Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

Proximity ligation assay (PLA) is a novel protein detection strategy based on the transformation of detected proteins into DNA sequences through a DNA ligation reaction (1,2,3,4,5,6). This conversion of protein into DNA strands is followed by DNA amplification and detection, for example, by quantitative PCR or rolling circle amplification (RCA). One further development of the basic proximity ligation strategy is the triple-specific proximity ligation assay (3PLA), in which three independent recognition events are required to give rise to a specific signal (3). 3PLA has been shown to detect as little as 100 molecules of the vascular endothelial growth factor (VEGF) in 1-µL samples, and very low concentrations were also detected of the biomarkers prostate-specific antigen (PSA) and troponin I (3).

In 3PLA, the binding of three proximity probes, identified as proximity probes 1, 2, and 3, results in the eventual formation of one amplifiable DNA sequence (Figure 1A). Initially, proximity probes 1 and 2 are blocked by oligonucleotides that hybridize to their ligatable ends, but upon binding of all three proximity probes to the same target molecule, the blocking oligonucleotides are displaced by proximity probe 3, which templates the ligation reaction of proximity probe 1, a cassette oligonucleotide, and proximity probe 2 to form a new DNA sequence that can be amplified and detected by real-time PCR. The blocking oligonucleotides ensure that the nontarget-mediated background signal is low. In the present study, all experiments were based on the 3PLA strategy.

Figure 1. The triple-specific proximity ligation assay (3PLA) reaction and formation of proximity probes. (Click to enlarge)

In previous studies (2,3), the most commonly used protocol for conjugation of antibodies to oligonucleotides to produce the proximity probes uses the high-affinity interaction of biotin for streptavidin (K_D approximately 10⁻¹⁵ M) (7). In this covalent streptavidin-biotin (B-STV) conjugation approach, single-stranded oligonucleotides are covalently attached to streptavidin molecules via maleimide conjugation chemistry (2,8). The purified conjugates can then be added to any biotinylated antibody to form functional proximity probes against a range of different target molecules (3,5,6) with no need for further purification. However, the conjugation of many different oligonucleotides to streptavidin molecules in this approach is tedious and time-consuming due to the coupling and purification procedure.

In the present study, we compared the B-STV conjugation approach to a self-assembly protocol that is solely based on the noncovalent interaction of streptavidin to biotins (B-STV-B). In the B-STV coupling approach, the covalently linked oligonucleotide-streptavidin constructs are added to biotinylated antibody at a ratio of 2:1 and allowed to form proximity probes (Figure 1B, panel i). The antibody conjugates are diluted in PLA buffer and used in PLAs (Figure 1B, panel ii). For the B-STV-B self-assembly, biotinylated oligonucleotides are allowed to bind streptavidin molecules at a 1:1 ratio. Subsequently, biotinylated antibodies are added at a ratio of 1:2, and proximity probes are allowed to form.

For the B-STV method of proximity probe conjugation, DNA oligonucleotides covalently coupled to streptavidin [100 nM diluted in 1× phosphate-buffered saline (PBS), 0.1% bovine serum albumin (BSA) buffer; Solulink, San Diego, CA, USA] were added to 100 nM recombinant streptavidin (Sigma-Aldrich, St. Louis, MO, USA) prepared in 1× PBS, 0.1% BSA buffer at a 1:2 ratio, incubated at 65°C for 1 h, and then allowed to cool down to room temperature. For conjugation to the antibody, 40 µL of this 100 nM streptavidin-oligonucleotide conjugate, 20 µL 100 nM biotinylated antibody (R&D Systems, Abingdon, UK) against either VEGF or tumor necrosis factor α (TNFα), and 40 µL 1× PBS, 0.1% BSA buffer were mixed and incubated at 37°C for 1 h and then at room temperature for 30 min.

For the B-STV-B method of proximity probe conjugation, 300 µL 100 nM biotinylated oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) and 300 µL 100 nM recombinant streptavidin, diluted in 1× PBS, were combined, incubated at 37°C for 1 h, and allowed to cool down to room temperature for 30 min. Eighty microliters streptavidin-oligonucleotide conjugates were then added to 20 µL 100 nM VEGF or TNFα biotinylated antibody solutions. The mixtures were incubated at 37°C for 1 h, followed by cooling to room temperature for 30 min. The B-STV and B-STV-B conjugates were diluted in PLA buffer [1× PBS, 1% BSA, 16 µg/mL sheared poly(A) bulk nucleic acid (Sigma-Aldrich), 1 mM free biotin, and 100 nM streptavidin] and either used directly in PLAs or stored at 4°C for up to 1 month.