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Molecule counting made easy This is a slower off-rate than would A common method for binding Technical limitations have thwarted be predicted in solution. Ideally, antiproteins to a surface is the biotin⫺ researchers from achieving the holy bodies could be removed from ligands streptavidin linkage, but the method grail of protein analysis: quantitative after detection to allow subsequent used in this study required no prelaanalysis of rare proteins in complex rounds of single-molecule counting biological sera. But in a new AC paper beling. A heterobifunctional crosswith different antibodies, but this linking system anchored the proteins, (2009, DOI 10.1021/ac901068x), proved tricky. “We had trouble getting first exposing the carboxyl groups on Robi Mitra and colleagues at the the antibodies off, which was surpristhe BSA surface and then immobilizWashington University St. Louis ing because we were worried School of Medicine and Helithat the off-rate would be cos Biosciences describe the too fast,” explains Mitra. counting of single molecules Future experiments will fofrom both purified protein socus on getting those sticky antilutions and rabbit serum on a bodies off the ligands, but in surface with low background the meantime, the researchers using fluorescently labeled used photobleaching to “erase” antibodies. antibody signals from the first The first step, according to round of binding, leaving the Mitra, was to find a good surdarkened antibodies behind. face for immobilizing proteins They counted 1497 antibodythat also minimized nonspecific bound proteins on the first adsorption of antibodies. “We round and then performed a did a systematic search of the photobleaching step; after a literature,” Mitra explains, to second round of antibody bindlook for the best noninteracting ing, they counted 1146 prosurfaces. Then, “we did a systeins. The fact that so many tematic comparison” of 12 proteins were detected twice surfaces. indicated that many ligands To compare the surfaces, the researchers exposed them to (Top) Single-molecule counting of endogenous IgG in rabbit serum were available for binding in the second round, making multiple fluorescently labeled antibodies at three time points using fluorescently labeled antibodies. (Botrounds of binding feasible. “Afand then washed them with tom) Single-molecule counting of total IgG levels compared to ter photobleaching, you can buffer. Using an inverted mithose levels obtained with ELISA. rebind a high percentage of croscope fitted with a total inproteins,” says Mitra. ternal reflection fluorescence illuminaing the protein ligands via their priThe researchers’ first attempt at protein tor, they detected any remaining mary amines. This method attached quantification was with purified Cy3-laantibodies on the surfaces. The clear ⬃1000 protein molecules to the surbeled proteins. After probing the surface winner was a surface chemically coated face per field of view. with Cy5-labeled antibodies, they deterwith bovine serum albumin (BSA); it To assess how well antibodies would mined the limit of detection to be ⬃55 showed the least nonspecific binding, a bind to the surface-bound ligands and at target molecules per 1000-m2 image, mere 3 pg/cm2. what rate they would dissociate, the researchers used a dual-color assay, labeling Mitra and colleagues next turned their which means that they are able to detect attention to the binding of protein ligands both antibody and ligand. Counting the proteins down to 100 pM. spots where the molecules co-localized reto the BSA surface. “This aspect of the When the purified Cy3-labeled protein vealed that ⬃70% of the target ligands were work was interesting in that they are not was spiked into rabbit serum, the limit of bound by antibodies. attaching antibodies directly to the funcdetection increased to 390 target molHad the other 30% disengaged from tionalized surface, as is commonly done, ecules per 1000-m2 image. Still, the the ligands before detection? Probably but are instead attaching antigens,” says abundance of serum proteins did not pronot, because, as it turned out, the reSteven Soper of Louisiana State Univerduce a significant background. searchers found that 90% of the sity. “This will allow one to create a more The method’s final test was to universal approach for doing surface-medi- antibody⫺protein complexes remained count endogenous unlabeled immunointact 48 hours later. ated immunoassays.” globulin G (IgG) from diluted rabbit 7130
ANALYTICAL CHEMISTRY /
SEPTEMBER 1, 2009
10.1021/AC901633N 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 08/11/2009
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blood at various time points after immunization. The amount of IgG detected increased 70% in four weeks, a result that matched those measured using ELISA, the gold standard of bulk protein measurements.
“I would have been happy enough to see it done in purified proteins,” says Tim Harris, an instrument specialist at the Howard Hughes Medical Institute’s Janelia Farm research campus. “But doing this in serum is a
huge accomplishment. This could be the definitive low-concentration protein detector.” —Erika Gebel
SEPTEMBER 1, 2009 / ANALYTICAL CHEMISTRY
7131
