# US 2010/0167422 A1 · Method of determining analyte concentration > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0167422 A1 - Document ID: US20100167422A1 - Status: Published application - Kind: A1 - Application number: 12/377,021 - Filed: 2007-09-12 - Published: 2010-07-01 - Earliest claimed: 2006-09-14 - Assignees: GE Healthcare Bio-Sciences AB - Inventors: Henrik Berling; Tanja Jarhede; Anita Larsson; Hakan Roos - Web page: https://patentgrep.com/patent/US20100167422A1 ## CPC - G01N 33/54306: {Solid-phase reaction mechanisms} - G01N 33/54366: {Apparatus specially adapted for solid-phase testing} - G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding} - G01N 15/1459: {the analysis being performed on a sample stream} - G01N 15/1484: {microstructural devices} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A method of determining the total concentration of an analyte in a fluid sample, wherein at least part of the analyte is present as a complex with an analyte-binding species. The methods includes the steps of: a) subjecting the sample to conditions that reduce the binding affinity between analyte and analyte-binding species sufficiently to dissociate substantially any analyte complex and provide substantially all analyte in free form, b) subjecting the sample to conditions that restore the binding affinity between analyte and analyte-binding species, and c) immediately after the binding affinity has been restored, and before any substantial re-complexing of the analyte has taken place, determining the concentration of free analyte in the sample. A method of determining the concentration of complex-bound analyte in a sample is also disclosed. ## Claims ### Claim 1 A method of determining the total concentration of an analyte in a fluid sample, wherein at least part of the analyte is present as a complex with an analyte-binding species, comprising the steps of: a) subjecting the sample to conditions that reduce the binding affinity between analyte and analyte-binding species sufficiently to dissociate substantially any analyte complex and provide substantially all analyte in free form, b) subjecting the sample to conditions that restore the binding affinity between analyte and analyte-binding species, and c) immediately after the binding affinity has been restored, and before any substantial re-complexing of the analyte has taken place, determining the concentration of free analyte in the sample. ### Claim 2 (depends on 1) The method of claim 1, wherein the determination of free analyte in the sample comprises contacting the sample with a solid support having an analyte-binding ligand immobilized thereon to bind analyte to the immobilized ligand. ### Claim 3 (depends on 1) The method of claim 1, wherein the conditions that reduce the binding affinity in step a) and restore the binding affinity in step b) comprise changing the pH-value of the sample. ### Claim 4 (depends on 1) The method of claim 1, wherein the complex is an acid-dissociable complex, step a) comprises acidifying the sample, and step b) comprises neutralizing the acidified sample. ### Claim 5 (depends on 1) The method of claim 1, wherein the complex is an immune-complex. ### Claim 6 (depends on 1) The method of claim 1, wherein the analyte is an antibody to a therapeutic antibody and the sample contains the therapeutic antibody. ### Claim 7 (depends on 1) The method of claim 1, wherein the concentration of analyte is determined by using a label-free detection technique. ### Claim 8 (depends on 7) The method of claim 7, wherein the label-free detection technique comprises evanescent wave sensing, particularly surface plasmon resonance (SPR). ### Claim 9 (depends on 1) The method of claim 1, wherein at least step c) is performed in a flow cell. ### Claim 10 (depends on 9) The method of claim 9, wherein step b) is also performed in a flow cell, and the flow cell comprises a detection area with immobilized ligand, and an inlet which via a junction is connected to first and second conduits, further wherein step b) comprises flowing a sample with dissociated analyte complexes in the first conduit and a fluid capable of restoring the analyte binding capability in the second conduit so that the two fluids mix at the junction of the flow cell inlet conduit and the mixed fluids pass through the flow cell over the detection area. ### Claim 11 (depends on 10) The method of claim 10, wherein (i) the distance between the detection area and the junction, and (ii) the fluid flow rates in the first and second conduits are selected such that when the mixed fluids reach the detection area, the analyte binding capability is substantially restored but re-complexing of the analyte has substantially not taken place. ### Claim 12 (depends on 10) The method of claim 10, wherein the fluid flowing in the first conduit is an acidified sample, and the fluid flowing in the second conduit is an alkaline fluid. ### Claim 13 (depends on 12) The method of claim 12, wherein, when the mixed fluids reach the detection area, the acidified sample is substantially neutralized but re-complexing of the analyte has substantially not taken place. ### Claim 14 (depends on 1) A method of determining the concentration of complex-bound analyte in a sample which comprises the steps of: a) determining the total concentration of analyte in a sample of claim 1; and b) determining the concentration of free analyte in the sample, the difference between the concentrations obtained in steps a) and b) representing the concentration of complex-bound analyte. ### Claim 15 (depends on 14) The method of claim 14, wherein the determination of total analyte in step a) and free analyte in step b) comprises contacting the sample with a solid support having an analyte-binding ligand immobilized thereon to bind analyte to the immobilized ligand. ### Claim 16 A method of determining the capability of an analyte to form a complex with one or more species in a sample containing the analyte, which comprises the steps of: a) subjecting the sample to conditions that reduce the binding affinity between analyte and analyte-binding species sufficiently to dissociate substantially any analyte complex and provide substantially all analyte in free form, b) subjecting the sample to conditions that restore the binding affinity between analyte and analyte-binding species, c) immediately after the binding affinity has been restored, and before any substantial re-complexing of the analyte has taken place, contacting the sample with a solid support having the analyte immobilized thereon, and d) analyzing the species bound to the immobilized analyte.