by Jeffrey M. Perkel
When the DNA microarray industry took off, it seemed only natural that protein arrays
would follow suit. Yet that hasn't been the case. Protein arrays present significant technical challenges not faced by so-called “gene chips,” namely the difficulty in producing so many distinct, heterogeneous proteins, keeping them properly folded, and ensuring their specificity.
Antibody array manufacturers in particular have been hamstrung by the availability of good antibodies, says Grigoriy Tchaga, R&D director for Clontech Laboratories. "There are thousands of antibodies, but when you start validating them for their performance on an array, a large number of them do not perform well," he says, whether because the antibodies recognize unfolded peptides rather than intact proteins, lose their activity when immobilized, or simply because of cross-reactivity and high background.
Nevertheless, a handful of manufacturers do offer commercial protein arrays, whether as off-the-shelf catalog products or as a service. Like DNA microarrays, these arrays come in all shapes and sizes to accommodate a variety of applications. They may comprise antibodies or antigens, be spotted on glass slides, nitrocellulose membranes, or in the wells of a microtiter plate, and can measure protein abundance, expression, or interaction.
ELISA Surrogates
The simplest arrays are antibody arrays specifically intended to multiplex ELISA reactions. Typically in ELISA, a capture antibody is immobilized on the bottom of the well of a 96-well microtiter plate. A biological sample is then placed in the well, where the desired antigen, if present, binds the antibody in a quantitative manner. Following a wash step, a second antibody to the antigen is added, which is detected either directly (via fluorescence) or indirectly (for instance, by the use of a third antibody coupled either to a fluorophore or enzyme). This type of assay is called a sandwich ELISA, as it results in the formation of an immobilized antibody-antigen-antibody complex.
Because each well of an ELISA plate holds just a single capture antibody, it can only test for a single analyte. That means more wells, more plates, and ultimately more sample is required to test a panel of analytes. That's where multiplexed ELISA protein arrays come in. These products array a small number of distinct capture antibodies at the bottom of the well, so that instead of one analyte per well, a dozen or more may be detected. The caveat is that, with singleplex ELISA, the only required hardware is a spectrophotometer to measure a color change in the well; with multiplexed assays, however, a more sophisticated detection system (often involving a CCD camera) is needed.
SearchLight arrays from Pierce, now part of Thermo Fisher Scientific, spot 16 or 24 capture antibodies (the former for chemiluminescent detection, the latter for infrared) per well. According to product manager Amy Willson, SearchLight is offered mainly as a service, in which customers request custom arrays, built from the company's menu of more than 200 validated antibody pairs, and supply the samples, and the company runs the assays for them.
"This is the most popular option," she says, because it spares customers from needing to acquire the detection instrumentation and analytical software necessary to collect and interpret the data.
Quansys Biosciences also offers microtiter plate-based Quansys ME Arrays with up to 25 antibodies per well.
RayBiotech offers glass slide-based quantitative arrays. Each Quantibody array includes 16 arrays per slide, with up to 40 antibodies per array, says Brett Burkholder, manager of marketing and business development, as on the Quantibody Human Inflammation Array 3. RayBiotech also offers custom arrays, drawing from a pool of over 200 human, 100 mouse, and 25 rat targets.
Primorigen Biosciences also offers a glass slide-based platform for quantitative ELISAs (called ThiNClear). But the company is preparing to roll out a fundamentally different platform. Called Spots-On-Dots™, the new platform "is a frameless microarray system," says President Chuck Oehler. Each Spots-On-Dots array is a plastic sheet, slightly smaller than a microplate, containing 96 nitrocellulose dots. Each dot contains 36 antibodies arranged in a 6 x 6 grid, which is developed via a colorimetric assay—hence the "spots."
According to Oehler, Primorigen is positioning Spots-On-Dots as a low-cost alternative to its competitors. He says the assay will use less reagent than a typical ELISA, and requires a minimal hardware investment. "You don't need all the bells and whistles you need for fluorescent scanners," he says. "If you have a flatbed scanner with 16-bit data, that's good enough."
Profiling arrays
Some manufacturers offer antibody arrays for expression profiling—that is, to assess which of a variety of proteins is present in a sample, and at what relative levels, by probing the arrays directly with fluorescently labeled or otherwise tagged biological samples.
One such product is the Antibody Microarray from Clontech Laboratories, which can detect 507 different proteins simultaneously. According to Tchaga, the product is typically used in a dye-swap experiment, in which two samples (a drug-treated sample and a control, for instance) are both labeled with each of two dyes. One array is probed with sample A labeled with dye 1, and sample B labeled with dye 2. The second array (there are two per kit) is probed with sample A labeled with dye 2, and sample B labeled with dye 1.
This method "allows us to compensate for low reproducibility of the dye labeling and the differential solubility of the antigens [in the two dyes]," Tchaga says. "In addition, it compensates for cross-reactivity on the antibodies and for differential amounts of antibody deposited on slides 1 and 2."
Sigma-Aldrich's Panorama product line is also designed for expression profiling. The company offers four relatively small arrays, ranging from a MAPK array with 84 antibodies, to a cell-signaling array with 224. According to Becki Davis, product manager for functional proteomics, a fifth product, the XPRESS Profiler725, encompasses all four of these antibody collections, plus in excess of 100 additional antibodies, to probe 725 different proteins per sample on a single array.
"These are qualitative to semi-quantitative discovery tools," says Davis, adding: "As with all protein expression profiling arrays we recommend downstream validation with ELISA or Western blots."
But she also notes the array can be a real time-saver. "It allows accurate and robust protein expression analysis in less than half a day," she says. "With Western blots, you could spend months to years profiling that many different antibodies to pinpoint where you really need to hone in."
RayBiotech offers an assortment of both glass slide- and membrane-based profiling arrays, in both sandwich ELISA and label-based formats. The company's Human L-Series arrays, for instance, probe the expression of 507 human, 308 mouse, or 90 rat proteins simultaneously, using one capture antibody per target. Detection is based on interaction of a labeled streptavidin with the biotinylated sample. RayBiotech's cytokine antibody arrays, on the other hand, use both capture and detection antibodies to detect over 174 human cytokines.
According to Burkholder, RayBiotech's membrane-based sandwich ELISA assay format "is still our best seller. It is very easy to use. If you can do a Western, you can use our product."
In January, Gentel Biosciences released its first screening product, the APiX Cancer Biomarker Array Kit, which profiles 47 cancer-related proteins from eight samples at once. Marketing manager Jared Browning says new products are on the way, including a glycobiomarker array to be announced at the upcoming American Association for Cancer Research meeting. "We expect many, many more content-based arrays under the APiX tradename," he says. (The company also offers slide-based quantitative arrays, such as a 17-plex cytokine array.)
Functional arrays
Invitrogen offers a protein array based not on antibodies, but rather on functional proteins. With more than 8,000 full-length human proteins arrayed on a nitrocellulose-coated glass slide, says Jennifer Cannon, business area manager, Invitrogen's ProtoArray is less a screening tool than a proteomics one, competing against such technologies as mass spectrometry and two-dimensional gel electrophoresis.
"Because we can penetrate a bigger portion of the proteome, we are looking at different types of competition with this product," she says.
Cannon says the product was originally developed for applications like mapping signaling pathways and identifying kinase substrates. But that is not what most users are doing, she says. Instead, the leading application appears to be biomarker discovery based on "reverse ELISA," in which researchers use the array to scan for the presence of autoantibodies in human serum.
"Cancer and autoimmune diseases elicit an immune response, generating a pool of autoantibodies that can be used as markers of the disease," she says. "This protein microarray tool is perfect for that."
The second most popular application, she says, is identifying protein targets that can bind to small molecule drug compounds, to explain pharmaceutical side effects, for example.
Cannon explains that each protein on the array is spotted in duplicate, and one sample may be tested per slide. A 9,000-protein array is in development, she adds, but no launch date has been announced.
