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FAC Advantages

There are numerous advantages to the FAC approach that differentiate it from many forms of bioassay - MS-dependent or otherwise. The FAC method offers thermodynamic and kinetic binding data from the breakthrough curves. As with the classical application of the FA method, the quality of the data is superb relative to other chromatographic or electrophoretic methods [9, 10]. It is an equilibrium method, as opposed to systems that rely upon the separation of bound from unbound, and this forms the basis of its accuracy.

The most significant figure of merit is the breakthrough volume and assuming a simple equilibrium model, this volume is used to determine the dissociation constant for the interaction being studied. These values may be measured by any appropriate detector and it is interesting to note that no detector calibration is required, nor does the accuracy of the volume depend on the efficiency of the column. A membrane, cartridge or high-efficiency microbore column can all basically provide the same V (and thus Kd) [11].

In other words, many different styles of affinity construct can be built, all of which can support breakthrough volume measurements. This is a liberating concept, as one can imagine making cheaper/simpler cartridges for high volume applications and more specialized columns for higher precision in follow-on measurements. It is also significant that the detector does not play a significant role in the assay, aside from monitoring breakthrough volumes. The detector is simply required to determine when breakthrough occurs and, as we will see, MS does provide some unique advantages in this regard.

A subtle but unique advantage to the method stems from the distinction between detecting the compound rather than the binding event. Essentially, FAC achieves molecular interaction analysis in a concentration-independent manner. Many assay types generate a signal that is in some way proportional to the amount of bound ligand. According to the law of mass action, this implies that the Kd of an interaction strongly determines successful detection, and means that a weak interaction cannot be detected as sensitively as a strong interaction. Low concentrations relative to the Kd of the interaction always generate the maximum breakthrough volume, as will be shown below, and so as long as the detector is sufficiently sensitive, the FAC method can detect interaction Kd values ranging from millimolar to picomolar without modifying the assay. This is an important advantage. Developments in MS detection ensure that we can "find" low abundance hits/ligands that may be present in the sample well below their Kd values. For example, a 10 mM Kd ligand present at a concentration of 1 nM would be difficult to detect with capture/wash methods as washing conditions would likely remove the bound ligand. This sets the method apart from biosensors in which the signal strength is directly related to the amount bound. While the high sensitivity of SPR-based biosensors can ameliorate this to a degree, there are clear advantages to removing the dependency on Kd value for detecting a binding event.

There are no inherent limitations to the nature of the interaction that can be probed with the FAC method. This too stems from an uncoupling of the binding event and the detector. The method can be applied to simple binary interactions between protein and small molecule, but also to protein-protein interactions, protein-cell interactions and virtually any interaction that can be modeled in a flow system. Some of the more elegant examples include drug interaction with whole cells [12] and membrane-bound receptors from brain homogenates [13]. Ultimately, the limitations are dictated by what can be detected from a stream of column effluent.

While it is possible for a FAC experiment to require excessive sample in order to equilibrate the column and generate a breakthrough curve, this can be easily prevented during column design. Lowering the binding capacity of the affinity column brings with it reduced sample requirements [10]. In practice, miniaturization to easily-constructed micro-cartridges supports sub-picomol amounts of immobilized protein and similar amounts of sample. This is comparable to modern biosensor technology in both operation and consumption of sample.

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