Antibody-oligonucleotide conjugates are used in immuno-PCR, a technique that combines the specificity of antibodies with the amplification power of PCR to increase target detection sensitivity by 100–10,000X. However, the standard chemistry and methods used to link oligonucleotides to antibodies can be difficult, time consuming, and require specific expertise.
Innova Biosciences (Cambridge, UK; https://www.expedeon.com/) has developed a conjugation system optimized so that both experts and novices can generate antibody-oligonucleotide conjugates easily and efficiently, using antibodies that are commercially sourced or prepared in the laboratory (Figure 1). The method is available as a kit that scientists can use themselves (Thunder-Link® Conjugation Kit) and as a service provided by Innova. The Innova technology is unidirectional, allowing only formation of antibody-oligonucleotide complexes, and never antibody-antibody or oligonucleotide-oligonucleotide complexes. Additionally, due to the optional unbound oligonucleotide removal protocol step, the final product is virtually free of any unbound oligonucleotides, ensuring high conjugation efficiency. The resulting bioconjugates significantly increase subsequent antibody-target detection sensitivity, which is key for many target applications. Antibody-oligonucleotide conjugates are used in a range of techniques, such as proximity ligation assays, proximity extension assays, and electrochemical proximity assays, in addition to immuno-PCR.
Figure 1. The Thunder-Link conjugation process. The antibody-oligonucleotide conjugation protocol consists of 3 main steps: activation of both the antibody and the oligonucleotide followed by desalting, after which the two are mixed and left to incubate overnight. If an unbound oligonucleotide removal step is necessary for the end application, this can be performed the next morning. Following this, the conjugate is ready to use.
The 5′ or 3′ end of any 20–120 base oligonucleotide sequence can be conjugated to an antibody. The oligo must include a terminal amine group, added during synthesis (Figure 2). (Note that the efficiency of antibody-oligo conjugation is slightly higher with 5’-aminated oligos. For more details, see Tips for good oligonucleotide design, below.)
While most commercial oligo suppliers offer aminated oligonucleotides, Innova and its customers who use the conjugation kits have found that oligonucleotide quality is important for optimal conjugation. Using high quality oligos also reduces background signal during subsequent conjugate use. Innova scientists have compared aminated oligos from a range of suppliers and have identified IDT HPLC purified reagents as providing conjugates of the highest quality.
Figure 2. Standard IDT amino modification. The functional amino group is attached to the custom oligonucleotide sequence by a 6-carbon linker.
High quality synthesis required for the highest quality oligos
The synthesis cycle of adding each base onto a nascent oligonucleotide is nearly 100% efficient. This “coupling efficiency” is affected by the reagents and instruments used, and therefore, varies with oligonucleotide manufacturers. IDT has pioneered oligonucleotide synthesis chemistry that delivers the highest coupling efficiency in the industry—99.25% across all oligonucleotide scales, at all of our manufacturing locations. IDT recognizes that even a small decrease in coupling efficiency reduces the proportion of the desired, full-length product in an oligonucleotide preparation. “Coupling efficiency is the most important metric we monitor in all of our manufacturing systems”, notes Jessica Alexander, IDT VP, Specialty and Manufacturing Services. For a 20mer oligo, a 0.2% reduction in coupling efficiency decreases the purity of that oligo by approximately 5%. The negative impact of low coupling efficiency on purity is much more profound for longer oligos. For example, a mere 0.1% decrease in coupling efficiency during synthesis of a 75mer oligo reduces purity of the crude oligonucleotide product by ~5%, and a 1% decrease reduces purity by 30%. Figure 3 shows how the proportion of full-length product varies with oligo length at IDT and industry standard coupling efficiencies.
Figure 3. IDT provides the highest coupling efficiency in the industry.
Further enhancing performance
Even at the high coupling efficiencies achieved at IDT, it is still necessary to purify these amino-modified oligonucleotides by HPLC to remove the small percentage of truncated products. IDT’s HPLC purified 20mers have an average purity of 90% (Figure 4), resulting in enhanced performance during bioconjugation.
HPLC purification is of particular importance for oligos with a terminal amino modification. Terminal amino groups act as strong nucleophiles that can indiscriminately react with other molecules in proximity, becoming blocked and ineffective for bioconjugation. HPLC also removes those oligonucleotides containing blocked amino groups.
Figure 4. HPLC purification increases purity of oligos for bioconjugation. Capillary electrophoresis traces of a 75mer oligonucleotide, modified at the 3’ end with an amino group, show the effects of HPLC purification. (A) Before HPLC purification, there is only 62.7% full length product, shown eluting at 18 minutes. Earlier peaks represent truncated products or sequences with small deletions. (B) The purified product, after HPLC, contains 98.4% full length oligonucleotide.
Figure 5 demonstrates the high level of sensitivity the Thunder-Link process provides over a standard ELISA assay. In this case, an HPLC purified, 5′ aminated 67mer oligonucleotide (IDT) was conjugated to an IgG antibody specific for the C-Reactive protein (CRP). The Thunder-Link assay provides 1000-fold greater sensitivity over a traditional ELISA assay.
Figure 5. The high sensitivity provided by immuno-PCR. Innova scientists conjugated an IgG antibody specific for C-Reactive protein (CRP) to an HPLC purified, 5′ aminated 67mer oligonucleotide (IDT) using the Thunder-Link procedure. This conjugate was then applied as the detection antibody in a sandwich assay format. A capture antibody was bound directly to the surface of a polypropylene-PCR plate, and samples were added after pre-incubation with the oligo-conjugated detection antibody. Following washing, bound DNA was quantified by real-time PCR. (A) The amplification curves shown here were generated with increasing amounts of antigen standard, showing that as antigen concentration increases, fewer amplification cycles are required to reach threshold fluorescence intensity. (B) This data is further plotted as cycle number required to reach the threshold signal vs. antigen concentration. In this assay, sensitivity was increased 1000-fold over a traditional ELISA assay.
Oligo conjugates for biological sensing and nanotechnology applications
Innova Biosciences also offers a kit (InnovaCoat® GOLD - Maleimide 20OD 40 nm Gold Particle Kits) and service for conjugating thiol-modified oligonucleotides to gold particles. In combination with a bio-polymer gold coating technology, the gold nanoparticle conjugation system allows sulfhydryl-oligonucleotides to be covalently attached to ultra-stable gold nanoparticles of high concentration. An example of this application would be the conjugation of antigen-specific aptamers to gold nanoparticles for use in lateral flow point-of-care detection systems.
These oligonucleotides must also be 10–120 nt and contain a terminal sulfhydryl group, which must be added during synthesis. Most commercial oligo manufacturers offer this modification. Free thiols must be excluded from samples and reaction buffers during this conjugation reaction, as they will compete for coupling sites. Innova scientists note that the resulting covalent conjugates are far more stable than those prepared by adsorption of thiolated oligonucleotides to bare metal surfaces.