METHOD OF DETERMINING ACTIVE CONCENTRATION BY CALIBRATION-FREE ANALYSIS
A method of determining active concentration of an analyte in a liquid sample, comprises the steps of: (a) contacting a laminar flow of the sample with a solid phase surface or surface area supporting a ligand capable of specifically binding the analyte at at least two different flow rates and under partially or completely mass transport limited conditions; (b) determining the initial binding rate dR/dt of analyte to the ligand at the ligand-supporting surface or surface area, and (c) fitting the initial binding rate data obtained in step (b) to a kinetic interaction model that includes a term for mass transport to obtain the active analyte concentration, wherein steps (a) and (b) are performed at a plurality of different dilutions of the liquid sample, and wherein in step (c) at least several of the plurality of dilutions of the liquid sample are in included in a global fit of initial binding rate data to the kinetic interaction model.
1 . A method of determining active concentration of an analyte in a liquid sample, comprising the steps of:
(a) contacting a laminar flow of the sample with a solid phase surface or surface area supporting a ligand capable of specifically binding the analyte at at least two different flow rates and under partially or completely mass transport limited conditions;
(b) determining the initial binding rate dR/dt of analyte to the ligand at the ligand-supporting surface or surface area, and
(c) fitting the initial binding rate data obtained in step (b) to a kinetic interaction model that includes a term for mass transport to obtain the active analyte concentration,
wherein steps (a) and (b) are performed at a plurality of different dilutions of the liquid sample, and
wherein in step (c) at least several of the plurality of dilutions of the liquid sample are in included in a global fit of initial binding rate data to the kinetic interaction model.
2 . The method of claim 1 , wherein the method is performed at two substantially different flow rates.
3 . The method of claim 1 , wherein the different flow rates are obtained by varying the flow rate during a single contacting cycle.
4 . The method of claim 1 , wherein at least three, and preferably at least five different sample dilutions are used.
5 . The method of claim 1 , further comprising contacting a laminar flow of the liquid sample with a plurality of solid phase surfaces or surface areas, each surface or surface area having a different ligand density, determining from the initial binding rates the initial binding rate corresponding to transport-limited interaction at the surfaces or surface areas, and from that binding rate determining the active analyte concentration.
6 . The method of claim 1 , wherein an interaction analysis sensor is used, preferably a biosensor.
7 . The method of claim 6 , wherein the interaction analysis sensor is based on mass-sensing, preferably evanescent wave sensing, especially surface plasmon resonance (SPR).
8 . The method of claim 1 , which is computer-implemented.
9 . A computer program product comprising instructions for causing a computer to perform the method steps of claim 1 .