High sensitivity detection of peroxidase activity
AssigneeBecton, Dickinson and Company
InventorsDaniel B. Wagner, Glen P. Vonk, Randy A. Hoke
Life of this patent
Estimating the term…
Abstract
A new class of peroxidase substrates consists of o-diaminobenzenes having a substituent in the 4 position. The new substrates may be used in immunoassays in which a peroxidase is the label, or in assays for the peroxidase itself. Immunoassays using other enzyme labels which cause formation of hydrogen peroxide may be followed using the substrate of the invention by adding the substrate and a peroxidase to the assay fluid and detecting color formed as a result of oxidation of the substrate by the formed peroxide.
Claims9
A method for determining an analyte in a liquid comprising:
- a) contacting a liquid suspected of containing an analyte with an antianalyte affixed to a solid support and a tracer including a second antianalyte having a peroxidase conjugated thereto whereby a bound fraction which includes said peroxidase is formed on said support;
- b) contacting said support with a liquid containing peroxide and an o-diaminobenzene having a substituent in at least one of the 4 and 5 positions selected from the group consisting of a lower alkyl group of 1 to 6 carbon atoms and a halogen atom, the peroxidase portion of said bound fraction catalyzing oxidation of said o-diaminobenzene by said peroxide;
- and
- c) determining said analyte by the appearance on said solid support of a stable insoluble colored spot formed by said oxidation.
A method for determining an analyte in a liquid comprising:
- a) contacting a liquid suspected of containing an analyte with an antianalyte affixed to a membrane and a tracer including a peroxidase whereby a bound fraction which includes said peroxidase is formed on said membrane;
- b) contacting said bound fraction with a liquid containing peroxide and an o-diaminobenzene having a substituent in at least one of the 4 and 5 positions selected from the group consisting of a lower alkyl group of 1 to 6 carbon atoms and a halogen atom, the peroxidase portion of said bound fraction catalyzing oxidation of said o-diaminobenzene by said peroxide;
- and
- c) determining said analyte by the appearance on said membrane of a stable insoluble colored spot formed by said oxidation.
A method for determining an antigen in a liquid comprising:
- a) contacting a liquid suspected of containing an antigen with a first antibody affixed to a membrane and with a second antibody having a peroxidase conjugated thereto whereby a bound fraction which includes said antigen, said antibodies and said peroxidase is formed on said membrane;
- p1 b) contacting said bound fraction with a liquid containing peroxide and 3,4-diaminotoluene, the peroxidase portion of said bound fraction catalyzing oxidation of said 3,4-diaminotoluene by said peroxide;
- and
- c) detecting said antigen by the appearance on said membrane of a stable insoluble colored spot formed by said oxidation.
Dependencies are inferred from the claim text.