METHODS FOR MULTIPLEX ANALYTE DETECTION AND QUANTIFICATION
First Claim
1. A method for detecting and quantifying two or more target analytes in a test sample, the method comprising:
- a) providing a reaction vessel having a microarray printed thereon, said microarray comprising;
i) a first calibration matrix comprising a plurality of first calibration spots, each calibration spot comprising a predetermined amount of a first target analyte,ii) a second calibration matrix comprising a plurality of second calibration spots, each calibration spot comprising a predetermined amount of a second target analyte,iii) a first capture matrix comprising a plurality of first capture spots, each capture spot comprising a predetermined amount of an agent which selectively binds to a first target analyte, andiv) a second capture matrix comprising a plurality of second capture spots, each capture spot comprising a predetermined amount of an agent which selectively binds to a second target analyte;
b) applying a predetermined volume of the test sample to the microarray;
c) applying a first fluorescently labelled antibody that selectively binds to the first target analyte and a second fluorescently labelled antibody that selectively binds to the second target analyte to the assay device, wherein said first and second fluorescently labelled antibodies each comprise a different fluorescent dye having emission and excitation spectra which do not overlap with each other;
d) measuring a signal intensity value for each spot within the microarray;
e) generating calibration curves by fitting a curve to the measured signal intensity values for each of the calibration spots versus the known concentrations of the first target analyte and second target analyte; and
determining the concentration for the first target analyte and the second target analytes utilizing the generated calibration curves.
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Abstract
The application refers to a method for detecting and quantifying multiple target analytes in a test sample using a single reaction vessel. The method uses a reaction vessel (a multi-well plate), which comprises a microarray of: (a) calibration spots, each having a predetermined quantity of the target analyte; and (b) capture spots, each having an agent (antibody) that selectively binds the target analyte. The captured analytes and the calibration spots are detected with fluorescently labelled antibodies specific for each different target analyte. The calibration spots are used to generate calibration curves that allow the measurement of the concentration of the different target analytes. The application also refers to a method for detecting and quantifying biomarkers that are useful for diagnosing rheumatoid arthritis. More specifically, the application discloses the use of rheumatoid factor (RF) and cyclic citrullinated peptide (CCP), as capture spots. Finally, based on the above method, it is proposed a method for diagnosing or monitoring rheumatoid arthritis.
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Citations
14 Claims
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1. A method for detecting and quantifying two or more target analytes in a test sample, the method comprising:
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a) providing a reaction vessel having a microarray printed thereon, said microarray comprising; i) a first calibration matrix comprising a plurality of first calibration spots, each calibration spot comprising a predetermined amount of a first target analyte, ii) a second calibration matrix comprising a plurality of second calibration spots, each calibration spot comprising a predetermined amount of a second target analyte, iii) a first capture matrix comprising a plurality of first capture spots, each capture spot comprising a predetermined amount of an agent which selectively binds to a first target analyte, and iv) a second capture matrix comprising a plurality of second capture spots, each capture spot comprising a predetermined amount of an agent which selectively binds to a second target analyte; b) applying a predetermined volume of the test sample to the microarray; c) applying a first fluorescently labelled antibody that selectively binds to the first target analyte and a second fluorescently labelled antibody that selectively binds to the second target analyte to the assay device, wherein said first and second fluorescently labelled antibodies each comprise a different fluorescent dye having emission and excitation spectra which do not overlap with each other; d) measuring a signal intensity value for each spot within the microarray; e) generating calibration curves by fitting a curve to the measured signal intensity values for each of the calibration spots versus the known concentrations of the first target analyte and second target analyte; and determining the concentration for the first target analyte and the second target analytes utilizing the generated calibration curves. - View Dependent Claims (2, 3, 4, 5, 12, 13, 14)
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6. A method for detecting and quantifying biomarkers diagnostic for rheumatoid arthritis, the method comprising:
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a) providing an assay device having a microarray printed thereon, said microarray comprising; i) a calibration matrix comprising a plurality of spots, each spot comprising a predetermined amount of one of;
a human IgA antibody, a human IgG antibody, and a human IgM antibody;ii) a first analyte capture matrix comprising a plurality of spots comprising a predetermined amount of rheumatoid factor; and iii) a second analyte capture matrix comprising a plurality of spots comprising a predetermined amount of cyclic citrullinated peptide; b) applying a predetermined volume of a serum sample to the assay device; c) applying a first fluorescently labelled antibody which selectively binds to IgA antibodies, a second fluorescently labelled antibody which selectively binds to IgG antibodies, and a third fluorescently labelled antibody which selectively binds to IgM antibodies to the assay device, wherein said first, second and third fluorescently labelled antibodies each comprise a different fluorescent dye having emission and excitation spectra which do not overlap with each other; d) measuring a signal intensity value for each spot within the assay device; e) generating calibration curves by fitting a curve to the measured signal intensity values for the each of the calibration spots versus the known concentration of the human IgA, IgG and IgM antibodies; and f) determining the concentration for each of captured rheumatoid factor-IgA, rheumatoid factor-IgG, rheumatoid factor-IgM, anti-cyclic citrullinated peptide-IgG, anti-cyclic citrullinated peptide-IgA, and/or anti-cyclic citrullinated peptide-IgM using utilizing the calibration curves; - View Dependent Claims (7, 8, 9, 10, 11)
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Specification