There are seemingly a hundred “right” formulas for building a protein chip – making the subject ripe for discussion at scientific meetings. The Northwest Microarray Conference, an intimate mid-August gathering organized by University of Washington researcher Roger Bumgarner and held in Seattle’s airy Benaroya Music Hall, proved no exception.
Ping Ao of Lee Hood’s Institute for Systems Biology, for example, described a surface plasmon resonance-based protein microarray his group has been developing over the past two years. In his West Coast academic uniform of shorts and Teva sandals, Ao explained how Surface Plasmon Resonance (SPR) works on a slide format: light from a laser shoots through an optical prism on top of a slide, then comes out the other side and shoots into a detector. A CCD camera below the slide measures the angle of refraction, which is changed if a protein is present on the area where the laser hits the slide.
While SPR technology is not new in the protein profiling world – Biacore uses this detection method in its platform – the problem has been to adapt it to a higher-density chip, Ao said.
The ISB group has so far cleared this hurdle by using glass slides with a strong coating and spotting probes 150 µm apart using an ordinary microarrayer. The system has been arrayed with oligonucleotides and proteins to probe protein binding. The signal read by the CCD camera is converted to an amplitude on a readout, with higher peaks representing more specific binding events, and lower peaks representing non-specific binding events.
Karin Hughes of Bothell, Wash.-based Prolinx said her company is also developing an SPR-based detection system for protein chips, to be used with its Versalinx attachment chemistry. This move is partly in response to the fact that probe labeling can bias the results of protein chip experiments, she said. Scientists have ignored this and other technical issues – such as the cross reactivity of antibodies “in the rush to get to protein microarrays,” and now it is time to take stock and tackle these issues, Hughes added.
NextGen’s ‘Stone Soup’ Consortium
The approach that NextGen Sciences, of Alconbury, UK, has taken in developing an optimal protein chip could be compared to the children’s story, “Stone Soup.” Just as the story’s main character went from house to house with a stone and a pot, asking each neighbor for just one ingredient missing from the soup and ended up with a rich stew, Nextgen has been developing a specialized breast cancer chip by assembling what it calls “a biochip consortium” of collaborators for all of the areas of expertise it lacks, said Linda Cammish, the company’s business development director, in a presentation at the conference.
“Where we don’t have experience in house to do something, we [look for] someone who has expertise” in that area, she explained.
The company has collected its protein chip ingredients from a far-flung array of collaborators: Cytomyx of Cambridge, UK, and the Harvard Institute of Proteomics in Cambridge, Mass., supply the company with full-length cDNA clones that it re-clones and expresses in its expression vectors to produce proteins; LabVantage of Bridgewater, NJ, supplies a Laboratory Information Management System to keep track of the proteins; Nunc, a Denmark-based division of Apogent, has provided NextGen with the exclusive rights to its protein chip substrate for use in ready-made protein chips; PerkinElmer has also provided its hydrogel protein chip substrates; Avidity, of Denver, Colo., is providing the “biotag” labels, which are attached to the capture proteins like balloons on a string; and Cambridge University researcher Carlos Caidas has seasoned the collaboration by applying his clinical and research knowledge to selecting antibodies and antigens for the biochips. Caidas will also be NextGen’s in house beta tester, using the chips it develops on clinical samples.
So what does Nextgen supply? “We source antibodies to targets,” said Cammish. This means finding proteins implicated in breast cancer and developing antibodies for them. To do this, the company stirs the pot of ingredients, sub-cloning the cDNA clones, which are selected from genes, then expressing them in an automated process it calls “the expression factory.” This factory combines bacterial, mammalian, and insect vectors to express the protein, and allows the researchers to “express hundreds of proteins in hundreds of vectors without having to go through a manual step-by-step process” to purify them, said Cammish, who has a PhD in peptide chemistry from Cambridge University.
So far, the company has developed a prototype breast cancer chip with 100 antibodies, which it is planning to introduce at the second annual Protein Microarray Technology meeting in Hamburg, Germany during the last week of September. Eventually, the company is aiming for a 500-antibody chip, which it plans to develop over the next three years, as well as a 1,000-feature antigen chip that it can check for cross-reactivity with the antibody chip, Cammish said.
The company, which has also developed the protein array workstation that PerkinElmer is now marketing, “would love to move into other [clinical] areas” outside of breast cancer, Cammish said. “We would love to do a prostate cancer protein biochip.”