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McGill Team Describes Novel, Antibody Array Method for Lower-cost Protein Detection

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NEW YORK (GenomeWeb) – A research team from McGill University has developed a low-cost alternative to standard fluorescence microarray assays, by creating a technique to array and analyze what the group calls silver enhanced sandwich immunoassays, or SENSIA, using standard flatbed scanners.

In their study, published last week in the Journal of Proteome Research, the group demonstrated that by using enzyme-mediated silver amplification, they could create sandwich immunoassay microarrays in which a signal is detected using a simple office-style flatbed scanner. This vastly reduces the upfront cost of this type of molecular detection compared to standard fluorescence microarrays, which require laser devices costing at least tens of thousands of dollars.

The group also described their initial validation of the SENSIA array approach compared to a conventional fluorescence microarray method, demonstrating that SENSIA had a comparable limit of detection and high concordance with the results of the standard assay.

Overall, the results suggest that SENSIA could be an inexpensive, but equally sensitive alternative to fluorescence microarray assays in a range of clinical applications, such as disease diagnosis and patient stratification.

David Juncker, the study's senior author and a professor of biomedical engineering at McGill, told GenomeWeb this week that his team is focused broadly on developing novel formats for antibody arrays.

"In genomics, arrays have been quite successful, but in proteins it has been quite laborious for various reasons," Juncker said. Previously, the group has tackled issues like cross reactivity, which have hindered the development of higher multiplexing of immunoassays.

The team's latest results reflect work somewhat tangential to their main efforts, he added. "We set out to show that using silver amplification we could perform an assay and amplify the binding into a readable signal in an equivalent way to fluorescent detection but using a regular scanner like one you might have in your office."

To do this, the researchers required a stable non-fluorescent reporter molecule that could be detected by this low-cost imaging tool.

According to the group, earlier research has suggested that single-plex immunoassays in conjunction with silver amplification could be detected using a flatbed scanner, but it was unclear whether silver amplification could be suitable for higher-sensitivity multiplex assays, or whether flatbed scanning technology has high enough resolution to image microarray spots.

After investigating several chemical and enzymatic silver amplification methods, the researchers settled on a method called HRP-mediated silver amplification and optimized this enzyme-based approach for detection in a microarray. The team developed an initial 15-plex assay covering a set of cancer-related proteins.

According to the authors, SENSIA uses essentially the same assay protocol as in a classic antibody microarray in a sandwich format, but replaces fluorescent labels with silver precipitation to amplify the signal and quantify the binding of target proteins without the need for a fluorescence scanner.

The cost of reagents is essentially the same, but the up-front cost of a detection tool is potentially reduced dramatically, from $30,000 to $50,000 or more for a laser scanner, Juncker said, down to the $100 range for a good quality flatbed scanner.

In the study, the team used a $100 scanner with an 8-bit signal depth and a lateral resolution of 1200 dpi, developing their own scanning protocol that used the machine's manual mode and four repeated scans to maximize resolution and reduce pixilation.

To validate the performance of SENSIA, Juncker and his colleagues compared their 15-plex assay to an equivalent fluorescence-based approach, fluorophore-linked immunosorbent assay, or FLISA, quantifying SENSIA's limit of detection and dynamic range compared to FLISA's.

According to the authors, the different detection strategies showed good agreement — enough so to indicate that SENSIA could serve as a high-sensitivity alternative to FLISA.

The limit of detection for FLISA was better than that of SENSIA for six proteins in the panel, while the opposite was true for the other nine. Overall, though, the limits of detection for both methods fell within a 10-fold difference for 13 of the 15 targets, close enough for the authors to conclude that they were sufficiently similar in their sensitivity to potentially be interchangeable.

The dynamic range also differed and was higher for 11 of the proteins with FLISA. But again, 13 of the 15 targets showed a less than 1.5-log difference in DR between the two methods.

According to Juncker, a logical application for SENSIA would be as a low-cost alternative for many of the same clinical applications that currently employ fluorescence-based protein microarrays.

Low-resource settings, where fluorescent scanners are not available but there is a need to perform these assays, could adopt SENSIA instead, using easily available flatbed office scanners.

Juncker said the team has been in conversations with some potential commercial partners interested in developing the technology in that vein.

The McGill group is now researching other applications of silver amplification. The group is working on how some of the same chemistry could be used to allow single-molecule detection, or could support digital immunoassays — a protein version of genomic approaches like digital PCR, which have the potential to be more sensitive than the average readout in a tool like SENSIA.

This research is being spearheaded by Gina Zhou, the first author of the team's SENSIA study and a graduate student in Juncker's lab.

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