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Antigen “density array” picks up more antibodies
the high-density form of the antigen, one bound to the low- and high-density forms equally well, and one had an inThe idiom about “not seeing the forest termediate density dependence. for the trees” is based on the idea that Gildersleeve reasoned that if his denthe density of trees, or details, can obsity array could detect these differscure one’s overall view of the world. ences among the monoclonal antibodBut on the scale of an antigen array, ies, then it could be a powerful tool to greater density might actually clarify the distinguish among the complex mix of view of a complex mix of antibodies, acuncharacterized polyclonal antibodies cording to research published in JPR in the serum of healthy (DOI 10.1021/pr9002245) patients. The researchers by Jeff Gildersleeve, Oyprobed the array with seinda Oyelaran, and colrum samples from 30 inleagues at the National dividuals and found that Cancer Institute. for a particular antigen, Researchers use antigen some individuals had an microarrays to characterantibody mix that bound ize diverse collections of to the high- and low-denantibodies, and carbohysity forms of an antigen drates represent an imequally well; others, howportant class of antigens, ever, had an antibody mix particularly in cancer. To Changing one’s spots. In a novel method for making arrays, the density of that strongly preferred date, carbohydrate array carbohydrates is varied by attaching different numbers of sugar antigens to only the high-density designs have all involved BSA. The BSA conjugates are spotted onto the array, which is then probed with form. “Those two types of attaching a monovalent antibody mixtures. people have different carbohydrate to the arpopulations of Tn antiray’s solid surface supbodies; we are seeing difport, typically a coated ferences that you could slide. But this approach NAc) residue R-linked to either a not detect with just a single antigen makes it difficult for scientists to vary threonine or a serine. density,” says Gildersleeve. the density, and therefore the presenWith these BSA molecules decorated Nicola Pohl, a carbohydrate chemist tation, of carbohydrate antigens in a with differing densities of antigen in at Iowa State University, says this is defined way. hand, Gildersleeve’s group next printed the message researchers should absorb “Density is known to be an importhem at high, saturating concentrations from this study. “These really elegant tant feature of antigen recognition, but to produce spots with uniform coverage. experiments show that you cannot exwhat is the best way to vary density on This ensures that density differences are pect to pick up every antibody with a an array surface?” Gildersleeve asks. between spots of different BSA conjusingle density of sugars,” she says. “My The traditional method has been to gates and not within a single spot. This hope is that a lot of people read this vary the concentration of the carbohyis a subtle, but beautiful, advantage of paper and say, ‘Ah! We have to be cogdrates in the printing solution that is the method, says James Paulson, a nizant of this density effect.’” Rebeing spotted onto the microarray chemical biologist at Scripps Research searchers must run experiments on slide. However, this method often Institute in La Jolla, Calif. “That is an traditional arrays at two or three differyields inconsistent results. extremely important insight that can be ent concentrations, she says. “You can present antibodies to these generalized to other array formats,” he arrays, but you don’t know exactly what Both Pohl and Paulson note that says. density and presentation of antigen the Gildersleeve’s specific method of BSA Next, the team wanted to know, “are antibody is responding tosit can be very conjugation for building density arwe producing densities that are biologihard to interpret the results,” says Gildrays has some limitations. But, they cally relevant with this method?” To test ersleeve. Instead, he and his group desay, that doesn’t detract from the imthat, they probed the array with three fined the density “before we even put it portant lessons: that there is a better plant lectin proteins and three monoon the array surface”. way to make a density array and that clonal antibodies, all known to bind to density must be factored into antigen Tn antigen. Even though the monoclonal Gildersleeve’s team synthesized array studies to capture the full specantibodies bind to the same antigen, neoglycoproteins by covalently attrum of antibodies in a mixture. they recognized density in very different taching carbohydrate moieties to —Kendall Powell wayssone strongly preferred binding to lysine residues of the protein bovine
10.1021/pr900497w
serum albumin (BSA). BSA has multiple potential sites for attaching sugars, so the group could vary the antigen density by changing the number of antigens per BSA molecule, from 3 up to 37. They used 11 different antigens, many of which were variations of the clinically important tumor-associated Tn antigen, which is an Nacetylgalactosamine (known as a Gal-
© 2009 American Chemical Society
Journal of Proteome Research • Vol. 8, No. 7, 2009 3221
