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Texas Biomed Researchers Develop Method to Quickly ID ELISA Antibody Pairs

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Researchers at the Texas Biomedical Research Institute have developed a simplified workflow that could dramatically shorten the time required to discover and implement new immunoassays.

According to Andrew Hayhurst, a Texas Biomed researcher and leader of the project, the technique can identify suitable antibody pairs for ELISAs in under an hour, making it an ideal rapid assay development process for purposes like pathogen or biothreat detection.

Detailed in a paper to be published in Nature Scientific Reports, the technique relies on site-specific biotinylation of antibodies to enable quick identification of appropriate antibody pairs.

Conventional assay development is complicated by the need to distinguish the capture antibody – which initially binds the analyte of interest – from the tracer antibody – which binds to the analyte once it has bound to the capture agent, and signals this binding event via, for instance, a conjugated fluorophore.

"For pairing usually you need to chemically immobilize one antibody, the capture, and then you need to modify the tracer by conjugating it to a [secondary tracer like a] fluorophore or an enzyme, and somehow distinguishing it so this secondary tracer doesn't bind to the capture antibody," Hayhurst told ProteoMonitor.

This typically has meant separating these two antibodies and then taking them through two different preparation pathways, which can prove tedious and time-consuming for researchers looking to identify good pairs from large repertoires of antibodies.

"If you've only got one or two antibodies, that's fine," Hayhurst said. "But when you start harvesting repertoires from llamas, for instance, that have been immunized with multiple antigens, you end up with hundreds of antibodies ... and this is a real burden."

He cited a previous project by his team in which they developed ELISAs to the seven different botulinum neurotoxin serotypes, drawing on a pool of 130 single-domain antibodies. Done by conventional methods, that process took them over a year. Hayhurst estimated they could bring that down to as little as one week using the site-specific biotinylation method.

Essentially, the wells of an ELISA plate are coated with neutravidin. The biotinylated capture antibodies are then applied to the plate, with the antibodies' biotin moiety binding to the neutravidin in the plate well. The analyte of interest is then added, followed by the tracer antibody, which also contains a biotin moiety. Then neutravidin charged with horseradish peroxidase is added as the secondary tracer, enabling readout of the reaction when it attaches to the unbound biotin in the tracer antibody.

The key, Hayhurst noted, is that the biotin in the capture antibody is securely bound to the neutravidin in the ELISA plate, meaning that it won't bind to the neutravidin-horseradish peroxidase tracer when it is added.

"We can harvest these antibodies from just a few milliliters of culture, with no purification, and just go straight into the assay – a done deal," he said.

Hayhurst cautioned that the result was not an assay refined enough to serve as a commercial end product, but rather a "stop-gap" assay for use either in time-constrained situations or as an upfront step to identify antibody pairs worth pursuing in a more rigorous fashion.

"I think if we were to take this kind of [assay] to market, it wouldn't be possible," he said. "People would say, 'Well, we need purified components.'"

Hayhurst noted that the method allows for large-scale expression of the antibodies to enable purification. But he envisions it would be most useful as a tool for selecting which pairs to purify in the first place.

Hayhurst and co-author Laura Sherwood undertook the project with the rapid development of assays against bioterror threats in mind, and used the method to develop a test for Zaire ebolavirus in a matter of days. The process could be sped up further by better automation, Hayhurst said.

"You can imagine that people with robotic capabilities could ram through huge panels of antibodies" using the technique, he said.

Beyond the method's usefulness for rapid pathogen and bioterror test building, Hayhurst said he sees two main uses for the technology. The first is small academic labs like his who have large panels of antibodies they need to run through but limited resources to do so.

"I think it's going to help a lot of small investigators without the resources for scaling up all of these purifications and such," he said. "For example, if you immunize a mouse with carcinoembryonic antigen and you're looking for single-chain [antibodies] against that cancer marker, to plow through all of these and put all of your efforts onto each and every antibody is going to be a hell of a chore. So I think for any antigen of choice this technique could really have an impact," in terms of helping small researchers prioritize which reagents to pursue.

The second is in "industrial concerns that have these huge single-pot libraries that can pull out thousands of different [potential] binders for a single protein and might be looking for a way of speeding up the process of pairing them," he added.

Hayhurst said that the researchers have patented the technique and are interested in commercialization opportunities but are not currently in talks with any specific parties.

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