Finding the right partner is not only a problem that haunts many startup companies. It also may be a major challenge for probe and target molecules on microarrays. For example, antibodies or their mimics could crossreact with the wrong partners, and bury the binding signals in significant amounts of noise. Nanotype, a fledgling outfit based outside of Munich, has a unique solution to this potential problem. The company uses its technology to add forces to the binding partners so that only the strong bonds remain intact.
Nanotype’s assay system, which is called C-FIT, for “congruent force intermolecular test,”consists of two arrays: in its protein version, the first chip carries capture antibodies that are each connected to a flexible polymer spacer, like a dog on a leash. These antibody-spacer combinations are spotted onto and covalently linked to the chip. Next, the sample fluid is added, and proteins bind to their respective capture antibodies, followed by fluorescently labeled secondary antibodies. But unlike a standard ELISA, these antibodies are connected to a second chip via a reference or force sensor complex and another flexible polymer spacer. The polymers not only keep the two chips at a distance of 40 to 60 nm, they also allow the antibodies to find their antigen partners. The reference complex consists in most cases of two short paired DNA strands, whose binding strength can be optimized by adding or removing base pairs.
When the two chips are pulled apart by a small macroscopic mechanical force, the flexible polymers extend until either the reference complex or the sample/antibody complex breaks apart, depending on which pair is stronger. If the antibody and antigen bind tightly, they stay connected on the first chip; if they react non-specifically with each other — less strongly than the reference complex — they separate. The remaining “true” interactions can be measured by the fluorescence that stays behind on the first chip. And because each secondary antibody can be associated with a different reference complex, the stringency can be different for each marker tested.
Gaub’s Piconewton Puller
This technology is the brainchild of Hermann Gaub, a researcher from the Center for NanoScience at Munich’s Ludwig-Maximilians-University. He developed a technique in the 1990s for applying pulling forces in the piconewton range to single biomolecules by atomic force microscopy.
Two years ago, Gaub and five others decided to spin out the company to commercialize this technology. After receiving seed funding of Ý200,000 from BioM, a German business incubator, and a total of about Ý50,000 from various government agencies and a local business plan competition, the company secured Ý3.25 million in venture capital in July 2001, allowing it to move into 4,600 square feet of space and increase its staff from eight to 15 within three months. Now the company has 18 employees, and is currently preparing a business plan for a new financing round later this year, in which it hopes to raise an additional Ý10 million.
On the technology end, the Nanotype scientists replaced the microscope and its single cantilever with the C-FIT chip, which can handle many interactions in parallel. Nanotype received a German patent in March on the C-FIT technology and plans to expand its intellectual property coverage to other countries.
The applications for Nanotype’s technology abound, and include DNA chips for SNP detection, but CEO Gunnar Brink thinks the C-FIT technology is especially suitable for putting ELISA-type assays on a chip in multiplex format. “We think that above 20 or 25, there is really not much technology around that can prevent cross-reactions and get rid of non-specific interactions,” he said. “If you put much effort and money into optimizing antibodies, you can do more multiplexing. But the costs can and will be prohibitive.” According to Brink, scientists have experienced problems, for example, in determining the phosphorylation state of more than ten kinases simultaneously on a chip.
But Steffen Nock, vice president for biochemistry of protein chip company Zyomyx, said that his company had not experienced problems with cross-reactivity when assaying for up to 30 different proteins in parallel. Choosing antibodies that do not crossreact, however, might not always be possible, especially when the number of markers to be detected in parallel grows into the hundreds. “There might only be one antibody for each one, … and if they cross-reacted, you might have to live with it,” said Dolores Cahill from Berlin’s Max-Planck-Institute for Molecular Genetics. However, little published data is available showing this to be problem in assays , she added.
While Nanotype’s competitors in the protein chip business have been developing their products for some time, Nanotype has so far only conducted proof-of-principle studies, both with antibody chips and with DNA chips for SNP detection. Neither has involved more than four or five different hand-spotted reagents on the first chip. The next step will be to show that the assay works universally with a variety of samples, Brink said, and he does not think it would be difficult to scale up the number of spots using an automatic microspotter.
Initially the company plans to develop antibody arrays for research purposes such as target validation and toxicity or metabolism studies. However, getting to real applications might not be so straightforward, and will rely on Nanotype’s ability to find suitable partners to develop customized or standardized arrays and provide content in the form of antibodies against validated markers.
So far, Nanotype has entered into collaborations with a large US East Coast pharmaceutical company, and with Xerion, another Munich based startup. With Xerion, Nanotype has agreed to develop a joint product in return for single-chain antibodies and validated markers for various disease areas. “If everything works out well, the result could be a joint product” several years down the line, said Brink. He added that the technology could also fare well in the diagnostic market, but others expressed caution that it has yet to show it is robust enough for this application. “It’s an interesting technology, but it has to be proven that it can fulfill diagnostics purposes,” said Thomas Joos, head of the Natural and Medical Sciences Institute at the University of T bingen in Germany. “To see whether there is an advantage over existing technology, they have to provide more data.”
Nanotype is currently working on several scientific publications detailing the technology and its application to protein profiling and SNP detection.