Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• High-quality affinity probes are essential for sensitive and specific protein detection, particularly for early disease biomarkers.
• Proximity extension assays (PEAs) enable high-throughput multiplexed protein detection in small sample volumes.
• Clonal affinity reagents like nanobodies (Nbs) offer advantages in reproducibility and standardization over polyclonal antibodies (pAbs).

Purpose of the Study:

• To explore nanobodies (Nbs) as an alternative to polyclonal antibodies (pAbs) for proximity extension assays (PEAs).
• To develop an efficient method for preparing high-quality, site-specifically conjugated Nb probes for PEA.
• To demonstrate the performance enhancement of Nb-based PEAs (nano-PEAs) for protein biomarker detection.

Main Methods:

• Site-specific conjugation of Nbs using Sortase A (SrtA) enzyme coupling to create oligo-conjugated Nb probes.
• Efficient removal of unconjugated affinity reagents post-conjugation.
• Application of purified Nb probes in PEA for selecting optimal binding reagent pairs and detecting interleukin-6 (IL6).

Main Results:

• An efficient and site-specific method for preparing high-quality Nb probes was established.
• Nb-based PEA (nano-PEA) demonstrated augmented assay performance for IL6 detection compared to pAb-based PEA.
• Validated Nb combinations were identified for competitive-free IL6 antigen detection via PEA.

Conclusions:

• Nanobodies are effective affinity reagents for proximity extension assays, offering improved performance.
• The developed site-specific conjugation method provides high-grade Nb probes suitable for sensitive protein detection.
• Nano-PEA represents a promising advancement for high-throughput, standardized protein biomarker analysis in diagnostics.