Homogeneous amperometric immunoassay

• US 5,346,832 A
• Filed: 12/18/1992
• Issued: 09/13/1994
• Est. Priority Date: 06/09/1989
• Status: Expired due to Fees

• Current Assignee: CIBA Vision Corporation (Novartis Ag)
• Original Assignee: CIBA Vision Corporation (Novartis Ag)
• Inventors: Manning, Brenda D., Hidaka, Miki, Uretsky, Laura S., Aizawa, Masuo
• Primary Examiner(s): Scheiner, Toni R.
• Assistant Examiner(s): Chin, Christopher L.
• Application Number:
  US07/993,224
• Time in Patent Office: 634 Days
• Field of Search: 422/82.01, 435/817, 436/518, 436/537, 436/538, 436/544, 436/806, 436/817, 436/818, 530/350, 530/363, 530/807, 530/812
• US Class Current: 436/518
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin
• CPC Class Codes: Y10S 530/807 Hapten conjugated with peptide or protein
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier

Abstract

A method is described for measuring the amount of analyte present in a sample containing the analyte using a homogeneous amperometric immunoassay. The analyte is chemically bonded to a suitable carrier molecule, which is also chemically bonded to an electroactive molecule. The electroactive molecule, such as ferrocene carboxylic acid, contains a redox center which is capable of transferring a charge to an electrode. A preferred carrier molecule is bovine serum albumin (BSA), while suitable analytes include digoxin, theophylline and HCG. The immunoassay is conveniently performed by applying a voltage to a set of electrodes.

Claims

1. A method for determining the concentration of an analyte present in a sample of biological fluid contained in an electrochemical cell equipped with a set of electrodes, said method comprising the steps of:

a) forming an electroactive complex containing an antigenic analyte chemically bonded to a carrier molecule, said carrier molecule also being chemically bonded to an electroactive molecule capable of transferring a charge to an electrode;

b) adding the biological fluid, predetermined amounts of the electroactive complex, and an antibody to the analyte to the cell;

c) applying a voltage across the electrodes and measuring the current in the fluid at different voltage levels to form a voltage/current relationship for the sample fluid; and

d) comparing the voltage/current relationship for the sample fluid with standard voltage/current relationships generated using known amounts of analytes to determine the concentration of analyte present in the sample.

2. The method of claim 1 wherein the analyte is selected from the group consisting of theophylline, thyroxine, digoxin and HCG.

3. The method of claim 1 wherein the carrier molecule is bovine serum albumin.

4. The method of claim 1 wherein the electroactive molecule is a ferrocene derivative.

5. The method of claim 4 wherein the ferrocene derivative is selected from the group consisting of ferrocene carboxylic acid and ferrocenylacetic acid.

6. The method of claim 5 wherein the ferrocene derivative is ferrocene carboxylic acid.

7. The method of claim 1 wherein the analyte is digoxin, the antibody is a anti-digoxin antibody, the carrier molecule is bovine serum albumin, and the electroactive molecule is ferrocene carboxylic acid.

8. The method of claim 1 wherein the working and counter electrodes are fabricated from platinum or graphite.

9. The method of claim 1 wherein the biological fluid is whole blood, plasma, serum or urine.

10. The method of claim 1 wherein a buffer is added to the fluid sample.

11. An electroactive complex comprising at least one antigenic analyte chemically bonded to a carrier molecule, said carrier molecule also being chemically bonded to at least one electroactive molecule.

12. The electroactive complex of claim 11 wherein the analyte is selected from the group consisting of theophylline, thyroxine, digoxin, and HCG.

13. The electroactive complex of claim 11 wherein the carrier molecule is bovine serum albumin.

14. The electroactive complex of claim 11 wherein the electroactive molecule is ferrocene carboxylic acid.

15. The electroactive complex of claim 11 wherein the analyte is digoxin, the carrier molecule is bovine serum albumin, and the electroactive molecule is ferrocene carboxylic acid.

16. The electroactive complex of claim 15 which is prepared by first covalently binding ferrocene carboxylic acid to bovine serum albumin, and subsequently covalently binding digoxin to the bovine serum albumin.

17. The electroactive complex of claim 11 wherein the chemical bonds are covalent bonds.

18. The electroactive complex of claim 11, wherein the carrier molecule is selected from the group consisting of bovine serum albumin, bovine gamma globulin, ribonuclease, cytochrome C, and insulin.

19. The electroactive complex of claim 11, wherein the carrier molecule is a molecule having a molecular weight less than 150,000.

20. The electroactive complex of claim 11, wherein the carrier molecule is selected from a molecule having one or more amino and/or one or more carboxyl groups.