Analytical measuring and evaluation method for molecular interactions
AssigneeGE Healthcare Bio-Sciences AB
InventorHans-Heinrich Trutnau
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Abstract
The invention relates to an analytical measuring and evaluation method for determining the interaction parameters between an analyte and a ligand, preferably in a biosensor. According to the inventive method, the concentration of the analyte is gradually changed at defined intervals ti and the initial association or dissociation rates or association and dissociation constants are determined. The invention further relates to a device for carrying out the inventive method.
Claims18
An analytical measurement and evaluation method for molecular interactions of chemical equilibrium binding pairs or binding systems, association and/or dissociation curves being acquired, and a first binding partner (“ligand”) having added to it a second binding partner (“analyte”), present in solution, which reacts reversibly with the ligand accompanied by complexation, the development over time of the complexation and its change
- 6 ( c t ( LA ) ;
- ⅆ c i ( LA ) ⅆ t )
- being detected and the reaction course being described, to a first approximation, with an exponentially proceeding function R(t), having an exponent k′on, or k′off, that tends toward an equilibrium value R′eq,
- 7 ⅆ R ( t ) ⅆ t
- indicating the association or dissociation rate of the analyte on the ligand,
- wherein at specific points in time ti the concentration cti(A) is raised or lowered, once or several times, stepwise;
- and from the respective differences between the equilibrium values R′eq,ass,i and R′st,ass,i or the differences between the values R′eq,diss,i and R′st,diss,i and the respective exponents k′on,i or k′off,i, the respective initial association or dissociation rates, and from those rates the respective association or dissociation rate constants kass or kdiss, can be ascertained.
The method as defined in one of the foregoing claims, wherein the initial rates are ascertained using the assumption of ideal conditions, in particular the assumption that no analyte depletion occurs upon association, and no analyte enrichment upon dissociation.
The method as defined in one of the foregoing claims, wherein the unknown analyte concentration c0(A) is ascertained at the beginning of the reaction.
The method as defined in one of the foregoing claims, wherein the dissociation rate constant kdiss is obtained from the initial association rates and the respective equilibrium association values of the function R(t), and the association rate constant kass from the initial dissociation rates and the respective equilibrium dissociation values of the function R(t).
The method as defined in one of the foregoing claims, wherein the method is carried out with affinity biosensors and/or array systems in solution and/or time-resolved ELISA or RIA techniques, known per se, or with other solid-phase-supported systems.
The method as defined in one of the foregoing claims, wherein in a cuvette system, the concentration of the solution in the measurement chamber is kept substantially constant during the association or dissociation process by continuous removal of a portion of the solution and addition of a solution having a corresponding concentration through one or more hollow needles.
The method as defined in one of the foregoing claims, wherein in the case of a substantially constant analyte concentration in each case for multi-step analyses, the values for kass and kdiss are ascertained from the relationship between k′on and the starting analyte concentration.
The method as defined in one of the foregoing claims, wherein with a constant concentration of the solution in the measurement chamber, a determination is made of that c0(A) value at which the association curve or a portion thereof intersects the dissociation curve or a portion thereof at the Golden Section of the overall association value or the respective partial value.
The method as defined in one of the foregoing claims, wherein the association rate constant kass is ascertained from the relationship between k′on and the equilibrium analyte concentration, and the dissociation rate constant kdiss is ascertained from the relationship between k′off and the equilibrium value of the function R(t) during dissociation.
An apparatus for carrying out the method as defined in one of the foregoing claims, having a sensor surface for binding the analyte, present in solution, to the ligand;
- having a measurement chamber on the sensor surface that is filled with the analyte in solution;
- having a respective hollow needle for the addition and/or removal of the solution and/or of a purging fluid;
- having an apparatus for mixing the solution in the measurement chamber;
- wherein the solutions in the measurement chamber are, at least partially, instantaneously exchangeable several times in a predefined quantity for solutions of a different concentration, the volume of the solution being kept substantially constant.
Dependencies are inferred from the claim text.