Questions and answers during the Biacore webinar on 07 November presented by Matt Jones.
This article is a recap of the questions raised during the live questions and answers (Q&A) session.
A: Biacore 8K has full software support for screening and characterization of small molecules and fragments. There are predefined run methods and evaluation methods for all types of fragment and LMW experiments such as clean screen, binding level screen, affinity analysis, LMW screen and kinetics (multi- and single cycle mode, parallel mode and 2D mode).
A: Yes, you can use all 16 flow cells for analysis if you want to. Just bear in mind that this leaves you with no reference. For optimal performance the system is designed to allow for only intra-channel referencing. Thus, you cannot use 15 flow cells for analysis and 1 flow cells as a common reference for the 15 active flow cells.
A: The plot shows the adjusted response values while unadjusted responses are shown in the sensorgram.
A: Fragments are usually defined by a set of rules, all of them involving the number three. This means that for example the molecular weight of a fragment should less than (or close to) 300 Da, the number of hydrogen bond donors is not more than three and there are not more than three rotatable bonds.
A: We know that secondary interactions and other types of unwanted fragment behavior manifest and are easier to detect at higher concentrations. So, by performing screening experiments at high concentrations we can easily identify such behaviors early in the process and deselect them from the screening set and thereby save time in subsequent experiments.
A: The follow up experiments were done in competition mode. Here we screened our fragments in the presence of a compound that is known to bind to the allosteric binding site that we were interested. The compound in this case was our positive control, Filibuvir. By running the assay in competition mode it is possible to assess if the fragments bind to the same site as the allosteric binder or to another site.
Q: Why is there a report point early in the injection during affinity screen and what fitting model was used?
A: The report point is placed early in the injection to minimize disturbances from any secondary interactions. Secondary interactions usually occurs at a slow rate and will therefore increase during the injection. The affinity data was fitted using the model Steady state affinity with constant Rmax. Due to solubility limitations, it is difficult to run the fragments at a high enough concentration to obtain a correct Rmax. To overcome this problem, a saturating concentration of an Rmax control compound is injected in separate cycles. This Rmax value can be entered in the model which is then used to estimate the Rmax of each fragment.
A: Yes, in this study all antibodies were purified. However, we have run studies on Biacore 8K in which we used targets in crude sample matrices, such as hybridoma or CHO cell membrane preparations. These were injected directly into the flow system of the instrument without any centrifugation or filtering.
A: Biacore 8K and Biacore S200 are both very sensitive instruments. There is no lower limit for molecular weight and they can both measure kinetics in a very wide dynamic range and at low RU levels. However, Biacore S200 has slightly lower background noise and a 40 Hz data collection rate. That means that you can capture faster binding events on the S200 and you also get better resolution at milli-RU response levels. Bear in mind that this is only needed for very low surface densities and for challenging targets with low activity. For most applications, the two instruments are equal with respect to sensitivity.
Q: You mentioned that SPR is better than biochemical assays for FBDD. What techniques are you preferring to?
A: I am referring to enzyme activity-based fluorescence intensity, fluorescence-based assays, and mobility shift assays.
A: I have basic knowledge of ITC but very little actual experience. ITC was not used in the study described in the webinar. Please let me know if you have more questions.
A: For the antibody screening study the reference surface was immoblized with a goat anti-rabbit antibody, the same as for the active surface. For the fragment screening studies the reference surface was unmodified (just the actual sensor chip).
A: Biacore 8K and Biacore S200 are both very sensitive instruments. There is no lower limit for molecular weight and they can both measure kinetics in a very wide dynamic range and at low RU levels. However, Biacore S200 has slightly lower background noise and a 40 Hz data collection rate. That means that you can capture faster binding events on the S200 and you also get better resolution at milli-RU response levels. Bear in mind that this is only needed for very low surface densities and for challenging targets with low activity. For most applications, the two instruments are equal with respect to sensitivity.