The enzyme-linked immunosorbent assay (ELISA) is a common biochemistry tool that is used to detect a specific ligand in a liquid sample. The basic principle involves immobilization of the ligand on a 96-well polystyrene plate and its subsequent detection using an antibody. We’ve spoken with experts from Rockland Immunochemicals, Expedeon, Boster Bio, and RayBiotech to learn more about four of the most common ELISA types.
Direct detection ELISA
The simplest variation of the ELISA strategy is direct detection. For this method, you first pour the sample containing the antigen of interest onto the plate and allow it to nonspecifically adhere to the surface. Next, you put in an antibody conjugated to a detection enzyme (e.g., HRP or AP) and allow it to bind the antigen. Lastly, you add the enzyme substrate, which produces a colorimetric signal. This signal reveals which wells contain the enzyme—and thus contain the antigen of interest.
“A direct ELISA has the advantage of being the quickest format since it involves only one antibody, avoiding any cross-reactivity issues with a secondary antibody,” say Georgia Sfyroera, Ph.D., quality control manager, and Yu-Hung Huang, Ph.D., R&D senior scientist, at Rockland Immunochemicals.
But of course, every advantage comes with a disadvantage. “Direct ELISAs have higher background compared to indirect ELISAs,” says Isabella Pupita, product manager from Expedeon, “and they have lower sensitivity, as secondary antibodies are not used.”
The direct detection method is “extensively applied for the titration of pure targets,” according to Sfyroera and Huang, as samples of high purity are able to directly coat the polystyrene plate without other antigens getting in the way. Pupita adds that this strategy can also be used to verify the presence of antibodies in a sample and is often utilized to diagnose infections and allergies.
Indirect detection ELISA
Indirect detection starts out the same way as direct detection. You pour the sample containing the antigen of interest onto the plate and then add the primary antibody after that. The difference is that, in this case, the primary antibody is unlabeled. Instead, you add a labeled secondary antibody. Lastly, you add the enzyme substrate, revealing which wells contain the antigen of interest.
More than one secondary antibody can bind to a single primary antibody. Therefore, the addition of secondary antibody to this strategy increases the assay’s sensitivity by amplifying the signal. “This ensures that lower concentrations of the target will be better detected than with a traditional direct ELISA,” Sfyroera and Huang explain.
“Furthermore,” Pupita adds, “the same secondary antibody can be used with different primary antibodies, reducing cost and improving flexibility.”
But like the direct assay, there are tradeoffs to these benefits. “A great disadvantage of indirect ELISAs is the cross-reactivity of the secondary antibody towards different primary antibody species or endogenous proteins, which increases the background signal,” says Pupita. “The use of secondary antibodies also requires more incubation and washing steps, making the assay more prone to errors.”
Generally speaking, the indirect detection method is used for the same types of samples as direct detection. “The direct detection ELISA is more convenient, while the indirect detection ELISA is more sensitive,” says Sijie Xia, VP of marketing and sales, from Boster Bio. “If the assay requires extra sensitivity, use the latter, and if not, use the former.”
Sandwich ELISA
Most commercially available ELISA kits use the sandwich ELISA strategy, which uses two different antibodies to detect an antigen as opposed to just one. In a sandwich ELISA, you first coat the polystyrene plate with a capture antibody. Next, you pour in the sample, allowing the antigen of interest to bind the capture antibody. Lastly, you add the other antibody and allow it to bind the antigen. The detection method can be either direct or indirect, depending on whether or not you use a secondary antibody.
“A sandwich ELISA offers higher specificity, as a pair of antibodies is required,” say Sfyroera and Huang. “For this reason, it’s highly recommended for the detection of molecules in complex samples, such as serum, urine, lysates, supernatants, etc. Using a pair of antibodies ensures the capture and the detection of the target from a matrix of other proteins.”
“However,” Pupita adds, “the antibody pair (if not readily available) could require optimization to exclude cross-reactivity between the capture and detection antibodies.”
Another disadvantage of the sandwich assay is that it isn’t suitable for just any antigen. “The sandwich ELISA is fitted for only large molecules that can be bound by more than two antibodies,” says Xia.
Competitive ELISA
When the antigen is small or has limited epitopes, competitive assays are an option. “Competitive ELISAs have the greatest flexibility, as they can be adapted to any of the assay formats mentioned above,” Pupita says.
In one basic variation of the assay, you coat the plate with the capture antibody and then add (1) the sample and (2) purified antigen that is conjugated to a detection enzyme. The purified antigen will compete with the sample antigen for binding with the capture antibodies, meaning that the lower the signal, the higher the antigen concentration in the sample.
“The competitive ELISA allows a direct measurement for the degree of biomolecule interference with multiple concentrations,” Sfyrorea and Huang explain. “Even though the ELISA does not have the robust output of a biosensor experiment, it still offers informative results that can be used for more detailed studies.”
“Also,” Pupita adds, “competitive ELISAs are less sensitive to sample dilution and matrix effects and are less prone to variability among duplicates, making the assay robust and consistent.”
An alternative to the sandwich and competitive ELISAs is the relatively rare cell-based ELISA, which compares the levels of a premeasured housekeeping gene (such as GAPDH) to the target protein inside the cell. “Please note that the cell-based format is not quantitative and can only be used to make qualitative measurements,” says Xia, “as we do not know how much housekeeping gene protein is in the matrix to start with.”
Due diligence
Before you purchase, “Consider the sensitivity, the reproducibility, and the accuracy of the kit,” Sfyroera and Huang recommend. “A careful look at the quality control data that accompanies the kit will ensure that the kit meets industry standard criteria.”
Unfortunately, this is a real concern. Some disreputable companies prey on customers who are desperate to find a kit that works for them.
“There are companies that claim to have tens of thousands of ELISA kits against pretty much everything in the proteome,” says Xia. “They then sell these ELISA kits that claim to detect the analyte but really detect something like TGF-beta.”
The situation has been reported in the academic literature, but Xia believes these articles aren’t getting enough press. Here’s an example of a paper reporting a false positive ELISA kit.
Specialized ELISA Products
Rockland Immunochemicals: BioQuantiPro CHO-HCP ELISA
BioQuantiPro™ was designed as a tool to support in-process testing of biopharmaceuticals during manufacturing. It detects HCP contaminants in crude cellular harvests, cell culture lysates, process intermediates, etc.
According to Sfyrorea and Huang, “Rockland also offers custom solutions to produce fit-for-purpose antibodies and robust immunoassays that can resolve antibody performance issues.”
Expedeon: CaptSure DIY ELISA
Using a combination of their CaptSure™ and LightningLink® technologies, Expedeon has developed a quick and easy method to rapidly develop in-house ELISAs, eliminating the need to perform expensive and tedious plate-coating procedures.
“Unlike standard ELISA systems, the CaptSure DIY ELISA kit has a ready-to-go assay plate, pre-coated with the CaptSure antibody, which is specific for the CaptSure peptide,” Pupita explains. “The LightningLink technology, supplied with the kit, enables users to quickly and easily conjugate their capture antibody to the CaptSure peptide and their detection antibody to HRP.”
Boster Bio: Plate-based Multiplex ELISA
Boster Bio’s multiplex ELISA kit can measure up to 18 analytes at once using as little as 25 microliters of sample, and the cost per analyte is as low as 30% of what it would be in a standard singleplex or bead-based multiplex assay, according to the company.
“In addition to our pre-made kits, you can pick and choose analytes from our large panel of prevalidated antibodies, and we will assemble and validate the assay for you free of charge. That way, you won’t have to spend the time calibrating your own multiplex assay, which could delay your research,” Xie says. “Additionally, we offer demo packages to validate the multiplex platform with your samples.”
RayBiotech: Immuno-PCR Ultrasensitive ELISA - IQELISA
IQELISA™ is an ELISA with an ultrasensitive detection strategy involving DNA amplification. The technique uses an oligonucleotide-linked antibody, PCR primers, and PCR master mix—allowing the researcher to quantify the product by real-time PCR. The method enhances detection sensitivity, especially in the lower detection limit.
“The robustness of the PCR detection method allows the researcher to easily convert an existing sandwich ELISA into an immune-qPCR-based ELISA, provided the detection antibody uses a biotin-streptavidin linkage,” says Valerie Jones, director, marketing and sales, from RayBiotech. “By incorporating an affordable reagent kit and a real-time PCR instrument, you can achieve immediate enhancement of sensitivity.”