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Calibration-free concentration analysis (CFCA) is an approach to measuring analyte concentration with Biacore systems that does not require any calibration standards. The method relies on measuring the rate of binding under conditions where the rate is limited by diffusion of analyte to the surface and is independent of the interaction kinetics. Diffusion-controlled binding rates are directly proportional to the analyte concentration and are independent of interaction kinetics.

CFCA gives an absolute value for analyte concentration in weight-based or molar units. Note that as with any interaction-based concentration assay, the values obtained represent the concentration of active analyte molecules, as defined by their ability to interact with the surface-bound ligand.

Uses

CFCA is useful for

• determining the concentration in absolute terms for analytes for which calibration standards are not available.
• determining the amount of active analyte in calibration standards (i.e. determining the activity of the standards) in order to validate a calibrated assay. The effect of partial activity in calibration standards on the results of a calibrated assay is illustrated here:
  
  Partially active calibration standards
  
  If the calibration standards used in a concentration assay are only partially active in terms of binding to the ligand, the assay results will be distorted.
  Consider the situation where the calibration standards are only 80% active. A nominal standard concentration of, say, 1 nM will give a response that actually represents only 0.8 nM of active calibrant. A sample that gives the same response will contain 0.8 nM active analyte but will be reported as 1 nM. The accuracy of the reported value will depend on the active fraction in the sample, as illustrated in the table below.
  
  Effect of partial activity of standards on the accuracy of calibrated concentration measurements.
  
  

  	Calibrant 80% active	Sample 1 100% active	Sample 2 50% active
  Calibrant concentration	1 nM	--	--
  True active concentration	0.8 nM	0.8 nM	0.8 nM
  Reported concentration	--	1 nM	1 nM
  True total concentration	1 nM	0.8 nM	1.6 nM

• determining the true active concentration of analyte for robust measurement of kinetic and affinity constants.

Limitations

The requirement for measuring binding under conditions of limiting analyte diffusion imposes the following limitations on the method:

• High ligand immobilization levels are desirable. As a rule of thumb, do not immobilize less than 25 RU per 1000 Da ligand molecular weight (e.g. for a ligand of 150,000 Da, immobilize at least 3,750 RU. Immobilization levels of 5-10,000 RU are preferable for average-sized proteins).
• The usable range for the measured analyte concentration is limited to about 0.5-50 nM. Samples outside this range should be diluted or concentrated as appropriate for reliable measurement.
• Analytes smaller than about 5000 Da cannot be measured reliably.
• Analytes with slow association rates (k_a < 10⁵ M^-1s^-1) and/or low affinity (K_D > 10^-6 M) are not suitable.

Surface preparation

Use a high level of immobilized ligand to help ensure mass transport limited analyte binding. As a rule of thumb, do not immobilize less than 25 RU per 1000 Da ligand molecular weight (e.g. for a ligand of 150,000 Da, immobilize at least 3,750 RU. Immobilization levels of 5-10,000 RU are generally preferable for average-sized proteins).

Samples

• CFCA is not suitable for analytes with molecular weight below about 5000 Da.
• The working range of the CFCA assay is approximately 0.5-50 nM, with best results at about 5 nM. If you have no idea of the sample concentration, prepare a 10-times dilution series in running buffer with a total of 4 samples (covering the original sample to 1000-times diluted). This will often give at least one measurable concentration, or at least give an indication of a suitable dilution.

Setup

• Run each sample at two widely separated flow rates (recommended 5-10 and 100 µl/min). Assay wizards for CFCA fulfil this requirement automatically. For custom methods, you will need to set the flow rates explicitly.
• Running the samples at several dilutions (serial dilution factor 5 or 10) can help in assessing the validity of the results.
• Run duplicate determinations if time and material allow.
• Include at least one blank sample analyzed at the same flow rates as the samples. Subtraction of the blank cycles improves the robustness of the evaluation.
• Provide values for the analyte molecular weight and diffusion coefficient at 20°C. These values are required for evaluation of the results. The diffusion coefficient value is automatically converted to the analysis temperature in the evaluation software. Diffusion coefficients can be obtained from the literature or calculated from the molecular properties of the analyte using the Diffusion Coefficient Calculator available on the Cytiva web site.