A team led by researchers at the Massachusetts Institute of Technology and Harvard Medical School have developed a synthetic biomarker assay that they say could improve early detection and monitoring of a variety of diseases.
In particular, the approach, which uses immunoassays run on inexpensive paper test strips as its read-out, could prove valuable in point-of-care and resource-constrained settings, Andrew Warren, an MIT researcher and one of the developers of the method, told ProteoMonitor.
Detailed in a paper published this week in Proceedings of the National Academy of Sciences, the method uses ligand-encoded reporters linked to nanoparticles by synthetic peptide substrates. Upon injection into a patient, these conjugates are targeted via the nanoparticles to the site of interest, where native proteases linked to the disease process under investigation cleave the peptide substrate, freeing the reporter molecules.
These reporters are then collected in urine samples and detected by immunoassay. By measuring them, the researchers are able to observe differences in protease activity between healthy and diseased subjects and identify signatures indicative of disease.
By using synthetic biomarkers, Warren and his colleagues hope to get around limitations – low abundance, in particular – inherent in endogenous protein markers. Because the researchers can control the level at which they introduce the synthetic markers, they can make certain they are abundant enough for easy detection. This makes them potentially useful in the case of ailments where existing markers are present only at low levels, or for which there are no known protein markers.
The PNAS paper follows on a study by the group published last year in Nature Biotechnology in which they used mass spec to detect the reporter molecules. By moving to immunoassay, the researchers aim to position the technology for use in point-of-care settings and developing countries where clinicians may not have access to mass spec, Warren said.
"This [represents] a modification so that the [synthetic markers] will work with a much simpler, more versatile system," he said. "Immunoassays are used throughout the clinic and there are many, many incarnations of them, so that's why we think this is an exciting technology," he said.
To convert the test to an immunoassay format, the MIT-Harvard team reformulated the reporters to include ligands specific to particular antibodies. They used for their analysis paper test strip immunoassays similar to those used in home pregnancy tests.
With the original mass spec version of the assay, the researchers looked at liver fibrosis and early stage colorectal cancer, finding in mouse studies that they could detect the former with an area under the curve of .91 and the latter with an AUC of .89.
In the PNAS study, they looked again at colorectal cancer and also used the approach for diagnoses of thrombosis. In a mouse model of colorectal cancer, the method was able to distinguish between diseased mice and healthy controls with an AUC of .90. In the case of thrombosis, it distinguished between diseased and healthy mice with an AUC of .92.
While the test performed quite well in separating diseased from healthy mice, Warren acknowledged that distinguishing between, for instance, colorectal cancer and a similar condition, is a more difficult problem.
"Here in this study we are just looking at discriminating disease from healthy controls," he said. "It's quite difficult to think about how to address other diseases that might occur."
The question is one the researchers are "definitely interested in, in terms of increasing the specificity of the test so that you're not going to get false positives if you, say, have a cold or something," he added.
The method's specificity for a given disease is based on two factors, Warren noted. The first is use of nanoparticles to guide the synthetic markers to the organ or bodily region of interest. To date, the researchers have had success using such particles to direct the markers to the liver and colon, he said, adding that they are currently looking at additional targets.
The second layer of specificity lies in construction of the peptide substrates so that they will be cleaved only by proteases linked to the target disease.
"Right now we are trying to look specifically at [diseases] where we know the proteolytic profile or where we know which proteases are most involved," Warren said. However, he said, "there are some diseases where that's not as deterministic, so those are a bit more difficult for us."
One potential way to address such diseases, he suggested, would be to expand the number of substrates used, allowing for profiling of a much larger number of proteases. This, however, could prove a challenge for the immunoassay version of the method, Warren noted.
To date, the researchers have managed to simultaneously measure four reporter ligands using the immunoassay platform. As with most immunoassays, however, there are limits to its multiplexing ability.
The original mass spec-based assay, however, could likely achieve significant multiplexing, Warren said, noting that he and his colleagues are continuing to develop this version of the technology, as well.
"The mass spec easily does [multiplexes of] 10, and with some slight redesign it could even do hundreds," he said. "So that is definitely an example of the benefit of the mass spec platform."
In a previous interview discussing the Nature Biotechnology paper, MIT researcher Gabriel Kwong, also a co-author on the PNAS study, said that in terms of applying the method to humans, the components used in the synthetic markers are fairly well established from a regulatory perspective, with US Food and Drug Administration-approved formulations and GMP guidelines in place.
Warren noted, however, that thus far the researchers had only performed preclinical mouse studies. "So there's a lot of work remaining to be done" before moving the technique into humans, he said.
The researchers have filed patents on the method, and Kwong and MIT researcher Sangeeta Bhatia, leader of the project, are working with the institute's Deshpande Center for Technological Innovation on a possible spinout company based on the technology.