Researchers say they have used a high-sensitivity mass spectrometry technique to improve the accuracy of sandwich ELISA. The team, from biopharmaceutical company Regeneron, hopes their work will help researchers better understand protein-protein interactions during preclinical development of a drug.
“Essentially, we used a mass spectrometry-based method, along with a computational method and interferometry, to identify a protein-protein binding site, geometry, and binding ratio within a short period of time,” explains Yue Su, PhD, a scientist at Regeneron.
Sandwich ELISA is a common technique in the biopharmaceutical industry, Su explains. To quantify antigens in complex samples, an antigen is “sandwiched” between capture and detection antibodies. But, to pair properly, the antibodies need to target different binding sites on the antigen. If the binding is not specific enough, there can be competition for binding sites or unwanted binding, she says.
To check the pharmacokinetics of the company’s antibody drugs, she explains, they use sandwich ELISA, but the team that developed the specific ELISA wanted to make more informed decisions to optimize their assay design, as well as enhance its specificity and sensitivity.
To assist in better defining binding sites, Su’s team decided to use hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful technique for studying protein-protein interaction and dynamics.
“If you imagine an antibody, an area that is bound and [an area that is] non-bound are protected differently. Therefore, [they are] exposed to the surrounding buffer environment differently, and we were able to identify the difference between these areas to investigate the antibody-antibody binding itself,” she explains.
The team combined HDX-MS with computational methods, such as AI, to understand the protein-protein interactions involved when using the sandwich ELISA, she says.
Now they hope that other researchers will adopt HDX-MS to help investigate sandwich ELISA and better understand protein-protein interactions more generally.
“To the best of our knowledge, this is the first time HDX-MS has been used to investigate the binding mechanisms in sandwich ELISA,” she says. “Hopefully, with new computational methods, such as AI, it can help facilitate our understanding of more protein-protein interactions.”
