ELISA Detection Strategies

When ELISA was first described in the literature, back in 1971, it used antibody-coated beads to capture an enzyme-labeled antigen from solution, and had a laborious manual workflow. ELISA has since become a plate-based, fully automatable technique, employing multiple detection strategies—each with its own advantages and disadvantages.

Choosing the right format

Modern, plate-based ELISAs can be configured in one of four main ways. “In a direct ELISA, the plate is coated with the antigen and a labeled primary antibody is used for detection,” explains CJ Xia, VP of Marketing and Sales at Boster Bio. “An indirect ELISA is similar but combines an unlabeled primary antibody with a labeled secondary antibody, which can provide signal amplification. During sandwich ELISA, the plate is coated with an antigen-specific antibody, then a different antigen-specific antibody is used to detect the bound antigen. All of these assays can be converted to a competitive ELISA, in which the sample antigen competes with a labeled reference antigen for antibody binding.”

Deciding between the different types of ELISA largely comes down to the aims of your research. Direct ELISA is often used for monitoring antibody: antigen interactions, such as during allergy testing or for checking antibody cross-reactivities, while indirect ELISA is well suited to determining the total antibody concentration in a sample. Sandwich ELISA is useful for analyzing complex sample types such as plasma or serum due to its high specificity and sensitivity, whereas competitive ELISA provides a means of studying small antigens with only one epitope available for antibody binding.

Fredrik Sundberg, Global Director Strategic Technology Partnerships and Customer Relations at Cytiva, reports that sandwich ELISA is generally the preferred format. “While sandwich ELISA often needs more optimization to identify matched antibody pairs and ensure there is minimal cross-reactivity between capture and detection antibodies, it has far greater specificity than ELISA formats that use just one type of antibody for antigen recognition,” he says. “For this reason, sandwich ELISA is popular for applications spanning early-stage drug discovery research through to biopharmaceutical manufacturing and clinical testing.”

Qualitative or quantitative analysis?

In addition to selecting a suitable ELISA format, researchers should consider the type of data output required. Vanitha Margan, Global Product Manager for Immunoassays at Bio-Rad Laboratories, notes that ELISA results can be qualitative, semi-quantitative, or quantitative. “Qualitative interpretation of ELISA data simply involves determining whether the antigen is present in a sample by comparison to a blank well that does not contain the antigen of interest—it’s a yes or no answer,” she says. “Semi-quantitative analysis instead provides a relative comparison of the antigen levels in different samples within an assay since the intensity of signal will vary directly with antigen concentration. During quantitative analysis, a known concentration of the antigen is serially diluted to produce a sigmoidal standard curve, then the concentration of unknown samples is calculated by comparison to the linear region of the curve.”

Depending on the nature of your research, you might wish to perform a qualitative or semi-quantitative ELISA first, before switching to a quantitative method. For example, a qualitative ELISA could help with identifying an appropriate sample type for compound screening purposes, then a quantitative ELISA could be used for ranking potential small molecule drug candidates based on their activity in the chosen sample material.

Selecting a readout

The ELISA readout is another important factor to consider. “In a research setting, colorimetric detection is the most common since assay development is relatively straightforward and there is no need for specialized instrumentation,” comments Xia. “However, when high sensitivity is required, detection methods based on chemiluminescence, electric signal, or qPCR can increase the sensitivity by as much as 1,000-fold.” Multiplexed ELISAs typically use either chemiluminescent or fluorescent detection when plate-based, and fluorescence for bead-based applications such as Luminex xMAP^® Technology.

When selecting a readout, it is critical to accommodate any unique characteristics of the chosen method. For example, fluorescence-based ELISAs require the use of black microplates to eliminate crosstalk, while chemiluminescent ELISAs must address the issue of substrate exhaustion. “During chemiluminescent detection, light emission occurs only during the enzyme-substrate reaction,” explains Sundberg. “Therefore, the signal ceases when the substrate becomes exhausted. If running large numbers of plates, researchers should implement measures to prevent signal decay prior to reading.” For ELISAs using colorimetric detection, adding a stop solution at a defined time point will prevent over-development of the reaction.

To multiplex or not to multiplex?

Another key decision is whether to multiplex an ELISA. “For researchers who have limited sample volume and time, and who want to measure several protein targets in the same well, multiplexing is the way to go,” says Margan. “To put this into context, measuring 48 cytokines in 38 samples would require 48 96-well plates, greater than 106 hours, and more than 1 mL of each sample for a conventional ELISA. On the other hand, using the Bio-Plex Multiplex System—a bead-based platform based on Luminex xMAP^® Technology—the same researcher only needs one 96-well plate, 3 hours, and as little as 12.5 µL sample.”

Comparison of Bio-Plex Multiplexing vs conventional ELISA. Image from Bio-Rad.

“Multiplex ELISAs can be especially valuable tools for discovery R&D, where it is often more informative to measure several proteins of interest early on,” comments Sundberg. “Yet, because targets are often present in sample material at very different concentrations, linearity over a wide range is essential for reliable data.”

Off-the-shelf or do-it-yourself?

A final factor to consider when deciding on an ELISA detection strategy is whether to purchase an off-the-shelf kit or to develop the assay in-house. For singleplex detection, it is highly likely that a commercial kit will be available, but Xia cautions against buying the first product that comes up in an online search. “When you need an ELISA kit, and you simply cannot find a legitimate company that you know offers it, don’t be desperate and buy from companies claiming to have kits against more than 10,000 targets in their portfolio,” he says. “Anyone who has developed an ELISA would know that to come up with so many products is impossible. At least if you do decide to risk your $700–$1000 on such a kit, perform a western blot using the detection antibody in the kit and make sure the band shows up at the right molecular weight. Do not risk your paper being pulled at a later date.”

If you choose to go down the DIY route, you may wish to consider outsourcing part of the process. “Developing an ELISA is labor-intensive and requires expertise,” reports Sundberg. “Consequently, there is a growing trend toward outsourcing primary and secondary antibody production, or even the entire development process, to an established supplier or service partner. If you need a process-specific ELISA, such as to test for process- and product-replated impurities like host cell protein or Protein A during later clinical stages, outsourcing ELISA development can be especially beneficial. Not only will it ensure higher specificity and sensitivity compared to using an off-the-shelf product, but it also safeguards long-term supply, which may be required for 10 years or even longer.”