Highly specific circular proximity ligation assay

#Quantitative detection of protein biomarkers in biological fluids is essential for diagnosis, monitoring and personalized treatment of disease. Despite considerable progress in recent years the clinical use of validated proteomic biomarkers remains limited (1). The benchmark for affinity-based protein measurements is defined by the enzyme-linked immunosorbent assay (ELISA) where affinity ligands (e.g. antibodies) are used in a sandwich format to detect and quantify the protein of interest (2, 3). ELISA generally involves several steps starting with sample incubation where the target analyte is captured on a surface precoated with primary antibodies, followed by washing steps and recognition with a secondary antibody, which facilitates detection with a colorimetric, fluorescent or luminescent label. ELISA offers reasonable sensitivity but requires a large sample volume, has limited dynamic range and frequently suffers from false positives due to nonspecific binding (4, 5). These limitations interfere with the discovery and validation of novel biomarker candidates that have the potential to enable early diagnosis and regular molecular monitoring of disease.

#Immunoassays combined with nucleic acid-based amplification and detection have facilitated new approaches and have extended the analytical sensitivity beyond that achievable with ELISA (6-9). One particularly promising approach is the proximity ligation assay (PLA) (10, 11). In PLA, pairs of affinity probes are individually conjugated to short, single-stranded DNA molecules to form proximity-probes that carry either a phosphorylated 5′-end or a 3′-hydroxyl group. When the probe pairs subsequently bind to their cognate target analyte in solution, the associated DNA strands are brought into close proximity and aligned by hybridization to a third bridging oligonucleotide. The free DNA ends are ligated, forming a new DNA sequence that is amplified and quantified using quantitative PCR (qPCR). It has previously been demonstrated that PLA provides femtomolar sensitivity and a wide dynamic range over 5 orders of magnitude, consuming as little as 1 μL of sample (12). One key benefit of PLA is that it addresses the widespread problem of cross-reactivity in antibody-based protein detection. Potential signals from cross-reactive antibodies are eliminated by tailoring the DNA sequences such that ligation only takes place when cognate proximity-probes are bound to the target analyte. This technology has been applied to detect more than 20 different biomarkers in clinical plasma samples using panels of seven multiplex PLA reactions (13, 14). Others have expanded the multiplexing capability even further (15).

#One of the limitations of affinity-based immunoassays, including PLA, is the so called hook effect in which the signal decreases at high antigen concentrations resulting in incorrectly low signals or even false negatives (16). The hook effect becomes predominant when the analyte concentration exceeds the concentration of proximity-probes (>>1 nM) and it effectively determines the upper quantification limit (17). The proximity-probe concentration can potentially be increased, but must be carefully balanced against a deteriorating signal-to-noise ratio from random background ligation events when probes and the bridge oligo come together by chance. The hook effect can ordinarily be avoided by sample dilution; however, dilution alters binding equilibrium. This does not present a problem for high affinity interactions but it may prevent low affinity antibodies to bind to their target analytes, thereby limiting the effectiveness of PLA to high-affinity capture agents (18). While incompatibility with low-affinity antibodies is a major drawback for PLA, it is not restricted to this assay alone. The availability of high affinity antibodies or other capture reagents is a general limitation for all sandwich immunoassays because the sensitivity is ultimately determined by the quality of the reagents used (19).

#The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

#FIG. 1. Schematic illustration of some of the features of the probe system used herein.

#FIG. 2. Schematic representation of traditional and circular proximity ligation assay. a) Traditional proximity ligation assay (t-PLA) detects proteins using pairs of antibody-DNA conjugates (red and blue) which are brought into close proximity upon 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 circular proximity ligation assay (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 to traditional PLA as 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).

#Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

#All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

#Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.