Since the first immunoassays were developed in the 1950's the variety of assay formats has increased dramatically. Technological and scientific advances, such as the development of quantitative lateral flow reader technology, have added to the complexity associated with the development of assays. However, they have become easier to use and significantly more sensitive.
Some analytes (or targets of interest) are present in samples at very low concentrations and clinicians increasingly demand earlier detection of diseases for effective treatment. Therefore many new techniques, methods of detection and assay formats have been developed to achieve higher sensitivity in diagnostic assays. Western blotting is an example of a method that can be used to detect analytes at the femtogram (10-15) level but it has not be adopted as a diagnostic tool because it is only qualitative and cannot be adapted into a multiplex format. The most common immunoassay format is the enzyme-linked immunosorbent assay (ELISA). ELISA assays are quantitative and are available in a multiplex format; however the limits of detection are usually around the nanogram (10-9) to picogram (10-12) level.
Immuno-PCR – enhanced sensitivity by combining molecular and immunological techniques
Numerous immunoassays have been developed and successfully used in diagnostics however, they have not achieved the desired sensitivity and accuracy of quantification required for some analytes. In 1992 T. Sano, C. Smith and C. Cantor modified an immunological technique (ELISA) by combining it with a molecular technique (real-time PCR) in an attempt to overcome the limitations associated with traditional immunoassays. In an ELISA assay the analyte of interest is detected colorimetrically using an enzyme-linked antibody and a substrate. Real-time PCR (qPCR) is a method for the quantitative amplification of a short strand of DNA.
This new method, termed immuno-polymerase chain reaction (immuno-PCR), benefits from the specificity of antibodies and the sensitivity of PCR. Several independent studies undertaken since the publication of this seminal paper have consistently demonstrated the advantages of this technique over traditional ELISA, some achieving more than a 50,000 fold increase in sensitivity over the equivalent immunoassay . Furthermore, immuno-PCR can be used in a multiplex format and offers accurate quantitation.
Problems conjugating antibodies to oligonucleotides
Immuno-PCR demonstrated that it could achieve significantly greater assay sensitivity yet it has still not replaced ELISA as the assay format of choice for the diagnostic industry. The main reason for this is the difficulty associated with linking oligonucleotides to antibodies. Several methods have been developed although they tend to be very complex, unreliable and often result in the cross linking. To overcome these problems an intermediate step is sometimes used (such as the streptavidin-biotin system) but the optimum method is one where the antibody is directly linked to the oligonucleotide . This is a complex technique requiring specialist knowledge, equipment and is very time consuming. Therefore, immuno-PCR's potential as a diagnostic tool has not yet been realised.
Innova Biosciences has developed a revolutionary new technology that enables scientists to conjugate their antibody directly to their oligonucleotide in just a few simple steps. This unique technology, the Thunder-Link® antibody-oligonucleotide conjugation system, offers many advantages over other methods, includes a positive control and a proprietary method to remove unconjugated oligonucleotides.
Thunder-Link® has already been used for the successful development of immuno-PCR assays and will hopefully help scientists to take advantage of this technique for the development of high sensitivity quantitative diagnostic assays.
A mouse monoclonal antibody specific for human CRP (clone C7) was purchased in unconjugated format from HyTest. The unconjugated antibody was conjugated to an oligonucleotide using a Thunder-Link® kit, and was used as detection antibody in a sandwich ImmunoPCR assay using a polyclonal anti-CRP antibody as capture reagent.
The top graph plots the number of qPCR cycles undertaken vs. fluorescence intensity generated by SYBR green containing qPCR probes at particular antigen concentrations. The bottom graph then converts this data to antigen amount vs cycle number to enable calculation of a standard curve.
The results show that the assay utilises 1000-fold less capture antibody, 100 fold less detection antibody and provides 1000-fold more sensitivity than the equivalent ELISA.
Want to know more? Take a look at the Thunder-Link protocol here.
- Download our new immuno-PCR application guide for a more indepth look into the developement of an Immuno-PCR assay
 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.
 Saito et al., 1999. Detection of Human Serum Tumor Necrosis Factor-? 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
 Lind, K. and Kubista, M., 2005. Development and evaluation of three real-time immune-PCR assemblages for quantification of PSA. Published by the Journal of Immunological Methods. Available at http://genexp.ibt.cas.cz/Lind88.pdf