US 2007/0166762 A1Published application

Method of compensation of dose-response curve of an assay for sensitivity to perturbing variables

AssigneeNovx Systems Canada Inc.

InventorsStephen Leonard, Samad Talebpour

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Abstract

The present invention provides assays and methods of compensating for changes in the dose-response curve of an assay where such changes are due to variations in a perturbing variable such as, but not limited to temperature. This is achieved by a two-step method, the first step of which involves measurements of the dose-response curve, and thus the individual assay parameters, at many different values of the perturbing variable, spanning the expected range of the perturbing variable. In the second step, unknown samples are assayed simultaneously with a known standard at a chosen analyte concentration. During this measurement, the value of the perturbing variable is unknown and the dose-response curve is therefore also unknown. The different dose-response curves from the first step are used to determine a mathematical relationship between the assay parameters and the assay signal of the known standard. With this relationship, in which the value of the perturbing variable is implicit rather than explicit, assay parameters that are valid for the unknown value of the perturbing variable can be obtained by substituting the value of the assay signal from the known standard (measured when assaying the unknown samples) into the mathematical relationship and solving for the assay parameters. The method enables an accurate determination of the analyte concentration even when the perturbing variable is changing or fluctuating from one sample measurement to another. Once the first step is completed, the second step can be performed repeatedly to measure unknown samples with accuracy.

Claims46

Dependent claims

A method of determining a dose-response curve of one or more assays, where said one or more assays are sensitive to the effects of a perturbing variable, comprising the steps of:

A) characterizing a dependence of said dose-response curve of one or more assays on said perturbing variable by the steps of,
i) measuring assay signals from a suitable set of known standards for said one or more assays at different values of said perturbing variable, and also measuring one or more other signals dependent on said perturbing variable, and whereby said one or more other signals dependent on said perturbing variable are measured for each of said different values of said perturbing variable;
ii) fitting said measured assay signals to a suitable functional form to produce a dose-response curve for each of said one or more assays at each of said different values of said perturbing variable and determining assay parameters from said fitting;
and
iii) establishing a mathematical relationship between each assay parameter of said one or more assays and each of said one or more other signals dependent on said perturbing variable;
B) when performing one or more assays of unknown samples, determining a dose-response curve that compensates for the effects of changes in the value of said perturbing variable by the steps of:
iv) re-measuring said one or more other signals dependent on said perturbing variable;
v) obtaining a value of each assay parameter of said one or more assays by substituting one of said one or more re-measured other signals dependent on said perturbing variable into said mathematical relationship between said each assay parameter and said one of said one or more other signals dependent on said perturbing variable, and repeating said substitution for each of said one or more other signals dependent on said perturbing variable to obtain one or more values of said each assay parameter;
and
vi) producing a compensated dose-response curve for each of said one or more assays of said unknown samples by averaging said one or more values of each assay parameter of said one or more assays.

7 more elements

A method of compensating a previously measured dose-response curve of one or more assays for the effects of a perturbing variable to which said one or more assays are sensitive, comprising the steps of:

A) characterizing a dependence of a dose-response curve of one or more assays on said perturbing variable by the steps of,
i) measuring assay signals from a suitable set of known standards for said one or more assays at different values of said perturbing variable, and also measuring one or more other signals dependent on said perturbing variable, and whereby said one or more other signals dependent on said perturbing variable are measured for each of said different values of said perturbing variable;
ii) fitting said measured assay signals to a suitable functional form to produce a dose-response curve for each of said one or more assays at each of said different values of said perturbing variable and determining assay parameters from said fitting;
and
iii) establishing a linear mathematical relationship between each assay parameter of said one or more assays and each of said one or more other signals dependent on said perturbing variable, and including obtaining a slope of said linear mathematical relationship;
B) periodically measuring said dose-response curves of said one or more assays by measuring assay signals from a suitable set of known standards for said one or more assays and fitting said measured assay signals to a suitable functional form to produce a dose-response curve for each of said one or more assays and when measuring said dose-response curves, also measuring said one or more other signals dependent on said perturbing variable;
C) when performing one or more assays of unknown samples, compensating for the effect of a change in said perturbing variable on a previously measured dose response curve for said one or more assays, where said one or more other signals dependent on said perturbing variable were measured with said previously measured dose-response curve for said one or more assays, by the steps of:
iv) re-measuring said one or more other signals dependent on said perturbing variable;
v) obtaining a compensated value of each assay parameter of said one or more assays by adding to each assay parameter obtained from said previously measured dose-response curve for said one or more assays a product of the following two terms:
1) said slope of said linear mathematical relationship between said each assay parameter and one of said one or more other signals dependent on said perturbing variable;
and
2) a difference between said one of said one or more other signals dependent on said perturbing variable re-measured with said assays for said unknown samples and said one of said one or more other signals dependent on said perturbing variable measured with said previously measured dose-response curve;
and repeating steps iv) and v) for each of said one or more other signals dependent on said perturbing variable to obtain one or more values of said each assay parameter;
and
vi) producing a compensated dose-response curve for each of said one or more assays of said unknown samples by averaging said one or more values of each assay parameter for each of said one or more assays.

12 more elements

Dependencies are inferred from the claim text.