An ELISA (enzyme-linked immunosorbent assay) is a popular technique for the quantification and detection of proteins, specifically antibodies. ELISA can quantify proteins, which is the reason that this method may be chosen instead of qualitative techniques, for example, western blotting.
The most basic steps in a typical ELISA are:
- The sample’s antigens are bound to a solid surface
- An antibody that will bind to the antigen is introduced. The antibody is connected to an enzyme.
- The substrate of the enzyme is introduced, which results in a reaction that displays an identifiable result (a color change for example).
ELISAs are comparatively simple, cost-effective to perform, and can also be automated. They are a frequently utilized method for this reason and will be known to the majority of researchers.
Similar to any common method, there are risks related to complacency through familiarity. In all laboratory techniques, some factors can go wrong. There are equally as many ways to ensure that the best possible results are achieved.
A range of competitive and sandwich ELISA kits are offered at St John’s Laboratory. More than 500 ready-to-go ELISA kits are available, including competitive and sandwich ELISAs. A researcher’s first step may be to check whether one of these kits is suited to their application.
Whether the plan is to employ a kit or produce an ELISA in-house, this article provides a few tips to assist any researcher in getting the most out of their technique.
1. Choose the Antibody Carefully
In an ELISA, the identification of the target protein is reliant upon the particular antibody-antigen interaction. The results should be better the more specific the binding is. It is essential to select the antibody carefully.
It is also essential to select an antibody with a high affinity for the antigen which should help to decrease non-specific binding.
2. Check the Antibody Concentration
If an application requires the development and optimization of an ELISA produced in-house, antibody titers must be performed to determine the concentration that creates the highest degree of specific binding.
If a secondary or detection antibody is used, this can additionally be titered with the capture antibody to determine the greatest ratio of signal-to-noise.
3. Be Consistent - It is about Accuracy and Precision
The standardization and consistency of the procedure is incredibly important with any assay. A ready-to-go ELISA kit should have been optimized and standardized for lot-to-lot consistency, as well as sensitivity, specificity and more.
Despite this, it is still important to attain consistency when utilizing the kits. Reading and adhering to the recommended process every time a kit is employed can help with this. Mixing components from various kits should also be avoided to attain consistency.
Whether a kit is employed or an ELISA is developed in-house, maintaining consistency in conditions such as humidity and temperature, and adhering to the same protocol every time, will help to enhance the reproducibility of the results.
The protocol should be checked first if results are inconsistent from one assay to the next. Varying temperatures throughout incubation, inadequate washing, or incorrect dilutions could also generate inconsistent results.
4. Wash Carefully with Buffer
The plate should be washed between each step of an ELISA to take away any non-specifically bound proteins. This washing is one of the elements that make an ELISA such an effective technique. Inadequate washing can result in problems ranging from inconsistent replicates to too much background.
If a kit is being utilized, ensure that the manufacturer’s washing guidelines are followed, including how many times the process must be repeated.
If the ELISA is being produced, pay attention to the washing step when protocol is established. Selecting an effective washing buffer from the range that is available is critical; the right selection can help to maximize signal and decrease background.
These are only some of the elements that could assist in getting the most out of any ELISA. Along with the ELISA kits offered by St. John’s Laboratory, its 30,000-strong catalog comprises of several antibodies appropriate for use in ELISA.
Look at the catalog to discover the range of applications and the validation status of St. John’s Laboratory’s antibodies.
About St John's Laboratory
St John’s Laboratory is your platform for purchasing commercially-validated and peer-reviewed primary and secondary antibodies.
Once established in 2013, St John's Labs knew that antibody validation was rapidly becoming a widespread concern for bioscience. In acknowledgement of these concerns, they invited a collective of industry experts, and business leaders to join the world’s First Antibody Validation Forum in 2014. See clips of the discussions which took place on their blog pages, or YouTube channel.
St John’s Laboratory believes the best way they can guarantee satisfaction in our products is through the provision of up to date testing data, as well as independent assessment - and thereby inviting all of their customers to take part in their Antibody Validation Project, where St John's Labs offer one of the greatest supplies of trial size antibodies available on the market.
St John’s Laboratory's vision is to give researcher partners as much control over their work as possible, by providing real up to date reviews and testing information. They encourage exploration of antibody specificity, species cross-reactivity and applications testing. They work alongside customers to carry out performance tests, so you can be assured that quality is the top of their agenda.
With St John's Laboratory platform, come and explore the performance of your required antibody before making a purchase and they’ll work to help you reduce the risk on your research, and save on essential research funds.
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