Catalyzed reporter deposition

• US 5,731,158 A
• Filed: 05/20/1996
• Issued: 03/24/1998
• Est. Priority Date: 03/29/1989
• Status: Expired due to Term

• Current Assignee: PerkinElmer Life Sciences, Inc.
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Bobrow, Mark Norman, Litt, Gerald Joseph
• Primary Examiner(s): Adams, Donald E.
• Assistant Examiner(s): STUCKER, JEFFREY J
• Application Number:
  US08/651,012
• Time in Patent Office: 673 Days
• Field of Search: 435/7.1, 435/7.5, 435/7.6, 435/7.7, 435/7.71, 435/7.72, 435/7.8, 435/7.9, 435/7.91, 435/15, 435/18, 435/21, 435/25, 435/28, 436/501, 436/518
• US Class Current: 435/7.5
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

The present invention concerns a method to catalyze reporter deposition to improve detection or quantitation of an analyte in a sample by amplifying the detector signal which comprises reacting an analyte dependent enzyme activation system with a conjugate consisting of a detectably labeled substrate specific for the enzyme system, said conjugate reacts with the analyte dependent enzyme activation system to form an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system.

In another embodiment the invention concerns an assay for detecting or quantitating the presence or absence of an analyte in a sample using catalyzed reporter deposition to amplify the reporter signal.

Claims

1. A conjugate comprising a detectably labeled phenol.

2. A conjugate according to claim 1 wherein the detectable label is selected from the group consisting of a fluorescent label or a member of a specific binding pair.

3. A conjugate according to claim 2 wherein the detectable label is fluorescein, tetramethyl rhodamine, coumarin or biotin.

4. A method for the detection or quantitation of an analyte in an assay which comprises using an analyte dependent enzyme activation system comprising at least one enzyme to react with a conjugate consisting of a delectably labeled substrate specific for the enzyme system to form an activated conjugate which covalently deposits substantially wherever at least one receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system, wherein deposited detectable labels either directly or indirectly generate a signal which can be detected or quanititated.

5. A method according to claim 4 wherein at least one enzyme of the analyte dependent enzyme activation system is selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases, and ligases.

6. A method according to claim 5 wherein the enzyme is selected from the group consisting of peroxidases, oxidases, phosphatases, esterases and glycosidases.

7. A method according to claim 6 wherein the enzyme selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and beta-galactosidase.

8. A method according to claim 7 wherein the enzyme is horseradish peroxidase.

9. A method according to claim 8 wherein the conjugate is selected from the group consisting of biotin tyramine, p-hydroxyphenylpropionylbiocytin, or fluorescein tyramine.

10. A method according to claim 4 wherein the conjugate is reacted with detectably labeled antibody.

11. A method according to claim 4 wherein the conjugate is reacted with a detectably labeled member of a specific binding pair.

12. A method according to claim 4 wherein the conjugate is reacted with delectably labeled streptavidin.

13. A method according to claim 4 wherein the detectable label is selected from the group consisting of enzymes, radioactive isotopes, fluorogenic, chemiluminescent, or electrochemical materials or a member of a specific binding pair.

14. A method for producing an activated conjugate comprising reacting a peroxidase enzymo with a delectably labeled phenol.

15. A method according to claim 14 wherein the detectable label is selected from the group consisting of a fluorescent label and a member of a specific binding pair.

16. A method according to claim 15 wherein the detectable label is fluorescein, tetramethyl rhodamine, coumarin or biotin.