The traditional, standard use of SPR is in the field of biological interaction analysis, otherwise known as BIA. The design of an SPR system gives them a high level of sensitivity to changes at the chip surface (see our tech notes for more detail). This means that we can track the interaction of molecules at the chip surface in fine detail, following numerous aspects of the process.
One of the most common SPR applications is in determining binding kinetics. In this case a molecule of interest, say a protein, is immobilised to the sensor chip. A solution containing a potential binding partner, perhaps the small molecular entity we are developing into a drug, is washed over the chip. We will see changes in resonance as the molecules interact, which provides information about the binding kinetics – switching the flow to buffer alone will then show the dissociation kinetics. Easy!
Thermodynamic calculations can be performed by leveraging the ability of XanTec SPR systems to operate at a wide range of temperatures, (up to 95°C with the correct module). Measurements of affinity constants at multiple temperatures can be used to calculate, via Van't Hoff thermodynamic analysis, changes in entropy and enthalpy during protein-protein interaction. Sound complex? Not really, it's an easy calculation to make using the data provided by XanTec's software.
Attempting to find the concentration of an analyte, for example in the media from your latest expression run? SPR allows simpler, faster techniques than those required for a typical ELISA. The secret lies in assaying solutions under mass-transport limited conditions (in which the analyte diffusing to the chip surface is the rate limiting step). In this situation, binding rate is directly proportional to analyte concentration, and so can be easily calculated. Best of all, it is a label-free method, saving you time and money.
Development of novel pharmaceuticals has long involved high throughput screening, in which thousands of small molecules are tested for their ability to inhibit a particular enzyme, process, etc. Sadly this method is not overly effective, and so many are focusing on fragment screening. In this technique thousands of even smaller molecules are tested for binding affinity, then linked together to make a synergistic, strong inhibitor.
As SPR is a highly sensitive test of binding affinity, it has become commonly used in fragment screening. Simply immobilise your enzyme of interest to the chip, use lab automation to load and run your samples, and then examine the data for interesting compounds.
Epitope Mapping of antibodies
Epitope mapping involves testing the ability of pairs of antibodies to bind to a target molecule – when both recognise to a similar epitope, interference will prevent simultaneous binding. This is commonly performed by ELISA, however this can be difficult to interpret and requires labelling of one antibody. A simpler method utilises SPR, immobilising one antibody to the chip, binding the target protein to it, and then washing the second antibody across. The SPR response curve shows binding (or lack thereof) unambiguously, while the ability to use unlabelled antibodies simplifies the experiment tremendously.
Selecting binding pairs for ELISA application
How does epitope mapping help further experiments? Suppose your group wants to develop an ELISA test, which requires both an immobilisation antibody and a detection antibody. Naturally, both of these antibodies need to bind different epitopes. Fortunately, it is easy to determine non-overlapping binding pairs using the data obtained from your SPR epitope mapping.
Screening for inhibitor specificity
Your group has spent months designing a diabody to target tumour cell antigens, but you need to know if specifically targets the antigen. Difficult? Simple! Immobilise your diabody to the chip, then pass a mixture of human proteins over, with and without added antigen. XanTec SPR systems then allow you to quantify just how much off-target binding is occurring, and this can then be minimised with further development.
Screening for cross-reactivity
The propensity of antibodies to bind to epitopes other than those they were raised against is known as cross-reactivity. This can be either a bonus, (e.g. vaccines against smallpox/cowpox), or a curse (when your new therapeutic antibody fails the tissue cross-reactivity test prior to Phase I). As such, determining cross-reactivity is an important part of the development process. SPR can provide many benefits in this process, ranging from mapping epitopes to quantifying the kinetics and binding specificity of these cross-reactive interactions.
Checking activity after purification
Your company has decided to improve downstream processing systems, and so are testing a number of new purification methods at lab scale. How do you verify, quantitatively, that the new system produces substrate-binding enzymes? By immobilising the substrate to a chip, the product from each purification method can be tested for binding affinity without the need for slow labelling reactions.