Antioxidant assay

• US 5,395,755 A
• Filed: 11/25/1992
• Issued: 03/07/1995
• Est. Priority Date: 06/12/1990
• Status: Expired due to Fees

• Current Assignee: British Technology Group Ltd.
• Original Assignee: British Technology Group Ltd.
• Inventors: Thorpe, Gary H. G. H., Whitehead, Thomas P.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Pyon, Harold Y.
• Application Number:
  US07/949,518
• Time in Patent Office: 832 Days
• Field of Search: 435/7.9, 435/28, 435/810, 435/968, 436/172
• US Class Current: 435/28
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

A method of assay of antioxidant activity of a biological fluid, which comprises monitoring the change exerted by the sample on a progressing luminescent reaction.

Claims

1. A method of assay of the antioxidant activity of a sample suspected of having such activity, which method comprises the steps of:

(a) initiating a chemiluminescent reaction and allowing said reaction to progress, thereby to generate a level of luminescence, said level being selected from the group consisting of (i) a rising level between 90 to 100% of maximum, (ii) the maximum, and (iii) a post-maximum substantially constant plateau level;

(b) adding said sample to said progressing chemiluminescent reaction, said sample causing said level of luminescence generated by said reaction to change when said sample has antioxidant activity;

(c) monitoring said change in the level of luminescence; and

(d) determining the antioxidant activity of said sample assayed by reference to that of samples of known antioxidant activity subjected to steps (a) to (c) above.

2. A method of determining the contribution of specific or particular classes of antioxidants in a sample, by comparing the antioxidant activity of the sample as measured by a method according to claim 1, before and after the specific or particular classes of antioxidants have been removed or extracted.

3. A method according to claim 1, in which the sample is a biological fluid.

4. A method according to claim 3, in which the biological fluid is mammalian serum or blood plasma, cerebrospinal fluid, synovial fluid or saliva.

5. A method according to claim 1, in which said chemiluminescent reaction is a peroxidase-catalyzed oxygen-dependent chemiluminescent reaction.

6. A method of assay according to claim 5, in which the chemiluminescent reaction is an enhanced chemiluminescent reaction.

7. A method according to claim 5, in which the chemiluminescent reaction comprises the reaction of a dihydrophthalazinedione (DPD) substrate.

8. A method according to claim 6, in which the enhancer is para-iodophenol.

9. A method according to claim 1, in which the time interval between the time when the sample is added and a predetermined level of luminescence is measured.

10. A method according to claim 1, in which said change monitored in step (c) is selected from the group consisting of

(1) a time period during which the level of luminescence falls to a trough level;

(2) a time period in which it remains at said trough level;

(3) a time period in which it recovers partially to reach a new substantially constant plateau level of lower level of luminescence than the first-mentioned substantially constant plateau level; and

(4) the shape of the curve during the time period (3).

11. A method according to claim 10, in which the chemiluminescent reaction is one in which the reaction takes place between a peroxidase, an oxidant and a dihydrophthalazinedione, in the presence of an enhancer and the sample is a biological fluid.