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Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.

ABSTRACT: Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.

SUBMITTER: Jalili R

PROVIDER: S-EPMC5798375 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.

Jalili Roxana R Horecka Joe J Swartz James R JR Davis Ronald W RW Persson Henrik H J HHJ

Proceedings of the National Academy of Sciences of the United States of America 20180116 5

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a ...[more]

PMID: 29339495

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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

Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.

Publications

Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.

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