Antibody-oligonucleotide conjugates (AOCs) are at the forefront of protein detection and analytical assays, enabling high-throughput and multiplexed analysis. As AOCs are highly tunable it is critical to optimize the conjugation chemistry for both single- and double-stranded oligonucleotides. Wiener et al have demonstrated how to optimize and potentially produce high-quality AOCs.
Challenges in Antibody-Oligonucleotide Conjugation
The ability to attach DNA oligonucleotides to antibodies in a controlled and consistent manner is critical for applications such as immuno-PCR, proximity ligation assays, and next-generation sequencing-based proteomics. Traditional conjugation strategies often suffer from variability in conjugation efficiency, steric hindrance effects, and loss of antibody binding affinity.
Optimizing Conjugation Conditions
Copper-free click chemistry was employed to achieve efficient antibody-oligonucleotide attachment, and to enable bio-orthogonal conjugation, minimizing the risk of non-specific modifications.
Other key reaction variables that influenced conjugation efficiency included:
- Molar ratios: The optimal oligonucleotide-to-antibody ratio (degree of antibody activation) was determined to prevent excess free oligonucleotide while maximizing conjugation yield.
- Oligo length – oligos with a length of 32 and 64 nucleotides, conjugated to the antibody with the same efficiency. Interestingly, introducing a secondary structure into the 32 nucleotides oligo reduced conjugation efficiency by ~50%.
- Temperature: Conjugation reactions were tested at 4°C, 22°C and 37°
- Conjugation periods: Times varied between 5 hrs up to 50hrs.
Of special note the ideal set of conditions is dependent on whether you wish to create a conjugate with single or multiple oligos per antibody. It is clear from this paper; optimizing and fine-tuning AOCs is not a trivial exercise.
Applications in Protein Analytics
The engineered AOCs were tested in Proximity Ligation Assays (PLA). AOCs were used in PLA to demonstrate antibody specificity of the AOC. Both the single and double stranded AOCs bound specifically to the target protein demonstrating antibody binding and specificity of the engineered AOCs were not compromised.
One critical finding was unpurified AOCs (both single and double oligos) resulted in high background signals in the PLA assay. The presence of unconjugated oligonucleotides and excess reagents led to non-specific interactions, significantly reducing assay specificity. Complete purification using size-exclusion and affinity chromatography effectively eliminated these contaminants, leading to cleaner signal readouts and improved assay sensitivity.
Conclusion
AOCs are sophisticated research tools which are highly tunable because of the oligo size and sequence. Different conjugation chemistries and conditions can be used to generate these products. At AbOliGos, we recognize the challenges associated with fine tuning AOCs which is why we are here to help. Our mission is to provide researchers with fast, reproducible, and high-quality AOC reagents tailored for cutting-edge applications.
Learn more about AbOliGo’s custom services.
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