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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
First Claim
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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.

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