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

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Proximity ligation assay (PLA) is a key technique for quantitative protein biomarker detection.
• Traditional PLA faces limitations in stringency, ease of use, and compatibility with low-affinity reagents.

Purpose of the Study:

• Introduce circular proximity ligation assay (c-PLA) as an improved method for protein detection.
• Compare c-PLA with traditional PLA to demonstrate its advantages in specificity, sensitivity, and reproducibility.

Main Methods:

• Developed c-PLA by ligating proximity probes to form a circular DNA molecule upon analyte binding.
• Utilized enzymatic removal of uncircularized DNA to enhance selectivity.
• Compared c-PLA and PLA using various biomarkers in buffer and human plasma.
• Employed surface plasmon resonance (SPR) and biolayer interferometry (BLI) for kinetic analyses.

Main Results:

• c-PLA demonstrated higher stringency, improved reproducibility, and enhanced sensitivity compared to traditional PLA.
• Both methods achieved limits of detection from femtomolar to nanomolar concentrations.
• c-PLA showed superior performance with low-affinity antibodies, attributed to antibody affinity variations.
• Lower background signal in c-PLA allows for increased probe concentration and high signal-to-noise ratios.

Conclusions:

• c-PLA offers significant advantages over traditional PLA, including enhanced specificity, sensitivity, and reproducibility.
• The assay format stabilizes complexes and reduces background ligation events.
• c-PLA facilitates the use of low-affinity reagents, expanding its utility in clinical protein detection, especially when high-affinity reagents are unavailable.