The enzyme-linked immunosorbent assay, better known as the ELISA, is a highly popular method for detecting and quantifying a target analyte within a sample. Although, as the name suggests, early ELISAs were dependent on the use of an enzyme-linked antibody to generate a signal, the technique has evolved significantly and alternative readouts are now available. Accordingly, the term ELISA is now broadly used to describe any plate-based immunoassay within which a component of the assay is adsorbed onto a solid surface. ELISA offers a multitude of advantages including high sensitivity and a quantitative readout; furthermore it is relatively simple to perform and is easily adapted to high-throughput screening, features that often make it the platform of choice for drug discovery programs.
ELISA offers a multitude of advantages including high sensitivity and a quantitative readout…
An ELISA can be configured in several different ways, but irrespective of the chosen method the assay will involve certain key steps, namely immobilization of the capture antibody or antigen on the microplate, blocking to prevent nonspecific binding, incubation with the test sample, and further incubations with relevant detection reagents. Wash steps are included throughout the process and, like every stage of the assay, should be carefully optimized to ensure that the readout is maximized while background signal is kept to a minimum. During the development of an ELISA, researchers will compare various parameters to identify those that generate an optimal assay window, and consideration should be given to the following:
- Plate coating—choice of coating buffer, concentration of capture reagent, incubation time and temperature
- Washing—choice of wash buffer; number, duration and volume of washes
- Blocking—nature of blocking reagent, concentration of blocking reagent, incubation time and temperature
- Sample incubation—sample volume, inclusion of relevant controls and standards, incubation time and temperature
- Detection method—colorimetric, fluorometric or chemiluminescent readout, requirement for multiplexing, use of biotin-streptavidin binding to boost signal
Successful and reproducible ELISAs depend on quality antibody reagents that exhibit high sensitivity and specificity, and Rockland Immunochemicals offers an extensive portfolio of immunoassay specific antibody products.
Camilo Moncada, director of custom research at Rockland, explains that “while an antibody can strongly recognize its target in a Western blot assay, this reactivity may not translate to a similar result in an ELISA. Rockland develops fit-for-purpose antibodies, and to assure that these function in the desired assay they need to be validated in the assay that they were designed for. Rockland’s antibody validation workflow includes extensive tests in various applications such as ELISA, IHC, and Western blot, giving us the ability to provide recommended antibody and assay specific protocols that help minimize optimization efforts for the end user.”
Lot-to-lot consistency and reproducible performance is guaranteed by applying strict release criteria for the final product. Important technical optimization steps in ELISA involve the determination of ideal concentrations for key assay components including antigens and antibodies, and optimization can, for example, be achieved by checkboard titrations testing various components at several concentrations.
“During the ELISA optimization process, the analyte should be in the same (or similar) matrix as that of the intended final assay. Moreover, negative matrix effects altering the performance of antibodies or properties of the antigen can be observed and therefore further matrix dilution might be required” adds Georgia Sfyroera, senior assay development scientist at Rockland.
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“One of the most common problems in ELISA is low signal-to-noise ratio and high background. In this case, screening several blocking and dilution buffers will help identify those with the best compatibility with the antibodies and samples being used.” Rockland’s catalog includes ELISA buffers and other supporting reagents used in the antibody validation and characterization process, offering the end user a reduced need for optimization and ensuring reproducibility between pre-release validation and application.
In situations where a researcher has already identified antibodies that show good specificity and selectivity for their target antigen, albeit in an alternative application, they may wish to evaluate those same reagents for their suitability for an ELISA.
“The FlexLISA® kits from Innova Biosciences, have been designed for quick and easy development and optimization of sandwich ELISA assays, and can be used to detect the presence of any antigen with a high affinity for any antibody pair in complex samples such as serum, plasma, or urine,” explains Giovanna Cacciola, senior scientist at Innova.
“FlexLISA relies on our Lightning-Link® antibody labeling technology, enabling you to conjugate your capture antibody to streptavidin and your detection antibody to HRP or alkaline phosphatase with just 30 seconds hands-on time. Each kit includes a microplate that has been pre-coated with biotin, and by exploiting the biotin-streptavidin interaction it’s possible to use up to 40 times less capture antibody, resulting in significant cost savings.” Cacciola adds that “with FlexLISA you can use antibodies of your choice, and since each kit allows you to conjugate up to three capture and three detection antibodies simultaneously, FlexLISA is ideal for screening different antibody combinations.”
The FlexLISA® reaction process.
While many researchers like to construct their own ELISA from the individual antibody components, a popular alternative is to purchase a ready-to-use kit such as those offered by Eagle Biosciences.
According to Dan Keefe, president of Eagle Biosciences, the use of pre-optimized kits can save significant amounts of time that would otherwise be spent on assay development. “Our ELISA kits are provided with detailed information that includes sensitivity, specificity, dynamic range, and suitable sample types, as well as typical standard curve data and a comprehensive protocol, giving our customers high levels of confidence in their sample analysis and enabling them to progress their research efficiently,” Keefe explains.
Standard curve data from Eagle Biosciences. The 25-OH Vitamin D ELISA assay test kit is designed for the determination of 25-OH Vitamin D in human serum or plasma samples.
Aviva Systems Biology recently launched a range of ELISA kits, enabling quantitative measurement of high-interest targets including cytokines, chemokines, growth factors, biogenic amines, serum/plasma proteins, and markers of infectious disease. Aviva’s ELISA catalog currently caters to over 8,000 assay targets, through a variety of assay types including sandwich, indirect and competitive ELISA formats, as well as in-cell assays.
According to Matt Landry, vice president of marketing and sales at Aviva, “a well-developed ELISA assay will be robust enough that small variations in assay processes will have minimal effect on results. An extensive optimization process has been carried out by our highly specialized team, saving our clients time and money, and providing them with a convenient, easy-to-use product. Our ELISA kits are supplied with fully validated protocols, optimized reagent sets, and comprehensive technical support to ensure guaranteed results and meet high-quality specifications for sensitivity and reproducibility.”
ELISA is an extremely powerful technique, applicable to an almost limitless range of targets, however it is essential to carefully optimize each stage of the assay, employ appropriate high-quality reagents, and follow consistent assay methods to achieve reproducibility. This affords reliable and accurate data to support the smooth progression of research programs.