Indirect colorimetric detection of an analyte in a sample using ratio of light signals

Claim 1

A method employing an enzyme immunoassay for measuring the concentration of an analyte in a sample by indirect colorimetric detection comprising: (a) combining an enzyme-labelled immunoconjugate, a sample to be tested for an analyte, and an antibody bound to a solid support so that the analyte binds to said bound antibody and to said conjugate to form an immunologic complex in solid phase; (b) admixing a liquid solution containing a chromogenic substance responsive to said enzymes with said immunologic complex to cause a reaction which activates the chromogenic substances said chromogenic substance being capable of absorbing increasing amounts of light at a first wavelength as the concentration of said analyte increases; (c) adding a substance to said admixture for causing attenuation of a light signal at or near said first wavelength; (d) directing incident light at a plurality of wavelengths into said admixture with said substance, said light including said first wavelength, a second wavelength of said light being spectrally removed from said first wavelength; (e) detecting light from the admixture at two different wavelengths and forming a ratio of said two detected wavelengths; and (f) comparing the magnitude of said formed ratio within the magnitude of a ratio associated with light detection when steps (a) to (e) are preformed with samples containing known concentrations of said analyte, where the concentration of the analyte in the sample is measured.

Claim 2

A method employing an enzyme immunoassay for measuring the concentration of an analyte in a sample by indirect colorimetric detection comprising: (a) combining an enzyme-labelled immunoconjugate, a sample to be tested for an analyte, and an antibody bound to a solid support so that the analyte binds to said bound antibody and to said conjugate to form an immunologic complex in solid phase; (b) admixing a liquid solution containing a chromogenic substance responsive to said enzymes with said immunologic complex to cause a reaction which activates the chromogenic substances said chromogenic substance being capable of absorbing increasing amounts of light at a first wavelength as the concentration of said analyte increases; (c) adding a substance to said admixture for causing attenuation of a light signal at or near said first wavelength; (d) directing incident light at a plurality of wavelengths into said admixture with said substance, said light including said first wavelength, a second wavelength of said light being spectrally removed from said first wavelength; (e) detecting light from the admixture at two different wavelengths and forming a ratio of said two detected wavelengths; and (f) comparing the magnitude of said formed ratio within the magnitude of a ratio associated with light detection when steps (a) to (e) are preformed with samples containing known concentrations of said analyte, where the concentration of the analyte in the sample is measured.

Claim 5

A method as claimed in any one of the preceding claims in which the analyte is selected human chrionic gonadotropin or luteinizing hormone.

Claim 13

A method for the detection of an analyte in a sample comprising: directing an incident light beam at a plurality of wavelengths into a liquid suspension containing an analyte of interest, said suspension being capable of attenuating the amount of light at a first wavelength scattered thereby as a function of the increasing concentration of the analyte present; detecting light scattered by said suspension at said first wavelength and at a second wavelength at which substantially no attenuation of light scatter occurs as the concentration of the analyte increases; forming a ratio of said two respective wavelengths an comparing said ratio with ratios of known amounts of said analyte to determine the amount of analyte in the sample.

Claim 14

A method for the detection of an analyte in a sample comprising: directing an incident light beam at a plurality of wavelengths into a liquid suspension containing an analyte of interest, said suspension being capable of attenuating the amount of light at a first wavelength scattered thereby as a function of the increasing concentration of the analyte present; detecting light scattered by said suspension at said first wavelength and at a second wavelength at which substantially no attenuation of light scatter occurs as the concentration of the analyte increases; forming a ratio of said two respective wavelengths an comparing said ratio with ratios of known amounts of said analyte to determine the amount of analyte in the sample.

Claim 15

A method for the detection of an analyte in a sample comprising: directing an incident light beam at a plurality of wavelengths into a liquid solution containing an analyte of interest, said solution being capable of attenuating the amount of light at a first wavelength proportional to the increasing concentration of the analyte present in the sample; detecting light signals from said solution related to said first wavelength and at a second wavelength at which substantially no attenuation of light occurs as the concentration of the analyte increases; and forming a ratio of said two respective wavelengths and comparing said ratio with ratios of known amounts of said analyte to determine the amount of analyte in said sample.

Claim 16

A method for the detection of an analyte in a sample comprising: directing an incident light beam at a plurality of wavelengths into a liquid solution containing an analyte of interest, said solution being capable of attenuating the amount of light at a first wavelength proportional to the increasing concentration of the analyte present in the sample; detecting light signals from said solution related to said first wavelength and at a second wavelength at which substantially no attenuation of light occurs as the concentration of the analyte increases; and forming a ratio of said two respective wavelengths and comparing said ratio with ratios of known amounts of said analyte to determine the amount of analyte in said sample.

Claim 18

A method employing an enzyme immunoassay for measuring the concentration of analyte in a sample by direct colorimetric detection comprising: (a) combining a peroxidase-labelled antibody conjugate, a sample to be tested for an analyte, and an antibody bound to the interior surfaces of a transparent plastic tube so that the analyte binds to said bound antibody and said conjugate to form an immunologic complex in solid phase; (b) admixing into said tube a liquid solution containing a tetramethylbenzidine and hydrogen peroxide to cause a reaction with said immunologic complex and to activate the tetramethylbenzidine for a colorimetric response; (c) adding to said admixture a particle-containing solution which terminates said reaction and causes a stable particulate suspension to result; (d) directing incident light at a plurality of wavelengths through said tube into said suspension, a first wavelength of said incident light being substantially at 450nm at which activated tetramethylbenzidine attenuates the amount of light scattered from said incident light as a function of the increasing concentration of the analyte present, a second wavelength being spectrally removed from said first wavelength and at which substantially no attenuation of light scatter occurs as the concentration of the analyte increases; (e) detecting light scattered by the suspension at said first and at said second wavelengths and forming a ratio of said two respective wavelengths; and (f) comparing the magnitude of said formed ratio with the magnitude of a ratio associated with light scatter detection when steps (a) to (e) are performed with samples containing known concentrations of said analyte, whereby the concentration of the analyte in the sample is measured.

Claim 19

A method employing an enzyme immunoassay for measuring the concentration of analyte in a sample by direct colorimetric detection comprising: (a) combining a peroxidase-labelled antibody conjugate, a sample to be tested for an analyte, and an antibody bound to the interior surfaces of a transparent plastic tube so that the analyte binds to said bound antibody and said conjugate to form an immunologic complex in solid phase; (b) admixing into said tube a liquid solution containing a tetramethylbenzidine and hydrogen peroxide to cause a reaction with said immunologic complex and to activate the tetramethylbenzidine for a colorimetric response; (c) adding to said admixture a particle-containing solution which terminates said reaction and causes a stable particulate suspension to result; (d) directing incident light at a plurality of wavelengths through said tube into said suspension, a first wavelength of said incident light being substantially at 450nm at which activated tetramethylbenzidine attenuates the amount of light scattered from said incident light as a function of the increasing concentration of the analyte present, a second wavelength being spectrally removed from said first wavelength and at which substantially no attenuation of light scatter occurs as the concentration of the analyte increases; (e) detecting light scattered by the suspension at said first and at said second wavelengths and forming a ratio of said two respective wavelengths; and (f) comparing the magnitude of said formed ratio with the magnitude of a ratio associated with light scatter detection when steps (a) to (e) are performed with samples containing known concentrations of said analyte, whereby the concentration of the analyte in the sample is measured.

Claim 21

A method employing an enzyme immunoassay for measuring the concentration of an analyte in a sample by indirect colorimetric detection comprising; (a) combining a peroxidase-labelled antibody conjugate, a sample to be tested for an analyte, and an antibody bound to the interior surfaces of a transparent plastic tube so that the analyte binds to said bound antibody and said conjugate to form an immunologic complex in solid phase; (b) admixing into said tube a liquid solution containing a retramethylbenzidine and hydrogen preoxide to cause a reaction with said immunological complex and to activate the tetramethylbenzidine for a colorimetric response; (c) adding to said admixture a first fluorophore for causing attenuation of fluorescence at or near the absorbance maxima of activated tetramethylbenzidine as a function of the increasing concentration of the analyte present in the sample; (d) adding to said admixture a second fluorophore which has substantially no attenuation of its flourescence as the concentration of the analyte increases; (e) directing incident light at a plurality of wavelengths through said tube into said last-mentioned admixture, said wavelengths including a wavelength at or near said absorbance maxima of activated tetramethylbenzidine, and including wavelengths for causing excitation of said first and said second fluorophores; (f) detecting fluorescence emitted by the fluorophores and forming a ratio of the two fluorescence wavelength; and (g) comparing the magnitude of said formed ratio with the magnitude of a ratio associated with fluorescence detection when steps (a) to (f) are performed with samples containing known concentrations of said analyte, whereby the concentration of the analyte in the sample is measured.

Claim 22

A method employing an enzyme immunoassay for measuring the concentration of an analyte in a sample by indirect colorimetric detection comprising; (a) combining a peroxidase-labelled antibody conjugate, a sample to be tested for an analyte, and an antibody bound to the interior surfaces of a transparent plastic tube so that the analyte binds to said bound antibody and said conjugate to form an immunologic complex in solid phase; (b) admixing into said tube a liquid solution containing a retramethylbenzidine and hydrogen preoxide to cause a reaction with said immunological complex and to activate the tetramethylbenzidine for a colorimetric response; (c) adding to said admixture a first fluorophore for causing attenuation of fluorescence at or near the absorbance maxima of activated tetramethylbenzidine as a function of the increasing concentration of the analyte present in the sample; (d) adding to said admixture a second fluorophore which has substantially no attenuation of its flourescence as the concentration of the analyte increases; (e) directing incident light at a plurality of wavelengths through said tube into said last-mentioned admixture, said wavelengths including a wavelength at or near said absorbance maxima of activated tetramethylbenzidine, and including wavelengths for causing excitation of said first and said second fluorophores; (f) detecting fluorescence emitted by the fluorophores and forming a ratio of the two fluorescence wavelength; and (g) comparing the magnitude of said formed ratio with the magnitude of a ratio associated with fluorescence detection when steps (a) to (f) are performed with samples containing known concentrations of said analyte, whereby the concentration of the analyte in the sample is measured.