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

• US 4,954,435 A
• Filed: 01/12/1987
• Issued: 09/04/1990
• Est. Priority Date: 01/12/1987
• Status: Expired due to Fees

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Krauth, Gary H.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US07/002,334
• Time in Patent Office: 1,331 Days
• Field of Search: 435/25, 435/28, 435/7, 436/517, 436/537, 436/805, 436/909, 436/518, 436/527, 436/581, 436/534, 436/544, 436/811, 436/818, 436/817, 356/320, 356/407, 356/341, 356/441
• US Class Current: 435/7.93
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/17 Systems in which incident light is modified in accordance with the properties of the material investigated where the material investigated is optically excited causing a change in wavelength of the incident light G01N21/63
• CPC Class Codes: G01N 21/51 inside a container, e.g. in an ampoule G01N21/53 takes precedence
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin

Abstract

The method of the present invention employs an enzyme immunoassay for measuring the concentration of an analyte in a sample by indirect colorimetric detection. An incident light beam at a plurality of wavelengths is directed into a liquid solution containing an analyte of interest. The solution is capable of attenuating the amount of light at a first wavelength received from this solution as a function of the increasing concentration of the analyte present. A light signal from the solution at the first wavelength is detected, and light at a second wavelength, at which substantially no attenuation of light signal occurs as the concentration of the analyte increases, is also detected. The ratio of the two respective wavelengths is formed and that ratio is compared with ratios of known amounts of the analyte to determine the amount of the analyte in the sample.

Claims

1. 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-labeled 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 by absorption 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.

2. The method of claim 1 wherein the analyte is selected from the group consisting of human chorionic gonadotropin and luteinizing hormone.

3. 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-labeled 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 first fluorphore for causing 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 fluorescence 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 wavelengths; 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.

4. 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-labeled analyte 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 competes with the enzyme labeled analyte conjugate for binding with said bound antibody to form a solid phase;

(b) admixing into said tube a liquid solution containing a tetramethylbenzidine and hydrogen peroxide to cause a reaction with said bound enzyme 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 450 nm at which activated tetramethylbenzidine absorbs an amount of light available for scatter 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.