Antibodies are the immune system's precision tools for recognising and neutralising viruses, bacteria and other foreign substances that can make us ill. These proteins circulate in the bloodstream and are built from chains of amino acids.
Yet pinning down the exact amino acid sequence of an antibody is surprisingly tough.
"If we can establish which antibodies are present and in what amounts, we can answer far more questions about the immune system than we can today. That knowledge could be hugely important for developing vaccines and immunotherapy," says researcher and shared first author Maria Chernigovskaya.
A shared reference for the field
In their new study, Chernigovskaya and colleagues ran a systematic benchmark and created a unique dataset for antibody research. A benchmark test evaluates how well a technology performs: how robust it is under different conditions and where it can be improved.
"Our study delivers the first large-scale benchmark for antibodies, combining several advanced methods to build the dataset," she says.
The result is an open "ground truth" reference that researchers across the field can use to test and refine antibody measurement and sequencing methods.
The study is published in Cell Systems.
Do the measurements match the reference?
The team assessed how accurately different methods could quantify antibodies in a sample and determine their amino acid sequence - in other words, sequence antibodies.
They mixed antibodies in known amounts, ran a high number of analyses, and compared the outputs against the ground truth.
"In an ideal world, our measurements should align with what we know about the dataset - the quantities of antibodies and how they're put together," Chernigovskaya explains.
"At low sample concentrations in particular, our study shows that current methods struggle to measure antibodies reliably," she adds.
Why antibodies pose a challenge
Antibodies are unusually difficult proteins to measure.
"They are highly similar to one another, and they also change continuously, because the immune system assembles them bit by bit. That really stretches the tools we have to study them," says Chernigovskaya.
The researchers combined genomics (which studies genes) and proteomics (which studies proteins), alongside mass spectrometry, to probe different aspects of antibodies.
Cutting antibodies into pieces
To read an antibody's exact amino acid sequence, the team first cut it into small fragments.
"Think of turning antibodies into puzzle pieces with a pair of scissors. We then use advanced methods to examine each piece," says Chernigovskaya.
"We also estimate how much of each fragment is present - which tells us about the abundance of different antibodies," she continues. "Finally, we try to reassemble the original antibody sequence to see the complete 'puzzle'."
What improved the measurements?
After several years of analysis and dataset development, three factors proved particularly important for successful measurements.
"We saw that the concentration of antibodies matters, and that the antibodies should be cut up in many different ways," she explains, and continues:
"Imagine different types of scissors that cut the antibodies in different places and in different patterns."
In addition, it was an advantage to use different computational tools and combine algorithms.
An open resource for researchers
The dataset is freely available. Both experimental and computational communities in academia and industry can use the dataset.
Professor Victor Greiff at the University of Oslo is last author of the study. He believes the dataset has a large potential to contribute to antibody measurement.
"This dataset is meant to be a community resource. If we want mass spectrometry-based antibody measurement to become as reliable and standardized as other omics technologies, we need open benchmarks that everyone can test against - from academic labs to industry," he says, continuing:
"Our hope is that this work helps drive better tools, better reproducibility, and ultimately better vaccines and immunotherapies."
The benchmarking is critically important to develop the field further
Researcher Tuula Nyman, also at the University of Oslo, is expert in the field of proteomics. She points out that mass spectrometry-based proteomics is the only method available for this type of studies of antibodies.
"The benchmarking done in the present study is critically important to develop this field further," Nyman states.
"This study is also a good example of a multidisciplinary study that can be done in our department. Mass spectrometry proteomics is still not always easily available to biological researchers and combining the computational expertise in Greiff's group with immunology and proteomics available at the Department of Immunology at UiO is a unique combination," the researcher, who is head of the Proteomics Core Facility, adds.
Reference
Chernigovskaya, M., Lê Quý, K., Stensland, M., Singh, S., Nelson, R., Yilmaz, M., ... & Greiff, V. (2025). Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases. Cell Systems, 16(11). The article can be accessed here.
