The principles of immunoassays
An immunoassay capitalizes on the specificity of the antibody-antigen binding found naturally in the immune system. Antibodies made by the adaptive immune response in the body are highly specific towards particular antigens. For example, this is why we receive vaccinations, to help our immune system build an antibody repertoire response towards parts of an antigen before we encounter it in a more pathogenic state. The immunoassay will use those highly specific antibodies to probe for molecules of interest when they are in mixtures with other molecules.
Applications of Immunoassays
Immunoassays can be applied to a situation where one wants to detect or isolate a molecule within a mixture. 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, then a reporter system is needed. Different types of detection/reporter systems are described below. If the goal of the immunoassay is to isolate a specific molecule then a separation system is needed. When the isolation is achieved by magnetic separation using a magnetic particle it is called a magneto-actuated immunoassay. The most common particle used in these assays is made of 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 one must decide which types of immunoassays best fits the goals of the experiment.
Steps and Parameters for developing and running an immunoassay
- Determine best immunoassay technique for the experiment, consider the sensitivity, throughput requirements, and cost. Types of immunoassays are described below. Here you will also consider what type of surface or environment you want your immunoassay to take place in.
- Decide on antibody-antigen pairs to be tested, and determine their availability commercially or produce reagents in the laboratory.
- Bind antibodies or antigens to a surface, such as a plate or a bead, depending on which target needs to be observed. Common techniques are the sandwich assay, the competition assay, or putting binding antigen first for antibody probing.
- Optimize blocking and washing steps to minimize non-specific binding.
- Incubate with the secondary molecule or secondary antibody for detection.
- Analyze data as appropriate for the type of immunoassay used.
- Validate your immunoassay procedure. There are many published guidelines for validation and they serve to ensure the consistency and accountability of your protocol and technique.
Five types of immunoassays
As biotechnology advances and our understanding of nanotechnology deepens we can expect that the immunoassay options available will expand. For now we can focus on these five types of immunoassay:
- Radioimmunoassay (RIA)
- Counting Immunoassay (CIA)
- Enzyme Immunoassays (EIA) or Enzyme-linked immunosorbent assays (ELISA)
- Fluoroimmnoassay (FIA)
It is also worth mentioning that there is currently a movement towards developing label free immunoassays. These clever technologies are founded upon physical principles such as constructive and destructive interference of light, resonance conditions, and how changes in effective refractive index alter these conditions. Label free assays enable the detection of antigen-antibody binding without the use of an additional light-emitting label. This allows for increased assay sensitivity and decreased working time.
The radioimmunoassay is perhaps the oldest types of immunoassays. Here, a radioisotope is attached to an antigen of interest and bound with its complementary antibody. Then a sample with the antigen to be measured is added. It 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 amount of radioactive signal is inversely related to the amount of target antigen. The health hazards of using radioactive substances caused a movement toward safer methods.
2. Counting immunoassay
In a counting immunoassay polystyrene beads are coated with many antibodies complementary to the target antigen. During incubation the beads bind to multiple antigens and group together into a large mass. Some beads remain unbound. The entire solution is passed through a cell counter and only the unbound beads are counted. The number of unbound beads is inversely proportional to the amount of antigen.
3. Enzyme-linked immunosorbent assay
In an ELISA the antibody is linked to an enzyme. After incubation with the antigen the unbound antibody is washed away. The bound antibody-enzyme attached to the target antigen is observed by adding a substrate to the solution. The enzyme catalyzes a chemical reaction of the substrate to produce a quantifiable color change. A practical example is a magneto-ELISA system for the detection of CD4+ cells for the diagnosis of AIDS.
In a fluoroimmunoassay the antibodies are labeled with fluorescent probes. After incubation with antigen the antibody-antigen complexes are isolated and the fluorescent intensity is measured.
5. Chemiluminescence immunoassay
The principles of a chemiluminescent immunoassay 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. This is the same principle as fluorescence. The difference lies in the mechanism of kicking the electron up to a higher energy in the first place. In fluorescence this is achieved with certain frequencies of light. In chemiluminescence this is achieved by a chemical reaction. These reactions require an emitter and a coreactant. A magneto-actuated chemiluminescence assay was developed to detect the presence of Zika virus in patient samples.
Label Free Immunoassays
The new photonics field has the potential to launch the development of an entirely new generation of label free assays. Photonics is centered upon the idea of using the technologies developed and perfected by the electronics industry in the production of microprocessors, and adapting them to light. So, instead of moving electrons around on silicon chips, we will move light around via waveguides on silicon chips. This idea will lead to the development of miniature lab-on-a-chip label free immunoassay devices. These devices will be functionalized with capture antibodies and will have a resonance condition of light. This resonance wavelength will be shifted upon a reaction between the capture antibody and the target antigen due to the change in refractive index. Simply measuring the shift in resonance wavelength will provide a readout of a binding event...no label, just antibody-antigen interaction.