Courtesy of Applied Biosystems
For labs equipped to measure surface plasmon resonance (SPR), determining how biomolecules interact with each other is simple. Load a glass slide into the benchtop instrument, give it some tubes, click the mouse a few times, and voila! – out come affinity measurements, on-rates, off-rates, and dissociation constants, all in real time.
"We can absolutely determine affinity, concentration, and all other things," says Stefan Lofas, vice president and CSO of Uppsala, Sweden-based Biacore
A few competitors have come and gone, Lofas points out, and a few...
WHAT IS SPR?
SPR occurs when light at a specific angle (the SPR angle) encounters a reflective surface such as gold, and some of the energy is transferred as an electromagnetic wave (the surface plasmon). Ordinarily the gold acts like a mirror, reflecting a bright rectangle. "But when the SPR effect comes into play, which means you've adjusted the angle of the light just right, you have the right thickness of gold and refractive index of glass, ... most of the light is absorbed, so now your rectangle is nearly black," explains Tim Burland, vice president for technology development of GWC Technologies, an SPR instrument manufacturer in Madison, Wis.
When the local mass of the surface changes, as when a ligand is attached, the surface plasmon wave characteristics become altered, changing the refractive index. "Now you would see this dark rectangle, and on it you would see some lighted areas where your probes were," adds Burland. Further binding of the probe to an analyte increases the local surface mass, thereby increasing its reflectivity, and a detector such as a CCD camera or diode array can capture these changes.
Unlike techniques such as ELISA or flow cytometry, SPR does not require the use of radio- or colorimetric labeling, and progress of the reaction can be followed in real time. Additionally, SPR offers exquisite sensitivity: Biacore instruments can detect direct binding of a single molecule as small as 100 Daltons, and can measure affinities in the millimolar to picomolar range.
NEW AVAILABLE IN TURBO
Courtesy of Biacore
Applied Biosystems introduced its 8500 Affinity Chip Analyzer in September 2003. This instrument was designed for "customers who are primarily involved in the quantitative affinity binding and ranking of antibodies in a higher throughput format," explains senior product manager Bob Beltz. The 8500 currently also supports such applications as protein-nucleic acid and protein-peptide interactions, with others in the works.
While Applied Biosystems' instrument cannot match Biacore's variety of chip surface chemistries – Biacore offers nine different surfaces to Applied Biosystems' three – the new system targets the drug-discovery market with its high-throughput format. The key strength of the system, says Beltz, is that it can measure binding to all the different ligands under exactly the same conditions. This, he notes, "provides really valuable comparative data, and it essentially eliminates run-to-run variability."
The 8500 can simultaneously examine up to 400 binding interactions on a single chip during a two-hour run and can measure binding constants in the micromolar to picomolar range; the minimum analyte size for kinetic measurements is 8 kD, says Beltz. Researchers use a commercial spotter to deposit ligands of interest onto Applied Biosystems' disposable gold-plated affinity chips. The company offers slides with protein A/G or streptavidin coatings, but users also can make their own surface chemistries on plain gold chips, using antibodies or biotin-labeled molecules. The chip is then placed in the instrument and exposed to a solution containing the binding partner.
Biacore's systems rely on microfluidics to deposit biomolecular ligands onto glass slides. The same channels then run solutions containing the binding partners of interest. Up to four different experiments can be run simultaneously on the Biacore 3000 (the company's most popular model), and the S51 instrument offers up to six microfluidics channels. An average run of a Biacore instrument takes around 10 minutes. The same chips can be reused perhaps 100 times or so, Lofas notes, allowing the ligands to be interrogated by hundreds of potential binding partners as well.
- Josh P. Roberts
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