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Detecting biomarkers of disease requires techniques that are sensitive and high-throughput. Which approach is most suitable for your experiment?
ELISA assays are widely used to quantify protein targets with minimal sample preparation. However, detecting and quantifying biomarkers in biological samples requires greater sensitivity, dynamic range and throughput than is provided by a traditional ELISA assay.
Several assay platforms are now available that provide improved performance across these parameters. These assay platform technologies can be divided into broad areas:
At the heart of all of these techniques is high performing antibody pairs, in which capture and detector antibodies bind specifically to their target in complex biological samples.
The technology relies on sophisticated fluid dynamics to manipulate <1 µL volumes within the chamber to assemble and then measure an immunocomplex. The exact details of the assay are dependent on the system you are using.
Microfluidic immunoassays allow for highly sensitive detection of target proteins allowing researchers to conserve precious sample.
How it works
In this system, the capture antibody is bound to a paramagnetic bead. After capture of the target protein, a labeled detector antibody is used for fluorescent readout. Each bead captures a single target protein and then the beads are dispensed into an array of wells – each small enough to trap only a single bead, which then emits a fluorescent signal.
The number of signal-positive wells is counted using a Simoa disc, allowing the detection of attomoles of target protein. The Simoa disc contains 24 flow cells, each with an array of 200,000 microwells, for a total of 4.8 million data points.Simoa disc, allowing the detection of attomoles of target protein. The Simoa disc contains 24 flow cells, each with an array of 200,000 microwells, for a total of 4.8 million data points.
The primary advantage of Simoa technology is increased sensitivity. This is of particular advantage when measuring very low abundance protein biomarkers like those present in the blood after traumatic brain injuries1.
How it works
The Gyros assay2 uses a network of multi-channeled microfluidic chambers. Each chamber contains a microscopic column containing streptavidin-coated particles. A biotinylated capture antibody is bound to the column and then the sample is applied. A detection antibody then binds to the target protein.
Solutions move through the channels of each chamber using a combination of capillary action and centrifugal force. The formation of the immunocomplex on the column is measured by fluorescent detection.
The use of very small volumes allows for reduced animal usage in assessing the pharmacokinetics of biotherapeutic agents. An article from Pfizer highlights the advantages of the Gyros system by using serial sampling of just one animal for a complete pharmacokinetic profile3.
Microarrays bind the capture antibody to a substrate and then use a variety of detection methods for quantification. The advantages are dependent on the detection method.
How it works
MesoScale Discoveries provides a unique ELISA-based technology that uses capture antibodies bound to wells of an electrode-embedded plate. The detector antibody is conjugated to electrochemiluminescent SULFO-TAG labels. Electrical signal stimulation of the tag generates visible light emission at 620 nm. The signal can be amplified to increase sensitivity and wells have multiple analysis regions that allow measurement multiple analytes per well.
The major strength of the platform is the dynamic range which provides linear measurements across a wide range of potential sample dilutions as demonstrated in a recent publication measuring the concentration of amyloid beta in mouse plasma4.
Proximity assays can be used to show interaction between the antibodies and proteins in a sandwich immunoassay based on how close the proteins are in solution, for example, when a target protein is bound by an antibody pair. Proximity assays can be highly sensitive as in the case of immuno-PCR or high throughput in the case of time resolved FRET.
How it works
Proteins of interest are conjugated to fluorophores; a donor and an acceptor fluorophore. When the proteins are in close contact – such as when two antibodies are bound to a target protein as in a sandwich immunocomplex – the fluorophores are brought together.immunocomplex – the fluorophores are brought together.
A laser or flash lamp is used to excite the donor fluorophore causing it to emit light at the excitation wavelength of the acceptor fluorophore. If the acceptor fluorophore is sufficiently close, then it will emit light at its emission wavelength. The signal from the acceptor dye is used to quantitatively determine the concentration of the target protein.
Although the assay principles are simple, the practice can be difficult due to background fluorescence from buffers, chemicals and proteins. In a modification of this technique called time-resolved FRET (TR-FRET), the signal is measured after emission from solution components and uncomplexed fluorophores has decayed. This requires that the FRET signal has long-lived emission signals: suitable fluorophores include europium3+ paired with allophycocyanin and terbium3+ paired with phycoerythrin.
TR-FRET is a homogeneous assay that does not require repeated washings to remove unbound antibody and sample from the immunocomplex, thus FRET is high throughput and is commonly used for screening assays during drug discovery.
How it works
In immuno-PCR, the detector antibody in a sandwich immunocomplex is labelled with biotin, which is then bound by streptavidin. The streptavidin then binds to single stranded DNA (ssDNA). This ssDNA is anchored to the immunocomplex which can then be amplified during real time PCR with a DNA intercalating dye. The amplification only occurs if the complete complex is formed5,6.
The amplification step in real time PCR results in the highest sensitivity of all the described techniques, which is useful in detecting very early stage cancer where detection of very low levels of biomarkers is required7.
In each of these technology platforms, advances in sensitivity, dynamic range and throughput relies on the performance of a highly specific antibody pair. Our matched antibody pair kits contain monoclonal and recombinant antibodies, which are screened for performance in biological samples for reliable performance whatever your assay format.