Material this artefact is greater when i there are

This preview shows page 21 - 24 out of 50 pages.

material. This artefact is greater when (i) there are big differences in the levels of immobilised material (e.g. >2000 RUs), (ii) the background signal is very large (e.g. >1000 RUs), and (iii) the - 21 -
binding response is much smaller (<10%) than the background response. These conditions are common when measuring very weak interactions, because high concentrations of analyte are injected, and with low molecular weight analytes, which give a very small response. A refractive index artefact can be detected by injecting a control solution with a similar refractive index to the analyte sample. If the control solution gives the same response in both flow-cells a refractive index artefact can be excluded. Refractive index artefacts are most easily avoided by taking care to immobilise the same amount of total material on both the control and ligand sensor surfaces. 5.4. Low molecular weight analytes
6. Qualititative analysis; do they interact?
- 22 -
Blocking experiments, using molecules known to block the interaction, are also useful negative controls. In contrast, if no binding is detected it becomes necessary to run positive controls to establish whether this reflects the absence of an interaction, a very low affinity, or an artefact resulting from defective ligand or analyte. The ligand is readily assessed by showing that it can bind to one or more mAbs or additional analytes should they exist. The analyte can be tested by injecting it over a surface with either a known interacting ligand or or mAb immobilised to the surface. 6.2. Qualitative comparisons using a multivalent analyte The binding of a multivalent analyte can be heavily influenced by the level of immobilised ligand, since the latter will influence the valency of binding. Thus when comparing the binding of a multivalent analyte to different ligands it is important that these ligands are immobilised at comparable surface densities.

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture