Traditional vs. Rapid ELISAs

Traditional ELISAs take the best part of a day to complete and often involve a lot of hands-on time. This article looks at some faster, easier alternatives and explains why you should consider using them for your research.

The gold standard for protein quantitation

Enzyme-linked immunosorbent assays (ELISAs) were first described by two independent groups in 1971.^1,2 They are now firmly established as one of the most popular immunoassay techniques due to the many advantages that ELISAs offer. According to Mark Stump, Associate Director, Molecular Assays at Cell Signaling Technology, these include ease of use, cost effectiveness, and the ability to handle any number of samples, as well as the capacity to provide quantitative data. “Today, a sandwich ELISA is considered to be the gold standard for protein quantitation,” he says. “Because a sandwich ELISA has the advantage of using a ‘two factor’ identification, meaning that both antibodies need to recognize the target, it offers highly specific analyte detection—provided the assay is based on well-validated reagents.”

A lengthy, multi-step workflow risks errors

A typical traditional sandwich ELISA workflow involves multiple steps. “First, a capture antibody is immobilized to the bottom of each well of a microtiter plate,” explains Andrew Ball, VP Assay Development Platforms at Abcam. “Next, the plate is blocked before the sample is added and incubated. After washing to remove any unbound material, an enzyme-labeled detector antibody is introduced and the sandwich complex is allowed to form. This is followed by another wash step, then an enzyme substrate is added and color development proceeds until being terminated by the addition of a stop solution. The entire process usually takes between three to five hours.”

“Of all the various steps in an ELISA, the incubations are the most time-consuming, while the wash steps are the most error-prone as they can cause cross-contamination between wells,” comments Stefan Jellbauer, Ph.D., Senior Manager, Product Management Immunoassays, Thermo Fisher Scientific. And the more steps that must be performed, the greater the risk of mistakes. “Each interaction between an operator and an assay creates additional opportunities to introduce error or imprecision,” says Ball. “For example, the more pipetting and washing steps involved, the more likely it is that small errors in precision or data artifacts from residual reagents can compound over time. These will lead to high CVs in the data, confounding statistical analysis and weakening the strength of the results.”

Advantages of rapid ELISAs

Rapid ELISAs, as the name suggests, evolved to provide researchers with a faster alternative to traditional ELISAs. However, speed is not their only advantage. “Beyond their swiftness, rapid ELISAs often incorporate simplified protocols that reduce the reliance on labor-intensive manual steps and minimize the potential for errors,” reports Daniel Austing, ELISA Product Manager at Bio-Techne. “Rapid ELISAs may also require smaller sample volumes, enhancing their suitability for scenarios with limited sample availability, or be designed to prioritize portability and user-friendliness, allowing for on-site testing in diverse settings. Importantly, the efficiency and convenience of rapid ELISAs contribute to streamlined workflows, making them valuable in applications where prompt and reliable results are essential.”

Available options

Off-the-shelf rapid ELISAs generally cut assay times to around just 90 minutes. Included among the available options are Abcam’s SimpleStep ELISA® kits, which currently cover over 1,400 targets and come as 96- and 384-well plate formats. “SimpleStep ELISAs are based on our CaptSure™ technology, wherein the antibody-analyte sandwich complex is formed in solution in a single step and anchored to the plate with an immunoaffinity tag,” explains Ball. “Only one wash step is required to remove any unbound material, reducing both hands-on time and time to results.”

Bio-Techne's ELISA QuicKits are another possibility. “The QuicKit ELISA protocol involves adding standards, samples, and controls to a pre-coated anti-tag microplate, followed by subsequent addition of an antibody cocktail consisting of a tagged capture antibody and an HRP-conjugated detection antibody,” reports Danica Purpur, Product Marketing Manager. “The plate is then washed, substrate solution is added, followed by stop solution, and the plate is read. The standardized components in QuicKits help minimize variability between assays, ensuring more consistent and reliable results.” Bio-Techne also offers an automated ELISA on the Ella™ instrument, which allows for multiplexed detection of up to 8 analytes.

Cell Signaling Technology has two rapid sandwich ELISA product lines: FastScan™ ELISA Kits and PathScan ELISA optimized for Rapid Protocol (PathScan® RP Sandwich ELISA Kits). “FastScan ELISA Kits are a solution-based sandwich assay, where the antibody-target sandwich complex is immobilized to the microplate via a proprietary anti-tag antibody, while PathScan RP ELISA Kits are a solid-phase sandwich assay with a capture antibody for the target protein pre-coated onto the microplate,” says Stump. “Having different ELISA formats allows us to develop kits using the format that our antibodies perform best in, ultimately resulting in better and faster assays for our customers.”

Instant ELISA™ Kits from Thermo Fisher Scientific are a further option. “Instant ELISA™ Kits come with all necessary assay components—including the capture antibody and lyophilized detection antibody, streptavidin-HRP, and sample diluent—already in the plate,” explains Jellbauer. “They also include serially diluted standards in separate strips, allowing all 96 wells to be used for samples. This approach reduces the number of steps to just 7, compared with 17 for a traditional sandwich ELISA, leaving less room for error and increasing the ELISA capacity by 20%.”

A technique that’s here to stay

Despite newer methods being developed during the past 50+ years, ELISA remains a go-to technique for scientific research. Whether you’re a traditionalist or you’re looking to speed up your workflow, there’s a product out there for you!

References

1. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971 Sep;8(9):871-4. 

2. Van Weemen BK, Schuurs AH. Immunoassay using antigen-enzyme conjugates. FEBS Lett. 1971 Jun 24;15(3):232-236.