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Immuno-PCR

Introduction

Immuno-PCR is an extremely powerful method of immunodetection which combines the specificity of an ELISA with the signal amplification of PCR. Since an ELISA requires the use of antibodies, it is adaptable to any protein, however its sensitivity is not adequate for the detection of analytes of low abundance. While real time PCR provides exponential signal amplification, it cannot be used directly for antigen detection. Immuno-PCR relies on the use of an antibody which has been conjugated to an oligonucleotide, and this conjugate acts as a bridge between the immunoreaction and DNA amplification.

Immuno-PCR

Schematic representation of a sandwich immuno-PCR assay. A capture antibody is immobilized on the surface of a microtiter plate and used to bind the target analyte. A second antibody, which has been coupled to an oligonucleotide, is then bound. The DNA is amplified and detected via real time PCR.

 

To find out more about immuno-PCR, why not download our free guide?

 

Immuno-PCR offers many advantages over an ELISA:

✓ Adaptable to the detection of any protein
✓ Provides exponential signal amplification
✓ Extremely low limit of detection (pg - fg)
✓ Suitable for small sample volumes
✓ Compatible with complex samples
✓ Fewer incubation steps than an ELISA, improved assay reproducibility
✓ Rapid time to results
✓ Wider dynamic range than an ELISA
✓ Amenable to multiplexing

 

Unfortunately, despite providing significantly greater sensitivity, immuno-PCR has not yet replaced ELISA as the assay format of choice for the diagnostic industry. The main reason for this is the difficulty associated with conjugating antibodies to oligonucleotides, a process which can be costly and time-consuming, and which requires expert knowledge. To overcome the issues that beset traditional methods of antibody-oligonucleotide conjugation, we’ve developed our ThunderLink® PLUS oligo conjugation system.

Thunder-Link process

The Thunder-Link® PLUS conjugation process.

 

Our ThunderLink® PLUS oligo conjugation system is an easy-to-use kit that enables stable antibody-oligo conjugates to be generated quickly and efficiently. The oligo is added to a vial of oligo activation reagent, and the antibody to a vial of antibody activation reagent, then both vials are incubated for 30 minutes at room temperature. During this incubation step two desalt columns are washed. The activated oligo mix is passed through one of the columns, and the activated antibody mix is passed through the other column. The antibody and the oligo are next incubated together for one hour at room temperature, and the conjugate is then ready to use. An optional clean-up step can be performed at this point to remove any unbound oligo, should this be necessary.

Immuno-qPCR data- qPCR cycles vs fluorescence intensity

 

Immuno-qPCR data - antigen amount vs cycle number

Immuno-PCR data generated using Thunder-Link®. A mouse monoclonal antibody specific for human CRP (clone C7) was purchased in unconjugated format from HyTest. The antibody was conjugated to an oligonucleotide using a Thunder-Link® kit, and was used as a detection antibody in a sandwich immuno-PCR assay with a polyclonal anti-CRP antibody as the capture reagent.

The top graph shows a plot of the number of PCR cycles undertaken vs. fluorescence intensity generated by SYBR Green containing PCR probes at particular antigen concentrations. The bottom graph shows this data following conversion to antigen amount vs. cycle number to enable the calculation of a standard curve. The results show that the assay utilizes 1000-fold less capture antibody and 100-fold less detection antibody, and provides 1000-fold greater sensitivity than the equivalent ELISA.

 

To learn more about antibody-oligo conjugation, why not download our free webinar?

 

Innova Biosciences offers a wide range of custom services and, when it comes to immuno-PCR, our experienced scientists can help you to optimize your antibody: oligo conjugate, and can also produce your chosen conjugate at a scale to meet the requirements of your particular assay. We also have a technical support team ready to answer your questions, so please do contact us if you require any further information.

And for advice on optimizing each step of your immuno-PCR assay, don’t forget to read our Tips for performing immuno-PCR application note.

 

References

[1] Sano, T., Smith, C. and Cantor, C., 1992. Immuno-PCR: Very Sensitive Antigen Dectection by Means of Specific Antibody-DNA Conjugates. Published by the American Association for the Advancement of Science. Available at http://blogs.bu.edu/clsmith/files/2010/05/Immuno-PCR_1992_Sano_Smith_Cantor.pdf.

[2] Saito et al., 1999. Detection of Human Serum Tumor Necrosis Factor-alpha in Healthy Donors, Using a Highly Sensitive Immuno-PCR Assay. Published by The American Association for Clinical Chemistry. Available at http://www.clinchem.org/content/45/5/665.full

[3] Lind, K. and Kubista, M., 2005. Development and evaluation of three real-time immuno-PCR assemblages for quantification of PSA. Published by the Journal of Immunological Methods. Available at http://genexp.ibt.cas.cz/Lind88.pdf

 

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