Factors to Consider for ELISA Development

Although modern-day researchers have access to a vast array of ready-to-use ELISA kits, they may still be required to develop an ELISA in-house—especially where the target analyte is little known or has only recently been discovered. This article shares tips for ELISA development from experts in the field.

A technique offering many advantages

ELISA has become an indispensable method for scientific research due to its sensitivity, ease of use, and compatibility with automation. Another reason for using ELISA is that, unlike many other immunoassay techniques, it provides quantitative results. Of the various formats that have been developed, sandwich ELISA is by far the most popular. Anne Sloan, Ph.D., Technical Scientist at Cell Sciences, suggests that this is because sandwich ELISA generally produces a higher signal to noise ratio than direct or indirect ELISA by using a matched pair of antibodies for target capture and detection. “The increased specificity of sandwich ELISA allows researchers to accurately detect even small amounts of a target analyte in complex matrices such as cell culture supernatant, serum, or other biological fluids,” she says. “This has led to its use for countless research and clinical applications.”

Consider validation from the outset

When developing an ELISA, it is important to think about validation from the outset. “Defining a validation strategy before you begin ELISA development can save both time and money by helping to identify any problems early on,” reports Dr. Tobias Polifke, Co-Founder and Managing Director at CANDOR Bioscience. “This should include determining the number of critical samples required for validation, establishing the method that will be used to evaluate dilutional linearity, and planning spike and recovery experiments for the various sample matrices involved. Parts of such validation experiments can be performed in parallel with assay development, where they may reveal sources of unwanted background signal or highlight a need to identify alternative antibody reagents. This approach greatly reduces the risk of having to go through a new development loop at what was supposedly the end of the development process.”

Tips for success

Every step of an ELISA protocol must be optimized to ensure reliable results. After sample collection and storage, plate coating is one of the earliest steps in the workflow which, for sandwich ELISA, should address the pH-dependent behavior of different antibodies. Sloan recommends comparing two widely used coating buffers—PBS at pH 7.4 and carbonate buffer at pH 9.5—to identify that which supports better antibody attachment to the plate, while Polifke notes that using a coating stabilizer such as Liquid Plate Sealer^® can improve assay reproducibility by preventing antibodies from denaturing as the plates are dried.

Next, plate blocking is essential to avoid off-target binding events. “Although BSA is still the most commonly used protein for surface blocking, numerous experiments have shown it to be inferior to more modern alternatives,” comments Polifke. “This is largely because BSA has a high molecular weight, which makes it difficult to produce a dense layer on the microplate surface. In contrast, newer blocking buffers comprise a wide range of molecular sizes and can yield more consistent results.” To illustrate this point, he points to data comparing the coefficients of variation (%CVs) of plates blocked with BSA and The Blocking Solution, a buffer based on fragmented casein that features modifications to enhance plastic binding. While the background measured in blank samples shows little difference between the two blocking solutions (data not included), %CV tells another story that highlights the importance of statistical validation.

Image: Comparison of %CV between BSA and The Blocking Solution following repeated measurements in human serum. n = 96.

Identifying a matched pair of capture and detection antibodies is key to the success of any sandwich ELISA. Yet, while a growing number of validated antibody pairs is available for purchase, these are not offered for every target. Tillmann Ziegert, Ph.D., CEO of Biorbyt, explains that where a matched antibody pair is unobtainable, performing some basic in silico research can help streamline antibody selection. “Taking the time to check that multiple epitopes are present on a protein analyte or ensure you have the exact location of the epitope recognized by a particular antibody will increase your chances of finding the most suitable reagents for your research,” he says. “Don’t be afraid to engage with suppliers about what you’re trying to achieve and where you’ve failed before. Often, excellent technical support is on offer, one only has to ask.”

Several popular strategies have evolved for testing potential antibody pairs. Sloan comments that these include using a monoclonal antibody for capture and a polyclonal for detection, which can provide signal amplification, and using the same polyclonal for both parts of the antibody sandwich, where the detection antibody is either biotinylated or enzyme-labeled. She also notes that applying a checkerboard layout across multiple plates to test different concentrations of each component is an efficient way of optimizing the signal-to-noise ratio and sensitivity. Polifke adds that diluting samples in a buffer designed to overcome matrix effects (e.g., LowCross Buffer^®) is also useful here since, by filtering out low-affinity binding events, it ensures only high-affinity antibodies make it through to subsequent rounds of testing.

Other considerations for ELISA development include the choice of control material and how it will be used. Ziegert remarks that while recombinant proteins are useful, endogenous protein is the gold standard. He also stresses that controls should be tested in matrices closely matching the diluted sample material to establish how they will perform in the final assay setting.

“Identifying a relevant control and building an understanding of how it behaves is vital to verify assay performance, as well as to direct any troubleshooting that may be required,” he says. “By making sure an ELISA is working as expected before going ahead with testing real samples, researchers can have greater confidence in their results.”

Ten top tips for developing a successful sandwich ELISA

• Perform parts of validation in parallel with optimization to save time, money, and critical sample material
• Compare different coating buffers to identify the best pH for your capture antibody
• Consider using a coating stabilizer to prevent antibody denaturation and increase the shelf-life of coated plates
• Measure both the background signal and %CV of blank samples (diluted in relevant matrix material) when identifying a suitable blocking buffer
• Choose matched antibody pairs, if available
• Use a checkerboard layout across multiple plates to streamline optimization
• Think about diluting samples in a buffer that overcomes matrix effects to ensure only high affinity antibodies are selected for further optimization
• Choose endogenous protein over recombinant material as the control wherever possible and compare control data to the International Standard, if available
• Remember to optimize factors such as incubation times, temperatures, and whether there is a need for plate shaking
• Don’t be afraid to speak with suppliers for help with assay design