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ANALYTICAL CURRENTS New and improved ELISAs Perchlorate in the environment Elevated perchlorate levels in the environment from sources such as missiles, rockets, and fireworks have health officials concerned because of the ion’s ability to disrupt the human thyroid. The accepted method for measuring perchlorate at trace levels is ion chromatography, which has low throughput (10 min/sample) and a reporting limit of ~4 ppb. There have been a few reports using electrospray ionization (ESI) MS, but these methods are not compatible with sulfate and phosphate, which are abundant in the environment. Roger Guevremont and co-workers at the National Research Council of Canada, the University of Alberta, and Perkin-Elmer SCIEX (all in Canada) have found a new perchlorate method that is compatible with sulfate and phosphate, yet still offers the specific detection of MS. At the center of the method is what is they are calling high-field asymmetric waveform ion mobility spectrometry (FAIMS)—a continuous-flow tech-
Always adaptable, the enzyme-linked immu-
nique for separating gas-phase ions at atmospheric pressure and room temperature. ESI is used to generate the ions, which enter the FAIMS analyzer where they are separated. The separation process relies on changes in ion mobility with an applied electric field. At low electric fields, the ion mobility is independent of the electric field, whereas at higher electric fields, it becomes a function of the electric field. If the ratio of the ion mobility values at high and low electric fields differs between two ions, they can be separated by FAIMS. As the ions come out of the FAIMS analyzer, they are transferred into the vacuum chamber of a triple quadrupole mass spectrometer. The ESI-FAIMS-MS method is sensitive enough to detect environmentally relevant levels of perchlorate (LOD ≈ 0.1 ppb). Moreover, FAIMS can separate perchlorate from bisulfate and dihydrogen phosphate before MS detection. (J. Anal. At. Spectrom. 2000, 15, 907–911)
nosorbent assay (ELISA) is being updated for the 21st century by Ruud M. T. de Wildt, Ian M. Tomlinson, and colleagues at the Medical Research Council’s Laboratory of Molecular Biology and Centre for Protein Engineering and at the U.K. Human Genome Mapping Project Resource Centre (all in the U.K.). The researchers describe a new highthroughput method for screening >18,000 recombinant antibody clones—all at the same time. The method is a variation of filter-based ELISA screening, in which the antibodies secreted by a genetically engineered phage are allowed to bind to a membrane that has been coated with a ligand or antigen. By using a robot to pick and array the clones, the researchers have made the method more robust, simultaneously eliminating the need for liquid handling, increasing the throughput, and spotting the same antibodies on up to 15 filters at once. In addition, unpurified bacterial, yeast, or mammalian cell lysates can be
ESI needle
used to coat the membrane in lieu of a puri-
High-voltage power supply
Liquid sample supply
fied ligand or antigen. The researchers note that, unlike conven-
Carrier gas in (Cin)
tional techniques, the new method produces a wide diversity of hits with only a single round of phage growth and selection. They also suggest that this procedure could be V(t) Sample gas out (Cout) Carrier gas out (Cout) Sample cone (OR)
used to select antibodies against targets in natural cell extracts and, by varying the concentration of antigen, it may also be possible to differentiate between high- and low-affini-
Roughing pump Skimmer cone
ty binding. Finally, they envision the method as a possible alternative to two-dimensional
Quadrupole mass spectrometer
Schematic of an ESI-FAIMS-MS instrument. (Adapted with permission. Copyright 2000 The Royal Society of Chemistry.) 680 A
A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 0
gel electrophoresis for separating proteins in proteomics application work. (Nat. Biotechnol. 2000, 18, 989–994)
