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Nanoparticle-Based Proximity Ligation Assay for Ultrasensitive, Quantitative Detection of Protein Biomarkers.

ABSTRACT: Proximity ligation assay (PLA) achieves extremely low limits of detection but requires the synthesis of antibody-DNA conjugates recognizing multiple target epitopes with appropriate proximity. In this work, we introduce a more generally applicable method by replacing antibody-DNA conjugates with nanoparticles which create ultradetectable PCR templates by capturing biotinylated oligonucleotides and catalyzing ligation. A competitive PCR readout was used to make the assay quantitative. We have demonstrated that NP-PLA can detect and quantitate human chorionic gonadotropin (hCG) levels as low as 2.6 fM (?0.1 pg/mL), nearly 1000 times more sensitive than the current state of the art ELISA.

SUBMITTER: Chen H

PROVIDER: S-EPMC6209106 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Nanoparticle-Based Proximity Ligation Assay for Ultrasensitive, Quantitative Detection of Protein Biomarkers.

Chen Hui H Crum Mary M Chavan Dimple D Vu Binh B Kourentzi Katerina K Willson Richard C RC

ACS applied materials & interfaces 20180911 38

Proximity ligation assay (PLA) achieves extremely low limits of detection but requires the synthesis of antibody-DNA conjugates recognizing multiple target epitopes with appropriate proximity. In this work, we introduce a more generally applicable method by replacing antibody-DNA conjugates with nanoparticles which create ultradetectable PCR templates by capturing biotinylated oligonucleotides and catalyzing ligation. A competitive PCR readout was used to make the assay quantitative. We have dem ...[more]

PMID: 30168312

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Project description:Infectious bronchitis virus (IBV) is a coronavirus responsible for major health problems in the poultry industry. New virus strains continue to appear, causing large economic losses. To develop a rapid and accurate new quantitative assay for diagnosis of the virus without DNA extraction, we selected highly specific single-stranded DNA (ssDNA) aptamers with a high affinity to IBV, using the systematic evolution of ligands by exponential enrichment (SELEX) technology for aptamer screening, followed by high-throughput sequencing technology. Two of these aptamers, AptIBV5 and AptIBV2, were used to establish homogenous and solid-phase proximity ligation assays (PLAs). The developed assays were evaluated for their sensitivity and specificity using collected field samples and then compared to the newly developed sandwich enzyme-linked aptamer assay (ELAA) and reverse transcription-quantitative PCR (qRT-PCR), as the gold-standard method. The solid-phase PLA showed a lower limit of detection and a broader dynamic range than the two other assays. The developed technique may serve as an alternative assay for the diagnosis of IBV, with the potential to be extended to the detection of other important animal or human viruses. IMPORTANCE Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. To better monitor the emergence of this virus, the development of a rapid and highly sensitive diagnostic method should be implemented. For this, we generated aptamers with high affinity and specificity to the IBV in an ssDNA library. Using two high-affinity aptamers, we developed a sandwich ELAA and a very sensitive aptamer-based proximity ligation assay (PLA). The new assay showed high sensitivity and specificity and was used to detect IBV in farm samples. The PLA was compared to the newly developed sandwich ELAA and qRT-PCR, as the gold-standard technique.

Dataset Information

Nanoparticle-Based Proximity Ligation Assay for Ultrasensitive, Quantitative Detection of Protein Biomarkers.

Publications

Nanoparticle-Based Proximity Ligation Assay for Ultrasensitive, Quantitative Detection of Protein Biomarkers.

Similar Datasets

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