The potential to detect a specific amount of protein that is present within a complex sample matrix has been an extremely valuable tool for many scientists. This process has led to the screening for immunity, the development of various diagnostic tests, detection of potential allergens in the food industry and the manufacture of novel vaccines. The different types of immunoassays that are available can offer an enormous amount of diversity, providing antibodies with high specificities that can target specific protein molecules to accurately detect their concentration present within a particular sample solution.
What is enzyme immunoassay?
Enzyme immunoassay (EIA) is also more commonly known as enzyme linked immunosorbent assay (ELISA). These are important tests that can be used to detect various antibodies or antigens that are present within a sample by forming an enzyme triggered colour change. Majority of the enzyme immunoassays are known as solid-phase assays. The 4 main types of ELISA are:
- Direct ELISA: When a sample or antigen is directly immobilised on the microwell plate, this is followed by the conjugated detection antibody binding to the target protein. A substrate is then used to generate a signal. There is a direct correlation between the amount of analyte present and the amount of signal produced. This method is simple and quick to perform, however, it can lead to high levels of background and may be less specific as only one antibody is used. This procedure is useful when investigating inhibitory/blocking interactions and when assessing antibody specificity and affinity.
- Indirect ELISA: This method requires an additional step for the amplification detection process. An unconjugated primary detection antibody is firstly bound to the specific antigen and then a conjugated secondary antibody is bound to the primary antibody. The substrate can generate a signal that is directly proportional to the quantity of antigen that is bound to the microwell plate. An advantage for this method is that is uses a secondary antibody for the amplification, however, this can create a potential for cross reaction being cause by the secondary antibody. This procedure is useful in detecting the amount of endogenous antibody.
- Sandwich ELISA: This is the most popular type of ELISA. In this method two specific antibodies (also known as matched antibody pairs) are used to sandwich the antigen. The signal generated is proportional to the quantity of analyte present within the sample. This method offers the highest sensitivity and specificity since there are two antibodies that are needed to bind the target protein, it can also be used on complex sample matrices. However, this method is more challenging to develop and has a much longer protocol. This procedure is useful in detecting the concentration of analytes present within biological samples.
- Competitive ELISA: This method is mostly used for smaller molecules, where it is not possible to sandwich the two antibodies. This is similar to the sandwich ELISA but instead the conjugate antigen is used to compete with the antigen for binding sites. Therefore, the greater the amount of antigen present then the fewer number of binding sites that will be available for the conjugated antigen to bind. The signal generated is inversely proportional to the amount of protein that is present in the sample. This procedure is ideal for detecting hormones and smaller molecules.
What is a radioimmunoassay?
Radioimmunoassay (RIA) is an immunoassay procedure that can be used to detect antibody and antigen complexes using radioisotopes. It is an in vitro assay that can offer high sensitivity and high specificity (even in minute concentrations). RIA is based on the competitive assay method and it uses a gamma-radioactive isotopes of iodine that is known as Iodine-125 (125-I) to label the antigen. The 125-I isotope can be prepared with high specific activity and it offers 100% isotopic abundance.
The unlabeled antigens from the serum compete with the radioactive antigens for the antibody binding sites. As the quantity of unlabeled antigens increases, then more of the radiolabeled antigens are displaced from the antibody – this will result in reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. At the end of this protocol, the antigens that are bound are separated out and the radiolabeled free antigens within the supernatant are detected using a gamma counter.
Similarities between ELISA and RIA
Some of the similarities that are shared between ELISA and RIA are listed below.
- Both procedures are based on immunoassay techniques.
- Both offer a highly sensitive and specific detection method.
- Both techniques rely on the formation of antibody and antigen complex.
- Both can be used to measure the quantity of unknown protein present within a given sample.
- Both play a critical role in diagnosing various diseases for many different types of organisms.
Difference between ELISA and RIA
Some of the differences that are shared between ELISA and RIA are listed below.
- ELISA detects antibody-antigen complex using enzymes whereas RIA detects antibody-antigen complex using radioisotopes.
- ELISA involves labelling the antibody whereas RIA involves labelling the antigen.
- ELISA assay is less sensitive when compared to RIA assay.
- ELISA procedure does not require a specific laboratory or trained staff whereas RIA will require a specific laboratory area to handle the radioactive material and specially trained staff.
- ELISA does not require special arrangements whereas RIA requires special arrangements for requisition, storage and the disposal of the radioactive material.
- ELISA does not involve the use of any radiation hazards whereas for RIA any radiation hazards will need to be documented and reported.
The use of immunoassays plays a pivotal role within any laboratory that is involved in bioanalytical procedures. Some of the major areas of research include: biopharmaceutical analysis, biosecurity, clinical diagnostics, food testing and environmental monitoring.
The measurement of a particular protein (antigen) is fundamental in the process of diagnosing a disease. Immunoassay methods such as EIA (ELISA) or RIA can provide a simple, quick and cost-effective procedure for analysis. Also the specificity and sensitivity is comparable, if not better than majority of the other conventional methods. The potential to automate, capacity for high throughput, the simultaneous analysis of many samples and the significant reduction in the analytical time has made the use of immunoassay procedures even more popular in recent years.
- ELISA Principle
- ELISA Protocol
- Analysing ELISA Data
- General ELISA FAQ
- ELISA Trouble Shooting FAQ
- ELISA Applications
- ELISA Sample Preparation
- Different ELISA Detection Strategies
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