Digital PLA

Abstract
The proximity ligation assay (PLA) is an advanced quantitative, sensitive and relatively inexpensive immunoassay technology that can be adapted for several assay formats, including protein detection by PCR. The binding of two antibodies, which are coupled to different, non-complementary oligonucleotides, to their target protein facilitates the enzymatic ligation of the oligonucleotides and the detection of the resulting amplicon by real-time quantitative PCR (qPCR) acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of fungal and bacterial pathogens, where PCR cannot readily identify viable, infectious or non-pathogenic strains. We shall discuss the design of innovative PLAs that use single monoclonal antibodies for the detection of Clostridium difficile, an important health care-associated pathogen. We also report for the first time the use of digital PCR to generate a robust dualplex digital PLA (dPLA) targeting the two major virulence factors TcdA and B. We conclude that PLA and dPLA have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is vastly enhanced.