ELISA: Principle and Protocol
Enzyme-linked immunosorbent Assay (ELISA) relies on the principle of antigen-antibody binding and its detection using a suitable enzyme. It is a plate-based immunoassay that detects and quantifies proteins, antibodies, hormones, etc., in biological samples. Since antibodies are produced by the immune system in response to pathogens, ELISA can help detect infectious diseases such as Ebola, Rotavirus, AIDS, Lyme disease, Syphilis, and Zika virus. ELISA can be of four major types: direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA, based on the method used for binding antigens and antibodies.
The ELISA assay has one of the simplest protocols involving a few steps. First, the microtiter plate wells are coated with a capture antibody (in sandwich or direct type) or the antigen (in competitive or indirect type). After washing the unbound antigen or antibody, the wells are blocked with a blocking buffer to prevent non-specific binding. The plate is then washed to remove the blocking buffer, followed by adding the sample, standards, and controls, as needed, in the wells. After incubation, the microtiter plates are washed to remove unbound molecules, followed by the addition of a detection antibody. After washing away the unbound antibody, a suitable substrate is added to facilitate the colorimetric detection of the antigen-antibody binding.
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Advantages of ELISA
Each type of ELISA has its advantages over the other types. However, ELISA as a technique has several advantages over other bioanalytical methods. It is a rapid test that generates results quickly. It yields an accurate diagnosis of diseases since it uses two antibodies. Antigens in samples do not need to be purified and can be detected directly in complex samples. It allows qualitative, quantitative, and semi-quantitative detection. It has a simple and uncomplicated protocol compared to other assays. It is a cost-effective and versatile technique that can be used in different fields, including the diagnosis of diseases, drug development, and environmental monitoring.
Owing to its diverse applications, ELISA is a routine experiment in bioanalytical laboratories. Various institutes and bioanalytical CROs offer ELISA assay development services and other Bioanalytical Lab Services that boost the drug development pipelines of pharmaceutical companies, medical device manufacturers, and various research organizations.
ELISA Assay Development: Best Practices
ELISA assay development involves meticulous planning and execution at each stage. As discussed earlier, ELISA method development requires a researcher to choose the most appropriate type of ELISA format guided by the target analyte and the experimental goals. For example, a direct ELISA is suitable for rapid antigen testing, while the indirect type is better suited for quantifying antibody levels in a sample. Thereafter, ELISA development involves optimization of the antibodies, namely, capture and detection antibodies. It is crucial to optimize the concentration of the capture antibody to achieve a strong signal that can be distinguished from background signals. The detection antibody must have a high affinity towards the antigen, and its concentration should be such that the signal produced can be easily detected.
ELISA assay development involves blocking buffers to prevent non-specific binding that can otherwise interfere with the output signal generated. Different blocking buffers of different concentrations should be evaluated to identify the optimized buffer and its concentration to obtain the maximum signal-to-noise ratio. ELISA development requires diluents that mimic the sample matrix as closely as possible to achieve good sample dilution linearity and a standard curve dynamic range. To obtain a standard curve with an acceptable dynamic range, it is crucial to use two to three-fold dilutions of the standard. Notably, dilutions that require small volumes of diluents should be avoided to minimize pipetting errors and inaccuracies. Each standard dilution should be measured in duplicate or triplicate for improved accuracy. Another crucial aspect of ELISA method development is substrate choice. The substrate chosen should be suitable for the target antigen and be sensitive enough to detect very low concentrations of the target antigens.
Tips for Developing ELISA with High Sensitivity and Specificity
In addition to the best practices elaborated above, a few aspects need consideration in ELISA Assay Development. These include the washing procedure, pipetting techniques, control wells, avoiding contamination, and methods to improve the consistency and reproducibility of the assay. The ELISA assay involves washing at different stages. A thorough washing helps ensure the removal of unbound reagents that can show non-specific binding, which contributes to the background signal. Using a plate washer can help to achieve consistent results.
In addition to the sample and standard, researchers can include control wells that help to ascertain the proper functioning of the assay and help prevent false positives and negatives in the results. Pipetting can be a common source of error in assays, leading to inaccurate and inconsistent results. A few tips to remember while pipetting are: changing tips between different samples to avoid contamination, holding the pipette at an angle to prevent the pipette tip from touching the bottom of the microtiter plate well, and using a multichannel pipette can help to enhance speed and consistency in pipetting. Researchers need to use consistent methods for sample extraction and processing. Samples should not be subjected to multiple freeze-thaw cycles to prevent their degradation. Lastly, conducting replicate experiments can help to ensure the reproducibility of results.
