METHODS FOR DETERMINING KINETIC PARAMETERS OF A REACTION BETWEEN ANALYTE AND LIGANDS

According to the present invention there is provided a method for determining kinetic parameters of a reaction between analyte and ligands, the method comprising the steps of, a) flowing a first volume of sample fluid (V1), which has a concentration (co) of analytes, over a test surface (3) of a flow cell (2) which has first ligands (4) attached thereto, for a first time period (tia); b) flowing a first volume of buffer solution (X/Γ) over the test surface (3) of the flow cell (2), for a first time period (tib); c) flowing at least a second volume of sample fluid (V2), over the test surface, for a second time period (t2a); wherein the second time period (t2a) is greater than the first time period (tia) and wherein the second volume of sample fluid (V2) has the same concentration (c0) of analytes as the concentration (c0) of analytes in the first volume of sample fluid (V2); d) flowing a second volume of buffer solution (V2′) over the test surface (3) of the flow cell (2), for a second time period (t2b); e) using a sensor to measure the binding on the test surface (3) during steps (a)-(d) to obtain a single measurement binding curve; f) flowing a first volume of sample fluid (V1), which has a concentration (Co) of analytes, over a test surface (3′) of a flow cell (2′) which is without first ligands, for a first time period (tia); g) flowing a first volume of buffer solution (X/Γ) over the test surface (3′) which is without first ligands, for a first time period (fib); h) flowing at least a second volume of sample fluid (V2), over the test surface (3′) which is without first ligands, for a second time period (t2a); wherein the second time period (t2a) is greater than the first time period (t1a) and wherein the second volume of sample fluid (V2) has the same concentration (c0) of analytes as the concentration (c0) of analytes in the first volume of sample fluid (V2); i) flowing a second volume of buffer solution (V2′) over the test surface (3) which is without first ligands, for a second time period (t2b); J) using a sensor to measure the binding on the test surface (3) which is without first ligands, during steps (f)-(i), to obtain a single reference binding curve; k) Using the single measurement binding curve and the single reference binding curve to determine the kinetic parameters.