Light scatter-based immunoassay
First Claim
1. A particle light scatter-based immunoassay for measuring an analyte in a fluid sample, comprising steps:
- (a) combining with said fluid sample a reagent set including first binding molecule-coated monodisperse microspheres and second binding molecule-coated colloidal particles smaller than said microspheres, or an immunoconiplex comprising said monodisperse microspheres and said colloidal particles, to form a mixture and allow a reaction to occur wherein at least one of said first and second binding molecules binds said analyte, said reaction being formation or decomplexation of said immunocomplex, so that the reacted mixture includes microspheres in uncompleted form, in said immunocomplex, or both, wherein the degree of binding of said microspheres to the colloidal particles in the reacted mixture, or the degree of decomplexation of the immunocomplex, is dependent upon presence or amount of said analyte in said fluid sample;
(b) illuminating the reacted mixture with an incident light source to produce individual light scatter signals for each of said microspheres;
(c) measuring said light scatter signals;
(d) determining a degree of variation of a statistical distribution of the measured light scatter signals from said microspheres, such that the degree of variation of said statistical distribution varies with the degree of binding of said microspheres with said colloidal particles in said reacted mixture; and
(e) correlating said degree of variation of said statistical distribution of said light scatter signals for said microspheres in said reacted mixture with the presence or amount of said analyte in said fluid sample.
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Abstract
Disclosed are an optical flow particle apparatus and method for conducting a particle light scatter-based immunoassay for simultaneously measuring the presence or amount of one or more analytes in a fluid sample which involves the use of a reagent set for each analyte including first binding molecule-coated monodisperse microspheres and second binding molecule-coated colloidal particles in which at least one of the first or second binding molecules specifically binds a respective one of the analytes. In the case where more than one analytes are detected, each monodisperse microperse microsphere of a particular reagent set has a light scatter signal resolvable from that of microspheres of any other reagent set. Changes determined in the distributions of the measured light scatter signals for individual microspheres of each of the particular reagent sets are indicative of the presence or amount of the respective analyte(s) in the sample.
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Citations
57 Claims
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1. A particle light scatter-based immunoassay for measuring an analyte in a fluid sample, comprising steps:
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(a) combining with said fluid sample a reagent set including first binding molecule-coated monodisperse microspheres and second binding molecule-coated colloidal particles smaller than said microspheres, or an immunoconiplex comprising said monodisperse microspheres and said colloidal particles, to form a mixture and allow a reaction to occur wherein at least one of said first and second binding molecules binds said analyte, said reaction being formation or decomplexation of said immunocomplex, so that the reacted mixture includes microspheres in uncompleted form, in said immunocomplex, or both, wherein the degree of binding of said microspheres to the colloidal particles in the reacted mixture, or the degree of decomplexation of the immunocomplex, is dependent upon presence or amount of said analyte in said fluid sample;
(b) illuminating the reacted mixture with an incident light source to produce individual light scatter signals for each of said microspheres;
(c) measuring said light scatter signals;
(d) determining a degree of variation of a statistical distribution of the measured light scatter signals from said microspheres, such that the degree of variation of said statistical distribution varies with the degree of binding of said microspheres with said colloidal particles in said reacted mixture; and
(e) correlating said degree of variation of said statistical distribution of said light scatter signals for said microspheres in said reacted mixture with the presence or amount of said analyte in said fluid sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
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57. A particle light scatter-based immunoassay for simultaneously measuring more than one analyte in a fluid sample, comprising steps:
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(a) combining with said fluid sample for each said more than one analyte, a reagent set including first binding molecule-coated monodisperse microspheres and second binding molecule-coated colloidal particles smaller than said microspheres, or an immunocomplex comprising said monodisperse microspheres and said colloidal particles, to form a mixture of fluid sample and all reagent sets together and allow a reaction to occur wherein at least one of said first and second binding molecules of each said reagent set binds a respective one of said more than one analyte, said reaction being formation or decomplexation of each said immunocomplex, so that the reacted mixture includes microspheres of each said reagent set in uncomplexed form, in said immunocomplex, or both, wherein the degree of binding of said microspheres to the colloidal particles of each said reagent set in the reacted mixture, or the degree of decomplexation of each said immunocomplex, is dependent upon presence or amount of each said more than one analyte in said fluid sample;
(b) illuminating the reacted mixture with an incident light source to produce individual light scatter signals for each of said microspheres wherein the light scatter signals produced for the microspheres of each said reagent set are resolvable from the light scatter signals produced for the microspheres of any other said reagent set;
(c) measuring said light scatter signals;
(d) determining a degree of variation of a statistical distribution of the measured light scatter signals from said microspheres of each said reagent set, such that the degree of variation of said statistical distribution varies with the degree of binding of said microspheres with said colloidal particles of each said reagent set in said reacted mixture; and
(e) correlating said degree of variation of eh said statistical distribution of said light scatter signals for said microspheres in said reacted mixture with the presence or amount of each respective one of said more than one analyte in said fluid sample.
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Specification