Immunoassays are tests that detect the presence of a specific molecule in a sample using antibody-antigen binding reactions. Antibodies bind to the specific structure of a particular antigen, making immunoassays highly specific: the antibody will only bind to a specific structure of a particular antigen. This makes antibodies effective reagents for detecting target molecules. As a result, immunoassays are a fundamental tool for hospitals, life science research and industry laboratories. Immunoassays come in a range of formats, and can be used to assess disease, track proteins, and detect environmental contamination.
What can be measured using immunoassays?
Immunoassays are useful when you want to detect or isolate a molecule within a mixture, typically a cell culture. The assay can be used to identify the presence of pathogens in a clinical sample, or it can be used to measure the amount of a target biomolecule. When using immunoassays to measure an amount of target, a ‘reporter’ system is needed.
Alternatively, immunoassays can be used to isolate a specific molecule. This can be done by magnetic separation using a magnetic particle. This kind of immunoassay is called a magneto-actuated immunoassay. Commonly, these assays use a particle with a core of magnetite that is coated with a biologically compatible material, and chemically modified by the attachment of antibodies. However, before designing a magnetic particle for an immunoassay you must decide what type of immunoassay best fits the aims of the experiment.
How many types of immunoassay are there?
There are at least four main types of immunoassay, with sub-types of each main format, and new techniques are being developed all the time. Usually, immunoassays are broken down into those that use labels and those that do not – called ‘label-free’ immunoassays. Labeled immunoassays are made up of an analyte (the target antigen to be measured), an antibody, and a label that allows the molecule to be detected.
The four most commonly used types of labeled immunoassays are:
- Enzyme Immunoassay (EIA) or Enzyme-linked immunosorbent assay (ELISA)
- Radioimmunoassay (RIA)
- Fluoroimmnoassay (FIA)
- Chemiluminescence immunoassay (CLIA)
Enzyme-linked immunosorbent assay
Enzyme immunoassays, or ELISAs, are simple, widely used tests that rely on highly specific antibody-antigen interactions. An ELISA is designed to detect and quantify soluble molecules such as antibodies, proteins and hormones. There are four main kinds of ELISA: sandwich, competitive, direct and indirect assays. These methods differ in how the antibody or antigen is attached to the solid plate, and how the signal is detected.
In a sandwich ELISA, for example, an antibody is immobilized on a plate. The sample containing the target antigen is added, which binds to the antibody and so is immobilized on the plate. Next, a second type of antibody is added, which also binds to the target antigen on the plate, forming a ‘sandwich’ with the target antigen in the middle. The second antibody is linked to an enzyme, called a reporter enzyme, which allows the binding reaction to be measured by creating a color signal. To create this signal, first any unbound antibody is washed away, and a colorimetric substrate is added. The enzyme catalyzes a reaction of the substrate, creating a color change. A stronger color signal indicates more target antigen is present. An example of this is a home pregnancy test.
Other types of ELISAs also have vital applications in clinical practice, such as magneto ELISA that is used for the detection of CD4+ cells for the diagnosis and treatment of AIDS.
A radioimmunoassay (RIA) uses radiolabeled antigens to measure concentrations of substances in body fluids such as blood and saliva. A radioisotope is attached to an antigen of interest and bound with its complementary antibody. A sample with the target antigen is then added, which competes with the radioactive antigen, kicks it out of the binding spot and replaces it.
After washing away unbound antigens the radioactivity of the sample is measured. The smaller the radioactive signal, the more target antigen is present. Compared to other immunoassay techniques, extra precautions are needed due to the radioactive substances involved, but RIA’s high sensitivity and specificity means that it remains in use today.
Fluorescent immunoassays (FIA) use a fluorescent compound as the detection reagent to detect and quantify a variety of compounds. FIA is widely used in the in vitro diagnostics (IVD) industry and has the advantage of being fast and highly sensitive compared to other methods. In FIA, antibodies are labeled with fluorescent probes. FIA fluorescent dyes illuminate in UV light and are used to detect a specific antigen-antibody binding. After incubation with the antigen, the antibody-antigen complexes are isolated, and the fluorescent intensity is measured.
The principles of a chemiluminescent immunoassay (CLIA) are the same as an ELISA or fluoroimmunoassay, but the reporter is different. Luminescence is the release of light due to an electron being kicked up to a higher energy state and emitting a photon as it relaxes down. CLIA uses a chemical reaction to kick the electron up to a higher energy, whereas FIA uses certain light frequencies.
Due to its high sensitivity and specificity, CLIA is used in a range of different fields, including environmental monitoring, disease diagnosis, life sciences and food safety. Newer techniques, such as magnetic-bead based CLIA have also widened its potential. For example, magneto-actuated chemiluminescence assays were developed to detect the presence of Zika virus in patient samples.
What are the advantages of label-free immunoassays?
The emerging field of photonic biosensors is driving development of a new generation of label-free assays that don’t rely on central laboratory facilities. Photonics is the science of detecting and manipulating light. By using technologies developed by the electronics industry in the production of microprocessors and adapting them to light, photonic biosensors combine photonic sensing with bio recognition technology to create label-free testing on-chip. Instead of moving electrons around on silicon chips, light is moved around on silicon chips via waveguides.
This technology has allowed the development of miniature lab-on-a-chip label-free immunoassay (LFIA) devices. These devices are functionalized with capture antibodies and have a resonance condition of light. This resonance wavelength will be shifted by a reaction between the capture antibody and the target antigen due to the change in refractive index. Measuring the shift in resonance wavelength provides a readout of a binding event. Label-free assays therefore enable the detection of antigen-antibody binding without the use of an additional label, resulting in increased assay sensitivity and decreased working time.
How to choose an immunoassay?
The large number of types and subtypes of immunoassays can make choosing the right immunoassay for your application challenging. Here are some things to consider when making your decision:
- Decide what sensitivity you need. This is determined by the concentration of antigens you’ll be working with: low concentrations need higher sensitivity to ensure detection. Also consider your throughput requirements and cost.
- Decide on the antibody-antigen pairs that you will test. You should check their availability commercially, or alternatively produce reagents in the laboratory.
- Decide what surface or environment you want to use. For example, you can bind antibodies or antigens to a solid plate or a magnetic bead, depending on which target needs to be observed.
Tips for a successful immunoassay
- Optimize blocking and washing steps. This will minimize non-specific binding.
- Ensure your analysis method is appropriate for the type of immunoassay used.
- Validate your immunoassay procedure. There are many published guidelines for validation of your protocol and technique to ensure they are reproducible and consistent.
- Check your sample is compatible with your immunoassay. Not all immunoassays will work for all sample types.
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