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ABRF and the American Society for Biochemistry and Molecular Biology (ASBMB) jointly sponsored the symposium, Antibody and Protein Microarrays for Highly Multiplexed Protein Analysis, at Experimental Biology 2003 in San Diego on April 15. Dr. Brian Haab of the Van Andel Research Institute organized the program.
The symposium focused on recent developments in antibody microarray technology—a promising technology for highly multiplexed protein analysis of biological fluids. The speakers were Ronald Niece of Research Resources & Technologies, Ruo-Pan Huang from Emory University, Stephen Kingsmore from Molecular Staging, Inc., and Brian Haab from the Van Andel Research Institute.
Dr. Niece provided the introduction to the series on emerging technologies acknowledging the support of ABRF, ASBMB, and the Education and Professional Development Committee of ASBMB. He presented a brief history of ABRF noting the areas of research activity and the very active electronic communication system. Much more information is available on the home page, www.abrf.org.
Dr. Huang described several implementations of microarrayed ELISA assays on nitrocellulose membranes. He demonstrated high sensitivity and low cross reactivity for several dozen cytokine assays in parallel. He then described several applications of the technology noting that the change in biological activity is often greater than the change in gene expression. Cytokine arrays were used to analyze conditioned media from multiple breast cancer cell lines, to study molecular mechanisms relating to glioblastoma, and to investigate secreted cytokine variations in response to vitamin E supplementation.
Dr. Kingsmore described his company’s platform for multiplexed assays of dozens of cytokines. The company uses rolling circle amplification (RCA) to achieve significant improvement in detection sensitivity over conventional fluorescence-based techniques, both for microarray assays and for bead-based assays using Luminex technology. Dr. Kingsmore described the extensive work in quality control, reduction of imprecision and reduction of cross-reactivity between antibodies. He described some of the applications of the technology the company has undertaken with academic and industrial collaborators. Seventy eight cytokines were measured in the cord blood of new-born babies to evaluate the possibility of diagnosing cerebral palsy earlier than the typical diagnosis age of 2 years, at which point treatment options are limited. Cytokine levels greatly varied with gestational age, highlighting the need for precise age-matched controls. In another study measuring the serum levels of 78 cytokines in 60 adolescents from birth to age 19, a significant elevation in most cytokines was present from age 8 to 12, just before puberty begins. Such novel insights demonstrate the value of the ability to multiplex protein measurements.
Dr. Haab described his laboratory’s implementation of antibody arrays, along with applications of the technology. The laboratory generally uses a single capture antibody assay, which detects labeled antigens, rather than a dual-antibody “sandwich” assay, as described by the other two speakers. Dr. Haab described two-color comparative fluorescence detection, in which serum samples labeled with Cy3 were each incubated together on antibody microarrays with a common Cy5-labeled reference pool of proteins. The analysis of these data is similar to the analysis of two-color cDNA microarray data. Twenty five serum proteins were measured in serum samples from pancreatic cancer patients and controls, revealing patterns of inflammation-related proteins. The healthy controls were clearly distinguishable from the disease samples, but the benign cases were not distinguishable from the cancer cases, indicating that antibodies targeting markers more specific for cancer would have to be added to the set. Finally, Dr. Haab showed that two-color RCA signal amplification of biotin-labeled and dig-labeled protein pools could enhance the sensitivity of the assay.
Prepared by Ronald L. Niece and Brian B. Haab