Exploiting a common molecular feature of the Ebola virus, researchers have developed a single-domain antibody-based system derived from llamas to capture and detect it.
“We’re using Ebola virus as a model emerging virus,” said lead author Andrew Hayhurst, an associate scientist at the Texas Biomedical Research Institute in San Antonio. “We think [our approach] could be a way into giving a rapid response to these new pathogens that we’ve yet to see.”
The development of a screening test for a virus normally takes months or even years as researchers immunize rabbits or mice with inactivated or recombinant viral components and then harvest a combination of antibodies.
In contrast, the approach described in the new study draws on a single-pot phage-displayed library of llama antibodies. By isolating the appropriate antibodies this way, a test can be developed in days rather than months, and the same antibody can be used for both capturing the virus and tracing it—driving down cost and labor.
Using one antibody for both capturing and tracing, an approach called monoclonal affinity-reagent sandwich assay (MARSA), isn’t new; it has already been used to detect several disease-relevant oligomers, including amyloid-β protein in Alzheimer’s disease and α-synuclein protein in Parkinson’s. But Hayhurst hasn’t found any other groups applying the approach to emerging viruses.
“Maybe it’s because people assume that viruses are constantly evolving and epitopes are constantly changing, but that’s not necessarily true,” Hayhurst said. The new Ebola virus assay targets a nucleoprotein that is important for viral assembly and therefore highly conserved.
In a biosafety level 4 containment laboratory, Hayhurst’s team exposes purified viruses to a library of a billion phage-displayed single-domain antibodies from llamas. The scientists then elute the phage-displayed antibodies, infect bacterial cells with antibody-displaying phage, and amplify the virus-specific antibody genes. They repeat the process, with each cycle further enriching antibodies specific for the virus.
After that, the researchers immobilize the virus-specific antibody on a plate, incubating it with the sample to be tested. Next, they apply their phage-displayed version of the same antibody, followed by an anti-phage antibody enzyme conjugate and a colorless substrate. If the virus of interest is present, a color change occurs.
Hayhurst developed the group’s single-pot llama antibody library seven years ago, diversifying the contact regions that usually bind antigens. Llamas produce antibodies that have a single-heavy chain and are easy to clone in bacteria. They are also temperature-stable, refolding even after their structure has been melted. That makes them ideal as a portable biosensor in tropical climates, he said.
Alone, one single-domain antibody has a weak affinity for a much larger antigen. But because the virus antigen contains repetitive sequences, many copies of the antibody latch on at the same time, making the signal stronger. As a result the sensitivity of the new assay approaches that of conventional quantitative PCR (qPCR), said Hayhurst.
However, the assay has not yet been tested with patient samples, though that’s the eventual goal. In the meantime, Hayhurst is hopeful that, with further optimization, the new assay could surpass the sensitivity of conventional methods and allow earlier detection of viral infection.
1. L.J. Sherwood and A. Hayhurst. Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation. published online 05 Apr 2013, PLOS ONE