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Label-free technology primer
The pharmaceutical sciences are charged with the twin missions of creating therapeutic products that help the sick regain their health and making money for the companies that sponsor them. So unlike purely academic pursuits, where prejudices and preferences are less obvious most of the time, the champions of one NCE, diagnostic or test procedure are likely to hew closely to the corporate bone. After all, as the saying goes, "He whose bread I eat, his song I sing." So, for the purposes of providing a sort of non-commercial primer on label-free assays, we asked Mann Shoffner, director of sales and marketing at SRU Biosystems Inc., to address the subject without describing his company's entries in the field (at least not at first).
First, we wanted to know what negatives attach to using labels—commonly radioactive or fluorescent. Label-free assays (LFA) differ in that they use native biological compounds, cells or proteins without modification. In addition, LFA are non-destructive and measure cell-based kinetics in real-time, while labeled assays provide only endpoint reads.
According to Shoffner, LFA allow researchers to use more "physiologically relevant" systems, enhance sensitivity, reduce false positives or negatives sometimes caused by the steric effects of tags and increase throughput by eliminating the time and effort involved in developing labeling reagents.
Shoffner says that at a very high level, there are two overarching LFA technologies: Optical, plate-based detection systems and impedance-based systems. For the record, he notes that GE's Biocore system is the gold standard in surface plasmon resonance (SPR) systems, but is not plate-based and is therefore less amenable to HTS applications. Impedance-based systems are used exclusively for functional cell-based assays, Shoffner says, and measure impedance changes of cells across the bottom of wells as they go through morphological changes. Impedance systems cannot be used for protein-protein binding studies. Also, because impedance systems measure the effect created by cells at the bottom of the well, cells in suspension don't work well.
Optical systems, on the other hand, measure changes in wavelengths at the bottom of wells caused by mass changes in cells or proteins deposited on a biosensor and can be used for both cell-based and in vitro biochemical assays. Also, optical systems are equally at home with adherent and suspension cells.
Shoffner notes that pharma would like to drive label-free assays more upstream in the drug discovery process and be able to use phenotypic assays to measure, say, insulin levels as opposed to glucose uptake. And here the commercial-free part of the story ends, as Shoffner introduces SRU's high-resolution LFA system that can pick up response at the single-cell level as opposed to measuring a single response for each well. For the kinds of assays pharma would like to run, Shoffner says, primary—not immortalized—cells are necessary, and they are both scarce and expensive. Doing some quick math, he estimates that the new SRU BIND scanner with single-cell resolution will allow a reduction in the total number of cells needed for a 10-plate, 384-well run from something like 200 million cells to about 3.5 million. He also points out that, unlike immortalized cell lines, primary cells are not monoclonal, they're heterogeneous. The new BIND system can distinguish among cell types and stimulate only the type of interest with a ligand. The system is scheduled to be launched at SBS in Orlando. Caliper Discovery Alliances & Services (CDAS), a Caliper Life Sciences CRO, has noted that its fast plate reading time of less than one minute for 384-well plates makes the BIND system easily incorporated into HTS applications. Software provides a means to adjust what wells are read and how often, so that assay development is readily accomplished using the same instrument and plates used for HTS.
Advantages of label-free
According to research conducted by HTStec (formerly HTS Technologies), an independent consultancy that was founded by Dr. John Comley in spring 2002, there are many potential advantages to label-free approaches. Most noteworthy is that they can provide direct monitoring of analyte binding to target molecules without modifying the molecules of interest with labels or by using reporter systems. However, from a cell-based assay perspective, HTStec's survey found it was the ability to work with primary or non-engineered cells that was perceived as the most significant advantage of cell-based label-free assays. This was closely followed by the ability to screen difficult targets, then the ability to generate biorelevant data, and only after these factors came elimination of artifacts associated with labels. The next factor rated was an alternative readout technology for further validation of hit or lead compounds, which is where label-free assays are generating quite a lot of traction currently, HTStec found. Interestingly, 34 percent of survey respondents stated they have already successfully used label-free detection to perform testing against a difficult cell-based target, the majority of which were GPCRs.