Biomarkers.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Protein reference materials are crucial for immunoassay standard curves.
• Current quality control (QC) methods lack precision and lot-to-lot consistency.
• Existing techniques rely on relative quantitation or spectroscopy without true quantity assessment.

Purpose of the Study:

• To introduce a novel digital Proximity Ligation Assay (dPLA) for precise QC of protein reference materials.
• To leverage the absolute quantification capabilities of digital PCR (dPCR) for enhanced accuracy.
• To improve lot-to-lot consistency and reliability in immunoassay measurements.

Main Methods:

• Developed dPLA by combining Proximity Ligation Assay (PLA) with dPCR.
• Conjugated unique oligonucleotide sequences to antibody pairs for target detection.
• Quantified ligated sequences using dPCR after immunocomplex formation, enabling background subtraction.

Main Results:

• dPLA demonstrated superior ability to differentiate small signal changes compared to conventional PLA.
• Achieved within 3% coefficient of variation (CV) for quantified standards across multiple runs.
• Showcased improved linearity and sensitivity with background subtraction, enabling quantification of low-concentration standards.

Conclusions:

• dPLA enables absolute quantification of reference materials, enhancing reproducibility and consistency.
• The method minimizes instrument-to-instrument variation caused by standard material discrepancies.
• Facilitates wider deployment of diagnostic platforms with improved lab-to-lab consistency.