A powerful method of immunodetection using Antibody-Oligo conjugates

 

Immuno-PCR is a method of immunodetection combining the specificity of an ELISA with the signal amplification of PCR. Enabled by antibody-oligonucleotide conjugates, iPCR detection assays have shown to perform 100-1000 fold more sensitive than traditional ELISA with much less antibody used.

An ELISA uses conjugated antibodies to detect any protein, meaning it can be highly specific; however it may not be sensitive enough to detect very low levels of protein. rtPCR cannot be used directly for antigen detection as it does not use antibodies, however it provides exponential signal amplification.

By combining these two assay formats using an antibody-oligonucleotide conjugate, immuno-PCR provides the best of both worlds.

Furthermore, it is possible to convert any type of ELISA to an immuno-PCR assay by replacing the detection antibody with an antibody-oligo conjugate.

 

Performing an immuno-PCR assay?

 

An antibody specific to the target antigen is immobilized on the surface of a microtiter plate. The washing removes any unbound antibody.  The plate is then blocked and the sample added, followed by further washing to remove any unbound sample. Then a second specific antibody, coupled to an oligonucleotide is added, followed by more washing steps, before the DNA is amplified and detected via real time PCR.

Figure 2. Schematic representation of the steps of an immuno-PCR assay.

 Advantages of immuno-PCR for immunodetection

  • Can detect any protein.
  • Provides exponential signal amplification.
  • Highly sensitive – can detect extremely low amounts of analyte (pg – fg).
  • Fewer incubation steps so improved assay reproducibility.
  • Provides the option of multiplexing.
  • Needs only small sample volumes.
  • Compatible with complex samples.

Despite the advantages of immuno-PCR, an ELISA continues to be the primary method of immunodetection. This is because the conjugation of antibodies to oligonucleotides has traditionally been associated with requiring special expertise and being an expensive and time-consuming process. oYo-Link Oligonucleotide Conjugation technology overcomes these challenges by enabling the quick and easy, site-specific labeling of oligonucleotides to antibodies with only 30 seconds hands-on time.

 

oYo-Link® Oligo Conjugation Technology

oYo-Link® Oligo Conjugation Technology enables efficient labeling of compatible antibodies with both ssDNA & dsDNA oligonucleotides, up to 80bp or longer, for use in immuno-PCR and other custom applications.

Unlike other oligonucleotide conjugation products available, oYo-Link allows site specific, covalent labeling of antibodies in the Fc region, assuring there are 1 to 2 oligonucleotides attached per antibody and yielding a homogenous mixture of antibody-oligo conjugates. Therefore, oYo-Link Oligo provides scientists with more control and precision over their experiments, removing any impact of decreased assay performance associated with traditional heterogeneous labeling products. Read more about the benefits of site-specific labeling here.

Figure 3. The oYo-Link site-specific labeling process attaches 1-2 oligos to Fc region of antibody using non-damaging UV light.

How does oYo-Link® technology work?

oYo-Link® reagents consist of low molecular weight (~6 kDa), high-affinity antibody-binding domains that possess a photo-crosslinker within their Fc-binding site. Upon illumination with non-damaging Black-light, oYo-Link forms a covalent bond with the antibody. This procedure is referred to as Light-Activated Site-Specific Conjugation (LASIC). Any label that is attached to oYo-Link will be covalently attached to the desired antibody.

Labeling is extremely quick with only 30 seconds hands-on time required and 2 hours total conjugation time.  Simply add oYo-Link to your antibody, illuminate for 2 hours and your antibody will be covalently labeled with your oligonucleotide and ready to use! There are no purification steps required post labeling, meaning 100% sample recovery.

You just provide us with your oligo sequence and we send you the custom oYo-Link Oligo reagent ready to use with compatible antibodies.

All Oligonucleotides are of the highest quality, manufactured by Integrated DNA Technologies, Inc.

oYo-Link 30 seconds hands-on time

Figure 4. The oYo-Link Oligo conjugation process requires only 2 steps and 30 seconds hands-on time. Conjugates are ready to use after 2 hours.

  Features & benefits of oYo-Link Oligonucleotide Conjugation Technology

  • Easy and rapid oligo-antibody labeling with under 30 seconds hands-on time – no high technical expertise required

  • Custom oligos supported, up to 80bp or longer, for ssDNA or dsDNA

  • Site specific labeling in the Fc region, so no loss in antibody functionality or batch-to-batch variability

  • Label as little as 1ug of antibody at a time – reduce overall antibody required = reduce overall cost and minimize wasted materials!

  • Label diluted antibody solutions – under 50 µg/mL – saving time as no need to concentrate your antibody prior to labeling

  • No purification steps so 100% sample recovery – no waste of expensive samples

  • No need for purification prior to conjugation – compatible with nearly all buffers, even those containing storage proteins and amines – save time and money

  • Aliquot and save for later – can be used for future reactions

Find out more about the oYo-Link Oligonucleotide Conjugation product here, or get in touch with our technical support team with any questions regarding the technology or immuno-PCR.

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