US 6,674,527 B2 · Ratiometric background correction for fluorescence polarization assays

Abstract

A method in which a multiplicative ratio approach is used to remove the effects of the unwanted background fluorescence when making fluoroescence polarization (FP) measurements rather than the conventional subtractive approach, thus preserving both the precision and accuracy of the FP measurements, is disclosed. The method comprises selecting an appropriate multiplicative ratio, then calculating the selected multiplicative ratio using sample measurements. The calculated multiplicative ratio is multiplied by an appropriate value in a standard FP measurement equation or an appropriate value in an equation derived from a standard FP measurement equation. After this, the corrected FP measurement is calculated. When such multiplicative ratios are applied to the appropriate value or values in an FP measurement equation, the effects of background noise can be reduced without decreasing the precision of the FP measurements.

Claims19

Dependent claims

A method for correcting for background fluorescence in one of a fluorescence polarization and a fluorescence anistrophy (FP/FA) measurement of a sample, comprising the steps of:

taking an initial FP/FA measurement of a sample, wherein the initial FP/FA measurement is uncorrected for background fluorescence;
calculating a ratio of an intensity of a background fluorescence to an intensity of a sample fluorescence (BTTR), wherein the sample fluorescence comprises at least a label fluorescence;
determining a FP/FA measurement of a background;
and
calculating a corrected FP/FA measurement using the calculated BTTR and the determined FP/FA of the background.

2 more elements

A method for correcting for background fluorescence in one of a fluorescence polarization and a fluorescence anistrophy (FP/FA) measurement of a plurality of samples, wherein each of the plural samples comprise an assay of at least a target substance and fluorophores, comprising the steps of:

measuring an intensity of parallel polarized light (I∥bkgrnd) from at least one well containing one or more assay components other than the fluorophores;
measuring an intensity of perpendicularly polarized light (I⊥bkgrnd) from the at least one well containing one or more assay components other than the fluorophores;
measuring a first sample intensity of a fluorescence of a first sample;
measuring a current sample intensity of a fluorescence of a current sample;
calculating a ratio of the measured first sample intensity to the measured current sample intensity (FTCR);
measuring an intensity of parallel polarized light (I∥crrnt) from the current sample;
measuring an intensity of perpendicularly polarized light (I⊥crrnt) from the current sample;
and
one of:
calculating a corrected FH measurement (FPcorrected) using following equation:
F ⁢ ⁢ P ⁢ c ⁢ ⁢ o ⁢ ⁢ r ⁢ ⁢ r ⁢ ⁢ e ⁢ ⁢ c ⁢ ⁢ t ⁢ ⁢ e ⁢ ⁢ d = 1000 * ( I ∥ crrnt - I ∥ bkgrnd * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) - ( I ⊥ crrnt - I ⊥ bkgrnd * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) ( I ∥ crrnt - I ∥ bkgrnd * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) + ( I ⊥ crrnt - I ⊥ bkgrnd * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) ;
⁢ and
calculating a corrected FA measurement (FAcorrected) using the following equation:
FA corrected = 1000 * ( I ∥ c ⁢ ⁢ r ⁢ ⁢ r ⁢ ⁢ n ⁢ ⁢ t - I ∥ b ⁢ ⁢ k ⁢ ⁢ g ⁢ ⁢ r ⁢ ⁢ n ⁢ ⁢ d * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) - ( I ⊥ crrnt - I ⊥ b ⁢ ⁢ k ⁢ ⁢ g ⁢ ⁢ r ⁢ ⁢ n ⁢ ⁢ d * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) ( I ∥ c ⁢ ⁢ r ⁢ ⁢ r ⁢ ⁢ n ⁢ ⁢ t - I ∥ b ⁢ ⁢ k ⁢ ⁢ g ⁢ ⁢ r ⁢ ⁢ n ⁢ ⁢ d * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) + 2 * ( I ⊥ crrnt - I ⊥ b ⁢ ⁢ k ⁢ ⁢ g ⁢ ⁢ r ⁢ ⁢ n ⁢ ⁢ d * F ⁢ ⁢ T ⁢ ⁢ C ⁢ ⁢ R ) .

11 more elements

Dependencies are inferred from the claim text.

Ratiometric background correction for fluorescence polarization assays

Life of this patent

Ratiometric background correction for fluorescence polarization assays

Life of this patent

Abstract

Claims19