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Researchers have relied on immunodetection techniques such as Western blotting, flow cytometry and Enzyme-Linked Immunosorbent Assay (ELISA) for years, but immuno-PCR is a relatively new method. By merging an ELISA with the Polymerase Chain Reaction (PCR), immuno-PCR provides extremely high levels of assay sensitivity.
An ELISA is an assay in which a molecule is adsorbed on to a solid surface, usually a microplate well. Broadly speaking, there are three different types of ELISA. An antigen-down ELISA is an approach where the antigen is bound to the microplate well. In contrast, a sandwich ELISA relies on first the adsorption of an appropriate capture antibody to the microplate well, then the antigen from a sample prior to detection with another antibody. A third format, the competitive ELISA, is useful when the analyte is small and cannot be bound by two different antibodies at the same time; it often utilizes an antigen-down setup. ELISAs are simple to perform, relatively cheap to run, and are easily adapted to high throughput screening.
PCR is a technique which is used to amplify a specific segment of DNA, and can be performed using only a very small amount of starting material. It employs a three-step cycling process: 1) heat-induced denaturation of dsDNA to separate the two complementary strands, 2) temperature reduction to allow binding of PCR primers, 3) use of DNA polymerase to produce a complementary copy of the target DNA sequence. During traditional PCR, the amplified DNA is run on a gel and stained with ethidium bromide. Real-time PCR (rtPCR), also known as quantitative PCR (qPCR), instead correlates production of the PCR product to fluorescence intensity, generating data as the assay happens. rtPCR is highly sensitive and affords a large dynamic range.
Immuno-PCR: The Best of Both Worlds
As long as appropriate antibody reagents are available an ELISA can detect any protein, however it may not be sensitive enough to identify proteins of low abundance. While rtPCR provides exponential signal amplification, it cannot be used directly for antigen detection since it does not utilize antibodies. Immuno-PCR combines these two assay formats via an antibody-oligonucleotide conjugate. You can convert any type of ELISA to an immuno-PCR assay, it is simply necessary to replace the detection antibody with an antibody-oligonucleotide conjugate.
As an example, a typical sandwich ELISA requires that an antibody specific to the target analyte is immobilized on the surface of a microtiter plate. Unbound antibody is removed by washing, and following a blocking step the sample is added. After incubation, any unbound sample is washed away then a second analyte-specific antibody is added. If a direct detection method is used, this antibody will be conjugated to a detection moiety such as an enzyme or a fluorescent dye. Indirect detection instead involves another round of washing steps, followed by the addition of a conjugated anti-species secondary antibody.
To convert a sandwich ELISA with direct detection to an immuno-PCR assay, the second antigen-specific antibody would simply be replaced with an antibody-oligonucleotide conjugate. After the final wash step, the DNA can be amplified and detected via rtPCR.
Conversion of an ELISA to an Immuno-PCR Assay
When developing an immuno-PCR assay, it is often sensible to adapt an existing ELISA. Each step of the assay should be optimized to maximize the specific readout while minimizing the background signal. It may be that factors such as the method of plate coating, the type of wash buffer and blocking solution, the length of the sample incubation step and the choice of antibodies are already known for the equivalent ELISA.
The success of any immunoassay depends on the quality of the antibody reagents but, when set up correctly, immuno-PCR offers many advantages over other assay formats:
- Highly sensitive – can detect picogram-femtogram amounts of analyte
- Needs only small sample volumes
- Compatible with complex samples such as serum or urine
- Provides the option of multiplexing
Generation of Antibody-Oligonucleotide Conjugates
Antibody-oligonucleotide conjugates can be expensive to produce since traditional conjugation methods require large quantities of antibody, with significant losses incurred during purification steps. Immuno-PCR is however becoming more widely accessible, since user-friendly antibody-oligonucleotide conjugation kits are now available.
Why Switch to Immuno-PCR?
Immuno-PCR affords significantly greater sensitivity than other immunodetection methods. This makes it ideal for detecting low abundance analytes in complex samples since the sample material can be diluted considerably, which reduces background effects and increases assay performance. The technique also affords consistent and reproducible data.
Immuno-PCR is, quite rightly, growing in popularity – why not give it a go? Provided the necessary antibody reagents are available, there are no limits to the power of this technique.Image Credit: Emma Easthope