Finding the right partner is not only a problem that haunts many startup companies. It might also be one of the major limitations of using protein capture arrays to profile hundreds of markers in parallel. That’s because the numerous detection antibodies could crossreact with the wrong partners — a common problem, at least according to Nanotype, a startup company based near Munich. By applying its unique technology, however, Nanotype believes it has a remedy: putting forces on the binding partners so that only the strong relationships remain intact.
The company of 18 employees was founded almost exactly two years ago as a spin-off from Hermann Gaub’s laboratory at the Center for Nanoscience at Munich’s Ludwig Maximilians University. The company has so far raised about Ý3.5 million in venture capital and government grants, allowing it to move into 4,600 square feet of space and increase its staff from eight to 15 within three months in mid 2001.
Testing Specificity with C-FIT
Nanotype’s platform technology is rooted in research by Gaub, who is one of the six founders. He developed a technique in the 1990s for applying pulling forces in the piconewton range to single biomolecules by atomic force microscopy. Nanotype has taken this technology a step further, replacing the microscope and its single cantilever with a chip that can handle many interactions in parallel, calling the new setup C-FIT for “Congruent Force Intermolecular Test.”
The assay system consists of two arrays: the first chip carries capture antibodies that are each connected to a flexible polymer spacer, allowing them to roam like a dog on a leash. They are spotted onto and covalently linked to the chip. The sample fluid is added next, and then 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 20-40 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.
Nanotype received a German patent in March on the C-FIT technology and plans to expand its intellectual property coverage.
Nanotype CEO Gunnar Brink thinks the C-FIT technology is especially suitable for multiplexing ELISA-type assays on a chip. “We think that above 20 or 25 [markers] 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, determining the phosphorylation state of more than ten kinases simultaneously on a chip.
A Common Problem?
Although Steffen Nock, vice president for biochemistry of protein chip company Zyomyx, said that his company has not experienced problems with cross-reactivity when assaying for up to 30 different proteins in parallel, choosing antibodies that do not cross-react, might not always be possible, especially when the number of markers to be profiled grows into the hundreds, said Dolores Cahill from the Max-Planck-Institute for Molecular Genetics in Berlin.
In contrast to Nanotype’s competitors, which have been working on their first products for quite a while, Nanotype has so far only conducted proof-of-principle studies, with both antibody chips and 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.
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. Initially the company plans to develop arrays for research purposes such as target validation and toxicity or metabolism studies.
So far, Nanotype has entered into collaborations with a large East Coast pharmaceutical company, and with Xerion, another Munich-based startup. The company’s pharma partnership, which began last December, provides Nanotype with a stream of monoclonal antibodies and revenue — in 2001 the pharma paid Nanotype Euro 56,000. With Xerion, Nanotype may develop a joint product based on Xerion’s single-chain antibodies and validated markers for various disease areas.
Although Brink added that the technology could also fare well in the diagnostic market, others are not so sure, cautioning that the technology has yet to prove the robustness and reliability required for diagnostic applications. “It’s an interesting technology, but it has to be proven that it can fulfill diagnostics purposes,” said Thomas Joos, head of the biochemistry department 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.”