Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B

Abstract

BackgroundThe proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection.MethodsWe prepared monoclonal and polyclonal proximity probes targeting Clostridium difficile toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions.ResultsThe performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA.ConclusionsPLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.

Wash-Free, Digital Immunoassay in Polydisperse Droplets

“…The ProQuantum high-sensitivity immunoassay uses the principles of proximity-based ligation and amplification technologies to detect target antigens. , Target-specific antibodies are conjugated to oligos. When these antibodies bind to their target antigen, the oligos are brought into proximity and can be ligated to create an amplification target.…”

“…Proximity-based assays require two or more reaction components coming into proximity for a signal to be generated. Therefore, only when a binding event takes place (such as antigen–antibody binding) will the reaction components be in close enough proximity to cause a secondary reaction that results in a signal when the target of interest is present. , For example, Dhillon et al demonstrated a multistep proximity ligation assay in bulk coupled to a downstream digital PCR to detect toxins from C. difficile …”

“…24,25 For example, Dhillon et al demonstrated a multistep proximity ligation assay in bulk coupled to a downstream digital PCR to detect toxins from C. dif f icile. 26 Currently, all commercial digital assay systems that use compartmentalization require the compartments to have uniform, or monodispserse, volumes, 27 increasing the costs and complexity of products due to precise microfabrication requirements, and limiting total addressable sample volumes due to the volume limitations of most microfluidic systems. To address our second goal stated above, we relaxed the monodispersity requirement and therefore removed the need to rely on complex microfluidic equipment to create droplets.…”

Antibody–Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents

“…To date, AOCs are mainly used for detection or therapeutic applications . The detection methods include immuno-PCR (iPCR), ,, proximity ligation assay (PLA), , proximity extension assay (PEA), electrochemical proximity assay (ECPA), − DNA-PAINT imaging, and protein arrays . They are based either on (a) the exceptional ability of PCR to amplify the number of DNA labels thus largely improving the detection limits or on (b) the transformation of readily accessible ON-arrays into otherwise difficult to access antibody arrays.…”

“…126 A similar approach was used by Pardridge and colleagues to prepare mAb-siRNA conjugates that showed an important (69−81%) decrease in luciferase gene expression in intracranial brain cancer in vivo. 127 The method was also applied to generate PLA probes for the detection of toxins produced by the clinically important bacterial pathogen C. diff icile 47 or to measure cytokines in a homogeneous test format with femtomolar detection sensitivities. 128 In 2014, Yin and colleagues applied AOCs to a newly developed DNA-PAINT imaging technique.…”