In a sign that protein chips are being increasingly integrated into mainstream proteomics, Charles River Proteomics’ service team is using Zeptosens’ protein arrays in the process of plasma proteomic analysis along with depletion processes using IgY antibodies, and traditional fractionation.
At a biochip conference last week at New York City’s Holiday Inn, Midtown, Sunny Tam, the manager of the company’s Worcester, Mass., proteomic services lab, detailed how the company is using protein arrays and these antibodies in plasma proteomics.
The company has no formal collaboration with Zeptosens — “we are only demo-ing their technology,” Tam later told ProteoMonitor. “But from some of the experiments we have done with them, within the array, and between slides, they are quite reproducible.” He said that the company is encouraging its service customers to sign on for experiments using the arrays.
Zeptosens, of Witterswill, Switzerland, confirmed that Charles River Proteomics provides its Zepto Mark Cell Lysate Arrays to its service customers, and that Charles River is itself a customer for the consumables associated with the arrays. Zeptosens introduced these arrays to the US market in February.
Tam said he chose to use the Zeptosens ZeptoMark Cell Lysate Arrays after evaluating a number of commercially available protein arrays. He has been working with protein arrays for five years, including during a stint at Genometrix where he was involved in the now-defunct firm’s protein microarray development. When he came to Charles River a year and a half ago, he was agnostic on the kind of protein array to use.
“We also tested the Zyomyx and the Schleicher & Schuell platform,” he added. But Zeptosens, he said, “has really put time and energy [in] to make sure the CV is very low between slides, within spots. They have done all that comparison and validation, [and] QC manufacturing.”
Recently, Tam and his colleagues in the service lab performed a study where they prepared the same type of tissues and arrayed lysates on the same slide, then did hybridizations on two different days with two different antibodies, and found them to produce consistent results.
The ZeptoMark Cell Lysate arrays that Tam’s team is using are reverse arrays, he said. Instead of probes being arrayed on a chip, protein lysates from the samples are arrayed on the chip. Although Zeptosens said the array can handle up to 286 different samples, typically Tam prints 32 or more samples at four dilutions each, and in duplicate. Analyte-specific antibodies are then added to the chip for detection of specific proteins.
In each assay, only a single antibody is added to the array, so the chip produces data on the presence of a single protein across multiple samples, rather than a number of proteins in a single sample, as other arrays would. Zeptosens offers about 50 different antibodies to customers for assays, according to Peter Oroszlan, the company’s director of business development. In April, the company licensed a number of phosphospecific an antibodies to kinases and other signaling molecules from Cell Signaling technology of Beverly, Mass. But customers can also provide their own antibodies. “You can use any antibody which you trust,” said Orozlan.
Zeptosens arrays are based on the company’s patented planar waveguide technology, which its founders developed at Ciba-Geigy before Zeptosens was spun out in late 1998 after Ciba and Sandoz merged to form Novartis.
This planar wave guide technology involves a “thin layer” of light, or evanescent field, created just above the chip surface. This layer excites only fluorophores that are bound to the chip, leaving those in the solution above dark. As a result, background fluorescence decreases and the signal-to-noise ratio improves, allowing users, for example, to omit washing steps to preserve delicate protein-protein interactions. To produce the evanescent field, a glass substrate is etched with parallel nanochannels and coated with a thin tantalum pentoxide layer. When illuminating the chip from below, the grating bends the light into the tantalum pentoxide waveguiding layer, where it propagates and creates the 200-300 nm thick evanescent field above the surface.
The advantage of the reverse array Cell Lysate Arrays, Tam said, is that the samples themselves can be archived after they are printed on the chip, whereas in traditional DNA or protein arrays, the sample must be discarded after the experiment. But the reverse array is traditionally less sensitive than the ordinary protein arrays which involve sandwich assays, in which binding between the probe antibody and target antigen is detected, he said. (The company said that the arrays can detect proteins at levels as low as 60 molecules per cell).
Depleting with Chicken Soup
To overcome this sensitivity obstacle, Tam and his colleagues at Charles River Proteomic Services have been focusing on the depletion process in plasma. They use a mix of three chicken IgY antibodies from GenWay that removes albumin, IgG, and transferrin. These antibodies, which do not interfere with human IgG or IgM, or with human anti-mouse IgG antibodies because of the evolutionary distance between mammals and chickens, are attached to microspheres. They are also oxidized, then covalently conjugated to UltraLink Hydrazide Gel, in order to orient them with the F(ab’) antigen-binding regions facing outward, and the Fc regions covalently coupled to the microbead. To perform depletions, they generally perform liquid chromatography using the Applied Biosystems Vision protein workstation, but are now working to validate a 96-well format to increase the throughput.
This mix-three can produce a four-fold amplification in detection of other proteins. But now, the company is in the process of validating for the protein arrays a “mix 12” from GenWay that removes the 12 most abundant proteins and can produce a 50-fold enhancement in detection of other proteins. “In the case of the protein array, for some of these interleukins or cytokines/ chemokines that express at picograms per ml—that’s really the bottom line of the dynamic range of plasma—in that case we want the detection limits to go as low as possible.” If this validation process continues to go well, Tam thinks he and his colleagues can make the reverse arrays as sensitive as sandwich arrays.
After depletion, the proteins are also fractionated with a PSL proteome systems multichamber electrolyzer, or an Invitrogen Zoom IEF Fractionator, which Tam said requires less initial amount of sample. Sometimes this is followed by sub-fractionation with a Beckman Coulter proteomelab PF 2D platform.
In the future, Tam would like to be able to print thousands of slides ahead of time, in order to lower the cost for individuals doing this proteomic analysis. “The mass production is important for the success of the protein array format,” he said.
In particular, he would like to see reverse arrays with protein lysates taken from tissues in different disease types, such as cardiovascular diseases, diabetes, and cancers. “We have such a powerful fractionation technology, we could print all these protein lysates on an array,” he said.
Users could validate drug targets using these arrays, he said. “We want to have significant penetration into the clinical market in terms of diagnosis and study, he said.”