US5149630A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Methods of assay
Assignee: Merck Serono SA
•
Filing: 1988-02-09
•
Granted: 1992-09-22
•
Expires: 2008-02-09
Abstract
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.
Inventors
- 👤 Gordon C. Forrest
- 👤 Hugh A. O. Hill
- 👤 Simon J. Rattle
- 👤 Grenville A. Robinson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-08 17:12
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54366; G01N33/54373; G01N33/5438; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/817; Y10S436/00; Y10S436/806; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
158 words
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.
Claim 2
Dependent Claim
158 words
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.
Claim 3
Dependent Claim
41 words
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.
Claim 4
Dependent Claim
26 words
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.
Claim 5
Dependent Claim
24 words
A method as claimed in claim 1 wherein said electron transfer chemical comprises an electron transfer mediator formed from ferrocene or a derivative thereof.
Claim 6
Dependent Claim
75 words
A method as claimed in claim 4, wherein the derivative of ferrocene contains at least one or more side chains of the formula --CHO, --(CH 2 ) n COOH or --(CH 2 ) m NR 1 R 2 (where n and m are each from 0 to 6 and R 1 and R 2 , which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms).
Claim 7
Dependent Claim
38 words
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.
Claim 8
Dependent Claim
16 words
A method as claimed in claim 1, wherein the ligand is an antigen or an antibody.
Claim 9
Dependent Claim
45 words
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.
Claim 10
Dependent Claim
35 words
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.
Claim 11
Dependent Claim
35 words
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.
Claim 12
Dependent Claim
35 words
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.
Claim 13
Dependent Claim
97 words
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.
Claim 14
Dependent Claim
97 words
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.
Claim 15
Dependent Claim
21 words
A kit as claimed in claim 12 which further comprises an electrochemical apparatus containing a working electrode and an auxiliary electrode.
Claim 16
Dependent Claim
198 words
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.
Claim 17
Dependent Claim
198 words
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.
Claim 18
Dependent Claim
41 words
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.
Claim 19
Dependent Claim
34 words
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.
Claim 20
Dependent Claim
24 words
A method as claimed in claim 14 wherein said electron transfer chemical comprises an electron transfer mediator formed from ferrocene or a derivative thereof.
Claim 21
Dependent Claim
73 words
A method as claimed in claim 17, wherein the derivative of ferrocene contains at least one side chain of the formula --CHO, --(CH 2 ) n COOH or --(CH 2 ) m NR 1 R 2 (where n and m are each from 0 to 6 and R 1 and R 2 , which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms).
Claim 22
Dependent Claim
38 words
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.
Claim 23
Dependent Claim
16 words
A method as claimed in claim 14, wherein the ligand is an antigen or an antibody.
Claim 24
Dependent Claim
45 words
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.
Claim 25
Dependent Claim
35 words
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.
References Cited (Backward Citations)
US4081334A
Cited
US4600690A
Cited
US4340448A
Cited
US4391904A
Cited
US4545382A
Cited
US4711245A
Cited
US4758323A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-09-22 | 1996-03-23 | 1996-09-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-09-22 | 2000-03-23 | 2000-09-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-09-22 | 2004-03-23 | 2004-09-22 | $7700.00 | Window Closed / Lapsed |