies the workflow by eliminating the need for preamplification. Circular ligation products are quantified by qPCR using primer sites spanning the newly formed junctions (P1 and P2).

Fig. 2.

Dose–response curves of t-PLA and c-PLA for detection of VEGF (A), GDNF (B), IL-6 (C), MIF (D), TNF-α (E), and IGF-II (F). The x axis displays antigen (Ag) concentration, and the y axis displays an estimated number of ligated molecules. The enhanced stringency for c-PLA is shown by a lower number of counts because of the rigor imposed by circle formation, background reduction through exonuclease treatment, elimination of preamplification, and tailored qPCR primer sites. Error bars denote 1 SD (n = 9), and the dashed lines denote limit of detection, defined as the mean signal of a blank sample +3 SD.

Fig. 3.

Isoaffinity analysis and corresponding c-PLA performance for six biomarkers. (A) Kinetic analysis reveals two distinct groups for K_d values: one group with high affinity (single-digit picomolar) and another group with affinity above 50 pM. (B) The differences in antibody affinities are directly reflected in the c-PLA dose–response curves, where analytes with low K_d values display limits of detection in the subpicomolar range, while the other group exhibits limits of detection in the midpicomolar range or higher.

Fig. 4.

Comparison of PLA methods at different probe concentrations for TNF-α. (A) c-PLA results offer a larger signal-to-background ratio than t-PLA. (B) Individual components for 100 pM signal-to-noise ratio. The greater signal-to-noise ratio for c-PLA is a consequence of higher stringency in c-PLA, which produces lower overall signals and larger differences between positive signal (100 pM) and negative background noise. (C) Dose–response curves showing that the higher signal-to-background ratios result in a more than 10-fold improvement in limit of detection (7 pM) for c-PLA when the probe concentrations are increased 10-fold compared with t-PLA (1×). Error bars denote 1 SD (n = 9), and the dashed lines denote limit of detection, defined as the mean signal of a blank sample +3 SD. Ag, antigen.

Fig. 5.

Performance of PLAs in human plasma. (A) Dose–response curves for VEGF detection show assay compatibility for both PLA methods in human and chicken plasma. The difference in limit of detection between the two matrices is attributed to endogenous VEGF levels in human plasma that are absent in chicken plasma. (B) Dose–response curves for TNF-α detection in human plasma showing improvement in assay performance for c-PLA over t-PLA. A 10-fold increase in probe concentration (10× = 2.5 nM) improves reproducibility for c-PLA, while there is no improvement for t-PLA. Error bars denote 1 SD (n = 9), and the dashed lines denote limit of detection, defined as the mean signal of a blank sample +3 SD. Ag, antigen.