The detection of low level protein biomarkers in clinical samples represents a fundamental challenge. Immuno-PCR (iPCR), introduced in 1992, achieves ultra-low level protein detection, by labeling antibodies with amplifiable DNA sequences. While the PCR detection of iPCR labels can be extraordinarily sensitive, analyte limits of detection often are limited in practice non-specific stickiness of the DNA reporters requiring extensive optimization and washing to minimize background. Proximity ligation assay (PLA; 2002) partially addresses this issue, since only the ligated DNA product can be amplified by PCR while the split DNA reporters cannot. PLA, however, requires the availability of antibodies recognizing target epitopes in adequate geometric proximity, each of which must be modified with DNA.<br/>In this work, we replace DNA labels with less-sticky nanoparticles capable of <i>creating</i> ultra-detectable DNA molecules. In Nanoparticle-proximity ligation assay (np-PLA), in which only one antibody modified with biotin is required. In ANC-PLA, protein biomarkers are first captured on the surface of a microplate; biotinylated detection antibodies conjugated to biotin bind to the microplate-captured protein of interest, then capture NN nm avidin nanoparticles. Key to the approach is our finding that avidin nanoparticles can catalyze the ligation of biotinylated DNA oligos into PCR-amplifiable reporter sequences. The inevitable non-specific binding of the smaller oligos creates no PCR background signal. Competitive PCR readout is used to make the assay quantitative, as well as ultra-sensitive. Initial experimental results have demonstrated that np-PLA is capable of detecting and quantitating human Chorionic Gonadotropin (hCG) down to fM levels.

Ultra-Sensitive protein detection with nanoparticle-proximity ligation assay

253rd National Meeting (2017)

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