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Improving immuno-PCR by optimizing antibody-oligo conjugation

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.

Definition: Immuno-PCR

Immuno-PCR is a technique similar to ELISA whereby an antibody is used to detect and quantify a specific antigen in a mixed sample. In immuno-PCR, however, the antibody is coupled to DNA, which is then amplified using real-time PCR. The technique is markedly more sensitive than ELISA, primarily because it combines the detection specificity of an antibody with the nucleic acid detection sensitivity of real-time PCR. First described in 1992 [1], the coupling of real-time PCR to immune detection has become the standard in recent years. The increased sensitivity provided by immuno-PCR allows lower limits of detection that surpass ELISA by 100- to 10,000-fold.

Reference
1. Sano T, Smith CL, Cantro CR (1992) Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody- DNA Conjugates. Science, 258:5079:120–122.

Simplifying bioconjugation

Innova Biosciences (Cambridge, UK; www.innovabiosciences.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.


Oligonucleotide requirements

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 the sidebar, Oligonucleotide Conjugation to Antibodies—Tips for Good Oligonucleotide Design.)

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.

Oligonucleotide conjugation to antibody—tips for good oligonucleotide design (for use with the Innova Thunder-Link® Oligo Conjugation System)

  • Place the amino (or a thiol) modification at either the 5’ or 3’ end of the oligonucleotide. Note that the 5’ end is preferred, as it results in higher purity oligos, which in turn slightly increases antibody-oligo conjugation efficiency.
  • Minimize base repetition (homopolymer regions).
  • Avoid stretches of >4 G bases in a row. These sequences can form a G-quadruplex or cruciform structures, which can lower your hybridization and coupling efficiencies. Read more about G-quadruplexes in the article, G Repeats—Structural Challenges for Oligo Design.

Profile: Innova Biosciences

Innova Biosciences is a growing, dynamic, ISO 9001:2008 business based in Cambridge, UK. At the core of Innova's business are bioconjugation and nanoparticle technologies including Lightning-Link®, InnovaCoat®, and Thunder-Link® brands. The expanding portfolio is sold to research laboratories, pharmaceutical companies, biotechnology companies, and diagnostic companies worldwide.

For this article we spoke with Dr Andrew Lane, the Executive Director for Innova Biosciences; Tom Speedy, Innova’s Head of Sales and Corporate Business; and Klaudyna Schmidt, the company’s Marketing Manager.

Product focus—oligos and modifications

Custom oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.

Learn more or order now.


Oligo modifications

Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? IDT will consider any modification you need. Just send your request to noncat@idtdna.com.

Further reading

G Repeats—Structural Challenges for Oligo DesignWhat are G-quadruplexes and how do they affect oligonucleotide synthesis and applications?

Oligonucleotide Modifications: Choosing the Right Mod for Your NeedsGuidelines on selecting oligonucleotide modifications and how they can help you in your research.

Oligo Modification—Post-Synthesis Conjugation Explained—Addition of NHS esters and amino and alkene modifications through click chemistry; answers to common questions about post-synthesis conjugation.

Review other DECODED Online newsletter articles on oligo handling and analysis, and oligo modifications or on PCR and qPCR applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.


Author: Ellen Prediger is a senior scientific writer at IDT.

© 2015, 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, seewww.idtdna.com/trademarks.


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