Methods of assay

• US 5,149,630 A
• Filed: 02/09/1988
• Issued: 09/22/1992
• Est. Priority Date: 01/26/1984
• Status: Expired due to Term

• Current Assignee: Merck Serono SA (Merck & Co., Inc.), Serono Diagnostics Ltd.
• Original Assignee: Gordon C. Forrest, Grenville A. Robinson, Hugh A.O. Hill, Simon J. Rattle
• Inventors: Forrest, Gordon C., Robinson, Grenville A., Rattle, Simon J., Hill, Hugh A. O.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Chin, Christopher L.
• Application Number:
  US07/157,100
• Time in Patent Office: 1,687 Days
• Field of Search: 435/7, 435/14, 435/810, 435/25, 435/817, 436/806, 436/808, 204/403
• US Class Current: 205/777.5
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• 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 436/808 Automated or kit

Abstract

An electrochemical specific binding assay of a ligand (e.g., antigen, hapten or antibody) wherein at least one of the components is enzyme-labelled, and which includes the step of determining the extent to which the transfer of electrons between the enzyme substrate and an electrode, associated with the substrate reaction, is perturbed by complex formation or by displacement of any ligand complex relative to unbound enzyme-labelled component.

The electron transfer is aided by electron-transfer mediators which can accept electrons from the enzyme and donate them to the electrode or vice versa (e.g. ferrocene) or by electron-transfer promoters which retain the enzyme in close proximity with the electrode without themselves taking up a formal charge.

The electrochemical apparatus will typically comprise two or three electrodes, including one working electrode onto which components may advantageously be immobilized.

The use of direct electrochemical measurement of the enzyme label avoids the errors and inconvenience of the known indirect measurement techniques.

Claims

1. In a homogeneous method of electrochemical assay of a ligand in a sample using an electrochemical apparatus having at least a working electrode and and auxiliary electrode and containing components comprising:

(a) the sample,

(b) a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners to the ligand, one of the components (b) being labelled with an oxidoreductase enzyme, and

(c) a substrate for the enzyme, the improvement which comprises said apparatus additionally containing an electron transfer chemical capable of aiding the transfer of electrons between the substrate and the working electrode via the enzyme as a result of oxidation or reduction of the substrate but which is not essential for the activity of said enzyme, and the method including the step of determining whether the said transfer of electrons is perturbed by formation of a complex of said ligand with at least one specific binding partner.

2. A method as claimed in claim 1, wherein said electron transfer chemical is an electron transfer mediator which accepts electrons from one of the enzyme and the working electrode and donates them to the other of the working electrode and enzyme.

3. A method as claimed in claim 1 wherein at least one of the components (b) and said electron transfer chemical is immobilised on the working electrode.

4. A method as claimed in claim 1 wherein said electron transfer chemical comprises an electron transfer mediator formed from ferrocene or a derivative thereof.

5. A method as claimed in claim 4, wherein the derivative of ferrocene contains at least one or more side chains of the formula --CHO, --(CH₂)_n COOH or --(CH₂)_m NR¹ R² (where n and m are each from 0 to 6 and R¹ and R², which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms).

6. A method as claimed in claim 1 wherein the perturbation in the transfer of electrons is determined from a perturbation in a peak current at a pre-selected constant potential of the working electrode relative to a reference electrode.

7. A method as claimed in claim 1, wherein the ligand is an antigen or an antibody.

8. A method as claimed in claim 1 wherein said electron transfer chemical is an electrode-immobilized electron transfer promoter which retains the enzyme in close proximity with the working electrode by being immobilized on said working electrode without taking up a charge during said electron transfer.

9. A method as claimed in claim 1, wherein the extent to which said transfer of electrons is perturbed is determined, the extent of perturbation being an indication of the amount of ligand in the sample.

10. A method as claimed in claim 4, wherein the extent to which said transfer of electrons is perturbed is determined, the extent of perturbation being an indication of the amount of ligand in the sample.

11. A method as claimed in claim 7, wherein the extent to which said transfer of electrons is perturbed is determined, the extent of perturbation being an indication of the amount of ligand in the sample.

12. A kit for carrying out a method of assay comprising in separate containers:

(i) at least one specific binding partner to the ligand or a specific binding partner to the ligand and at least one further reagent selected from ligand analogs and specific binding partners to the ligand, one of the components (i) being labelled with an oxidoreductase enzyme,

(ii) a substrate for the enzyme and

(iii) an electron transfer chemical capable of aiding the transfer of electrons between the substrate and an electrode via the enzyme as a result of oxidation or reduction of the substrate.

13. A kit as claimed in claim 12 which further comprises an electrochemical apparatus containing a working electrode and an auxiliary electrode.

14. In a heterogeneous method of electrochemical assay of a ligand in a sample using an electrochemical apparatus having at least a working electrode and an auxiliary electrode and containing components comprising:

(a) the sample

(b) a specific binding partner to the ligand or a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners to the ligand, one of the components (b) being labelled with an oxidoreductase enzyme, and

(c) a substrate for the enzyme, the improvement which comprises said apparatus additionally containing an electron transfer chemical capable of aiding the transfer of electrons between the substrate and the working electrode via the enzyme as a result of oxidation or reduction of the substrate but which is not essential for the activity of said enzyme, and the method including the steps of providing means for separation of any enzyme-labelled complex containing said ligand relative to any other enzyme-labelled component, or where an enzyme-labelled ligand analog is present as component (b) providing means for separation of any complex containing said ligand analog relative to non-complexed enzyme-labelled ligand analog, and determining whether the said transfer of electrons is thereby perturbed.

15. A method as claimed in claim 14, wherein said electron transfer chemical is an electron transfer mediator which accepts electrons from one of the enzyme and the working electrode and donates them to the other of the working electrode and enzyme.

16. A method as claimed in claim 14 wherein at least one of the unlabelled ligand analog, unlabelled specific binding partner to the ligand and said electron transfer chemical is immobilised on the working electrode.

17. A method as claimed in claim 14 wherein said electron transfer chemical comprises an electron transfer mediator formed from ferrocene or a derivative thereof.

18. A method as claimed in claim 17, wherein the derivative of ferrocene contains at least one side chain of the formula --CHO, --(CH₂)_n COOH or --(CH₂)_m NR¹ R² (where n and m are each from 0 to 6 and R¹ and R², which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms).

19. A method as claimed in claim 14 wherein the perturbation in the transfer of electrons is determined from a perturbation in the peak current at a pre-selected constant potential of the working electrode relative to a reference electrode.

20. A method as claimed in claim 14, wherein the ligand is an antigen or an antibody.

21. A method as claimed in claim 14 wherein said electron transfer chemical is an electrode-immobilized electron transfer promoter which retains the enzyme in close proximity with the working electrode by being immobilised on said working electrode without taking up a charge during said electron transfer.

22. A method as claimed in claim 14, wherein the extent to which said transfer of electrons is perturbed is determined, the extent of perturbation being an indication of the amount of ligand in the sample.