Calibration Free concentration analysis

Dear SPR Pages,
Can someone explain what is Calibration Free concentration analysis? How does it get concentration as opposed to using a UV spec or a Bradford technique in determining protein concentration? Also, how would it compare? If I got 1 mg/mL using a UV spec. Would I this CFCA to be close to 1 mg/mL also? Kindly comment.

Thanks.

Calibration Free Concentration Analysis (CFCA) (1, 4, 5) (2, 6) makes it possible the measure the active concentration of a ligand without a calibration curve. The method makes use of the mass transport limitation (km) calculated from the molecular mass (Mw), which occurs when high-density ligand surfaces are used. By injecting the analyte at two different flow rates (e.g. 10 – 90 µl min-1), the active analyte concentration can be calculated from the slopes of the curves. The advantage of the CFCA over more traditional concentration measurements is that CFCA measures the ‘active’ concentration. This means that only functional compounds are measured even in a crude solution. However, not all interactions are suitable for CFCA. The interaction should have a rapid association (ka > 5 x 105 M-1s-1) and have a reasonable affinity (KD < 10-7 M). The method assumes a 1:1 interaction and the molecular weight of the interactant must be known to calculate the diffusion coefficient. The accuracy is mainly dependent on the diffusion coefficient of the analyte but compared to other concentration measurement the CFCA method is very accurate (3).

Kinds regards
Arnoud


1. Chavane, N., Jacquemart, R., Hoemann, C. D., et al.; At-line quantification of bioactive antibody in bioreactor by surface plasmon resonance using epitope detection. Analytical Biochemistry (378) 2: 158-165; 2008.
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6. Sigmundsson, K., Masson, G., Rice, R., et al.; Determination of Active Concentrations and Association and Dissociation Rate Constants of Interacting Biomolecules: An Analytical Solution to the Theory for Kinetic and Mass Transport Limitations in Biosensor Technology and Its Experimental Verification. Biochemistry (41) 26: 8263-8276; 2002.