Biacore X100 reference portal

The amount of ligand to immobilize for any application depends in the first place on the relative molecular weights of the ligand and analyte and on the sensitivity of the Biacore system. Since the SPR response is directly proportional to the mass concentration of material at the surface, the analyte binding capacity of a given surface is related to the amount of ligand immobilized as follows (assuming a 1:1 binding stoichiometry):

For example, if the ligand molecular weight is 100,000 daltons and the analyte molecular weight is 50,000 daltons, immobilizing 5,000 RU of ligand will give a theoretical analyte binding capacity of 2,500 RU assuming that the ligand is 100% active. Note that this is the theoretical binding capacity, which can strictly be reached only at infinite analyte concentrations and infinite contact time. In practice, the maximum observed response (the effective binding capacity) is also affected by the activity of the ligand, the kinetics of analyte binding and limitations on the maximum contact time and available analyte concentration. The theoretical binding capacity can however be useful as a guide to how much ligand to immobilize and also as a reference for assessing the activity of the surface.

Guidelines

General guidelines for choosing a suitable immobilization level differ according to the purpose of the analysis:

• Concentration measurements
  For concentration assays with macromolecular ligands and analytes, immobilize as much ligand as is reasonably possible (typically 10,000–20,000 RU).
  
• Specificity measurements
  Aim for medium to high immobilization levels (typically 3000-10000 RU) so that the response to analyte binding can be seen clearly.
  
• Kinetic and affinity measurements
  Kinetics: The amount of immobilized ligand should be kept as low as possible (maximum analyte capacity is less than 30 RU) with an acceptably measurable response.
  Affinity: Aim for an immobilization level that gives a clearly measurable response over the whole range of analyte concentrations.
  

For concentration assays with macromolecular ligands and analytes, immobilize as much ligand as is reasonably possible. For protein ligands of average molecular size (of the order of 50,000–150,000 daltons), immobilization levels of 10,000–15,000 RU are typical. The analyte binding response at a given sample concentration is directly related to the level of immobilized ligand, so that a high immobilization level will enable measurements at lower analyte concentrations.

If the ligand is large and the analyte is small, responses will generally be low so that the sensitivity of a direct concentration assay may be limited even at high ligand immobilization levels (20,000 RU or higher). Concentration assays for small molecules are generally better designed as inhibition assays to overcome this limitation.

If the ligand is small and the analyte is large, it may be necessary to moderate the amount of ligand immobilized in order to avoid steric crowding effects at high analyte concentrations. This is also true for assays that use capturing or enhancement reagents, where a multi-molecular complex is built up on the sensor surface during the course of an assay. If several of the components are large, high levels of immobilized ligand can result in crowding and steric hindrance between binding molecules at a subsequent stage in the assay, limiting the observed response.

Aim for medium to high immobilization levels (typically 3,000-10,000 RU) so that the response to analyte binding can be seen clearly. If the immobilization level is too low or the immobilized ligand is only partially active the response will be low and may be misinterpreted as “no binding”. Specificity can be determined with partially inactive ligand provided that the analyte response can be measured with confidence.