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With CF Chip on the Market, Thermo Unit Looks at OIA Tech for SARS Test

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Hoping to build on the market potential of its recently released cystic fibrosis multi-analyte-specific reagent, a Thermo Electron unit may soon begin marketing a chip-based diagnostic test for a variety of respiratory viruses to major reference laboratories, a researcher familiar with the underlying technology told SNPtech Reporter.

The development also means that Thermo Biostar, and by extension Thermo Electron, may soon begin competing against companies such as Sequenom, Applied Biosystems, and especially Third Wave Technologies in the molecular-diagnostics space.

“We can now take things that Thermo Biostar currently sells as diagnostic tests and we can increase the sensitivity of those tests 100-fold to 1,000-fold,” said David Ward, professor of genetics and molecular biophysics and biochemistry at Yale. Ward is also a co-author of a paper detailing the use of Thermo Biostar’s core Optical ImmunoAssay technology as a SNP-analysis tool.

The object of Ward’s study, which appears in the Sept. 15 issue of the Proceedings of the National Academies of Science, was to leverage the OIA technology so reference labs could use it to detect the presence of several respiratory viruses, including SARS and influenza A and B, he said. The question that remains, however, is what kind of diagnostic product Thermo Biostar will develop.

Thermo Electron currently does not have a SNP-detection product. Rather, it sells the OIA technology in the form of a biochip to the reference-lab market through a partnership with Quest Diagnostics. This deal gave Quest exclusive rights to incorporate the cystic fibrosis MASR into its home-brew testing methods.

Noel Doheny, director of business development and strategic marketing at Thermo Electron’s clinical diagnostics division, which oversees the Biostar unit, confirmed the company’s strategy for its OIA technology. “There is interest on our part in the respiratory pathogen field,” he said. Thermo Electron currently has “a wide range of tests” in this disease field that are based on antibody-detection using its OIA technology, “and we have an interest in the DNA area. … The obvious extension is to go to multi-analyte nucleic acids on the same platform.”

He said Thermo Electron has not determined a timeline to commercialize such a test. Doheny also stressed that “the current regulatory environment does not lead me to believe that an ASR path is one that will likely be a long-term solution.” He cited the US Food and Drug Administration’s ongoing debate over the status of ASRs and MASRs [see 5/23/03 and 7/17/03 issues of SNPtech Reporter].

Breathe easy

The respiratory chip was developed following the success of a partnership Thermo Electron penned with Quest Diagnostics. This deal, which is independent from the respiratory-chip collaboration, centered on a cystic fibrosis MASR called CF Portrait that was designed to identify parents at risk for having children with the disease. The chip, which entered the market in late July after a short delay, is currently being used at Quest Diagnostics’ Nichols Institute, in California.

Thermo Biostar designs and manufacturs the CF Portrait based on the OIA technology, which automates the detection of nucleic acid hybridization. Quest, which uses the OIA platform with its own DNA-extraction and -amplification methods, has exclusive rights to incorporate the CF Portrait chip into its home-brew testing methods, and the Nichols Institute has exclusive rights to further develop the chip as part of a testing system for laboratories worldwide.

The OIA technology is based on a proprietary polymer placed on the chip that allows biological materials to be attached onto its surface; when researchers place a capture reagent on the chip, it will reflect light and emit a gold color, Ward said. However, in the presence of a biochemical reaction — say, an antigen is bound by an antibody, or a hybridization reaction occurs — the light wavelength that is reflected from that surface changes as you change the thickness of the film.

“The color change is caused by the deposition of mass on the surface,” added Doheny. “What the whole technology is about is getting specific mass enhancement to occur in the presence of a target. No target, no mass,” he said.

The CF Portrait wafer is cut into 64-spot chips that are placed in a 96-well microtiter plate. After a patient’s DNA is collected in a doctor’s office, it is obtained by a reference lab — in this case Quest Diagnostics — which amplifies the sample using PCR. The DNA is then hybridized using normal and mutant probes, the technology generates a signal seen as purple spots on the gold background of the chip. The plate then moves to Thermo Electron’ NucleoSight imaging station where the genotyped data are analyzed.

The DNA chip that Yale and Thermo Biostar developed is based on this technology. Ward, who described the tool in the PNAS study, said the .7 cm2 chip is coated with Thermo Biostar’s polymer to create a thin-film optical biosensor. Allele-discriminating and aldehyde-labeled oligos are arrayed and covalently attached to a hydrazine-derivatized chip surface. Target sequences — for example, PCR amplicons — are created with biotinylated primers. Those biotinylated amplicons are then hybridized to the labeled oligonucleotides.

After a stringent wash, researchers are able to observe the ligation of biotinylated detector probes to “perfectly matched capture oligomers” as a color change on the chip surface — gold to blue/purple — after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. (Doheny stressed that the CF Portrait product does not use a ligation method.)

PCR fragments are tested in 30-40 minutes, “up to several hundred SNPs” can be assayed on the chip, Ward wrote, and SNP scoring can be performed by eye or with a digital-camera that is based on density. “It’s a simple assay where, by eye, you can detect the presence of a biochemical reaction, because the light that’s reflected from the surface changes as a function of the thickness of that surface,” said Ward. “The eye is a very good optical detector, and in this platform it can detect thicknesses in the range of 20 to 30 angstroms.” Doheny, however, prefers the camera, and said that Ward “makes a great point: There’s a certain number of spots you want to look at. If you get too many on a chip, you’d go batty doing it,” Doheny said.

In the study, Ward called the assay “extremely robust,” and wrote that it “exhibits high sensitivity and specificity, and is format-flexible and economical.” Specifically, in studying mutations linked to venous thrombosis through genotyping/haplotyping African-American samples, Ward’s team “document high-fidelity analysis with zero misassignments in 500 assays performed in duplicate,” he wrote.

In his interview with SNPtech Reporter, Ward said additional adjustments made to the chip after the PNAS paper was published have increased the sensitivity of the chip surface “by a thousand-fold.” “This would make the chip more useful in diagnosing rare pathogens,” he said, adding that Thermo Biostar and Yale are discussing ways in which these adjustments can be applied to new diagnostic products.

Ward also said that his lab has been working with the Chinese government on developing the product as a “fieldable” chip for SARS that can be used by small hospitals without PCR machines.

“If you’re looking at the detection of something like SARS, where early detection is important, and therefore you need to detect small numbers, we hope that we’ll be able to do that for diagnostics,” Ward said. “And because you don’t require an instrument to read it means that you can look for infectious-disease agents [without an instrument] in an inhospitable environment.

“Virtually all existing techniques for SNP identification have as a preface the application of PCR,” Ward said. “Because the optical biosensor doesn’t count molecules — all it does is count thickness in a layer — the more thickness you can add per hybridization event or per antibody-binding event, the more sensitive the assay can become. So we calculated that we should be able to do SNP analysis on total genomic DNA without having to do PCR,” Ward explained.

“That’s the direction” that this technology is headed, he added. “We’re not there yet, but we’re getting darn close.” He stressed that the technology will require another 10-fold sensitivity to get there. He said this is likely 6-12 months away.

— KL

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