Protein microarrays are one aspect of highly automated, large-scale biological screening technologies, the other is nucleic acid arrays. As DNA arrays reveal their origin in Southern blotting protocols, protein microarrays reflect their genesis in Western blotting, in which protein mixtures are separated on acrylamide gels, transferred to nitrocellulose, and reacted against specific antibodies.
Protein blots, however, are specialized and labor intensive, highly individualized for characterizing proteins by their molecular weight and other properties. Unlike DNA arrays, much of their success hinges on the availability of quality antibodies that will react strongly and with high specificity against the target proteins. At the recent “PEPTalk” meeting held in California, participants argued the merits of various approaches to building and operating protein microarrays. These include separating, detecting, and characterizing the proteins under study.
Looking from the Opposite Direction
Protein microarray applications run the gauntlet from basic science to drug discovery to diagnostics and personalized medicine, as Antonia Holway, Ph.D., director, microarray applications at Aushon Biosystems explained. The company employs a variety of materials and surfaces in its arrays to achieve these goals.
Protein arrays can be implemented in two ways—in the forward-phase mode, the antibodies are printed on the array surface, followed by reaction with the antigen samples; whereas, in reverse-phase mode, antigens are printed, to be followed by reaction of the microarrays with the panel of primary antibodies. With the forward-phase approach, hundreds of antibodies can be printed on a high-content slide for use as a screening tool. This is basically a qualitative assay, demonstrating differences between two samples. Quantitative forward-phase arrays, similar to multiplex ELISAs, are also possible and powerful.
“Reverse-phase arrays can provide high-throughput, multidimensional protein measurements,” stated Dr. Holway. In the reverse-phase mode, the antigen, which is printed on the solid substrate, could be any one of a variety of biological materials, including cell lysates, tissue lysates, body fluids, or recombinant proteins. Here, the investigators can look at hundreds or even thousands of samples on a single slide.
As an example of a study using the reverse-phase microarray approach, Dr. Holway discussed work by Ian Summerhayes of the Lahey Clinic Medical Center, whose goal was to identify histone deactylase inhibitors that can modulate the expression of different tumor and invasive suppressor genes. Summerhayes printed out lysates of nine different bladder carcinoma cell lines treated with histone deacetylase inhibitors. He found that different classes of these agents share similar potential to upregulate the expression of important tumor and invasive suppressor genes involved in bladder tumorigenesis.
In another series of investigations, Bennedetta Accordi and Giuseppi Baso of the University of Padua in Italy studied the phosphoproteomic profiles of children affected by B- and T-cell acute lymphoblastic leukemia using reverse-phase arrays, in which they screened the cancer cells for proteins known to be involved in many pathways including cellular proliferation. “Here the goal was to identify key alterations related to disease outcome and drug resistance and even to provide targets for new drugs,” Dr. Holway explained.
Yet another application described by Dr. Holway has been developed by 20/20 Gene Systems, and concerns an early detection approach for non-small-cell lung carcinoma. Using a reverse-phase array with phage-expressed proteins as the antigen, a blood test for early detection was developed by measuring the levels of multiple antibodies generated during early stages of the disease. Preliminary results suggest that it may be possible to detect anticarcinoma antibodies in the blood of subjects as much as three to five years before radiographic screening methods are effective.
Dr. Holway concluded her talk by discussing her company’s efforts to deal with protein array fabrication challenges. “With our technology we are able to print on a wide range of binding surfaces while handling a variety of buffers and viscosities using extremely low sample volumes,” she stated. “This gives us the ability to produce quality arrays in high volume.”