Patent US3858506A successfully refreshed from Google Patents.
Patent EP0125139A2 successfully refreshed from Google Patents.
EP0125139A2
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EP
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Assay techniques utilising specific binding agents
Assignee: Medisense Inc
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Filing: 1984-05-08
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Granted: —
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Expires: 2004-05-08
Abstract
Abstract This specification discloses assay techniques and especially those for the detection measuring and monitoring of complex, usually biologically-arising, molecules or part-molecular structure often in complicated admixture with other similar molecules. The techniques of the invention utilise specific binding agents, i.e.g complex molecular species which have a specific reaction, usually based on electrostatic or hydrophobic forces, with one or more active sites on a given type of molecule only. One limb of this invention is concerned with immunoassay techniques for ligands and analytes to be carries out in vivo or in vitro. It should be noted that these ligands include species which are not onlyperse antigenic, but may be made so by crosslinking them to a suitable carrier. Another limb of the present invention is concerned with; firstly techniques for investigaging DNA or RNA sequencing, and secondly with other types of reactions in which specific, non-immunological binding occurs. The specification further discloses equipment or materials useful in the above techniques, either as such or possibly as usable coordinated kits of parts. The specification particularly discloses, an assay system comprising (a) a ligand-antiligand pair or analyte-specific binding agent pair capable of specifically binding together, (T.A) (b) a mediator (F) enzyme (GOD) and corresponding substrate (GLUCOSE) triplet, whereby conversion of the substrate into a product by the enzyme generates electrical charge which is transferred via the mediator to an electrode; wherein at least some of one of the pair and at teast some of one of the triplet are chemically cross-linked (GOD-T., so as to inhibit at least a part of the said charge transfer when the ligand and antiligand pair or analyte-specific binding agent pair are bound together and, wherein the addition of either one of the said pair or one of the said triplet changes the rate of charge transfer to the electrode.
Inventors
- 👤 Hugh Allen Oliver Hill
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-06 14:48
CPC Classifications
A; A61; A61B; A61B5/00; A61B5/145; A61B5/1468; A61B5/1486; A61B5/68; A61B5/6846; A61B5/6847; A61B5/6848; C; C12; C12Q; C12Q1/00; C12Q1/001; C12Q1/004; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6825; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54366; G01N33/54373; G01N33/5438
Claims Analysis
Total Claims: 35
Claim 1
Independent Claim
97 words
l.A method of assay in which (a) at least one specific binding reaction stage between reactive species comprising a ligand and an antiligand is carried out in association with an enzyme/mediator system electrochemically linked to a level of a substrate to which the enzyme is catalytically active so that the binding reaction affects the electrochemical availability of at least one of the components of said system and (b) the effect on the said electrochemical availability is detected or measured to give a signal from which the presence or amount of the said reactive species can be established.
Claim 2
Dependent Claim
17 words
A method as claimed in claim 1 in which the specific binding reaction takes place in solution.
Claim 3
Dependent Claim
19 words
A method as claimed in claim 1 in which the specific binding reaction takes place at a solid surface.
Claim 4
Dependent Claim
85 words
A method as claimed in claim 1 in which an unknown amount of a ligand species to be determined is subjected to a specific binding reaction with a known amount, known to be in excess, of a species antiligand therefor itself chemically linked with the said mediator compound which antiligand species in its unbound state only is available for charge transfer from the enzyme/substrate reaction, whereby the subsequent electrochemical activity of the excess unbound mediator/antiligand species provides a measure of the amount of ligand species.
Claim 5
Independent Claim
141 words
A method of homogeneous enzyme immunoassay which comprises: (a) (i) mixing a sample containing ligand to be assayed with a known amount, known to be in excess, of an antiligand whereby some ligand-binding sites on the antiligand are left free; (ii) mixing with the resultant mixture a known amount, known to be in excess, of the ligand chemically linked with a mediator compound whereby all of the previously free ligand-binding sites are occupied and some ligand-linked mediator remains electrochemically available; (b) mixing an enzyme and a substrate therefor in a liquid mixture; (c) contacting the enzyme/substrate mixture with the mixture from stage (a); and (d) contacting the resulting mixture with a sensor electrode; whereby the charge transferred to the electrode is dependent upon the amount of available unbound ligand-linked mediator and thus permits derivation of the amount of the original ligand.
Claim 6
Dependent Claim
14 words
A method as claimed in claim 5 in which the mediator is a ferrocene.
Claim 7
Dependent Claim
15 words
A method as claimed in claim 6 in which the ferrocene is a carboxyl ferrocene.
Claim 8
Dependent Claim
23 words
A method as claimed in claim 4, 5 or 6 in which the substrate/enzyme system is chosen from glucose/glucose oxidase and glucose/glucose dehydrogenase.
Claim 9
Dependent Claim
174 words
9 A method as claimed in claim 1 in which (i) (a) an unknown amount of ligand species X to be assayed and (b) an amount of a species X = E, in which X is chemically linked with an enzyme E without destroying the enzymatic activity thereof, are mixed in solution (ii) the mixture is contacted with a substrate S for the enzyme and a mediator compound M in the presence of a sensor electrode to which a measured charge is thereby transferred in dependence on the enzymatically catalysed reaction (iii) antiligand A is contacted with the solution to set up competitive equilibrium binding reactions of the general nature of reactions I and II whereby part of the enzymatically active species X = E is converted to the enzymatically inactive species A - X = E thereby altering the extent of reaction and the measured charge at the electrode and (iv) there is derived from the decrease in charge or rate of decrease in charge a measure of the concentration of analyte X.
Claim 10
Dependent Claim
165 words
A method as claimed in claim 1 in which (i) (a) an unknown amount of ligand species X to be assayed and (b) an amount of a species X = M in which X is chemically linked with a mediator M with destroying the mediator activity thereof are mixed in solution (ii) the mixture is contacted with a substrate S and an enzyme E catalytically active thereon in the presence of a sensor electrode to which a charge is thereby transferred for measurement in dependence upon the enzymatically catalysed resction (iii) antiligand A is contacted with the solution to set up competitive equilibrium binding reactions I and II whereby part of the species X = M is converted to the inactive species A - X = M thereby altering the extent of reaction and the charge measured at the electrode and (iv) there is derived from the decrease in charge or rate of decrease in charge a measurement of the concentration of ligand analyte X.
Claim 11
Independent Claim
156 words
A method of immunoassay for a species, which comprises: (a) covalently linking an analyte X and an enzyme E to give an enzymatically active species X-E; (b) mixing with the covalently linked species X-E a suitable reactive ligand A to form an enzymatically inactive species A-X-E with its specific binding sites fully occupied; (c) contacting the species A-X- E with the species X to be assayed, so as to set up a competitive specific binding reaction with species X and achieve the equilibrium so that the amount of species X-E is a measure of the amount of species X to be assayed; and contacting the mixture with a substrate for X= E , and transferring charge from the consequent enzymatically catalysed reaction to a sensor electrode by means of a mediator compound to give a measure of the amount of enzymatically active species X= E present, from which the amount of species X can be derived.
Claim 12
Dependent Claim
14 words
A method as claimed in claim 9 in which the mediator is a ferrocene.
Claim 13
Dependent Claim
14 words
A method as claimed in claim 12 in which the ferrocene is a carboxyferrocene.
Claim 14
Dependent Claim
25 words
A method as claimed in claim 9, 10 or 11 in which the substrate/enzyme system is chosen from a glucose/glucose oxidase and a glucose/glucose dehydrogenase.
Claim 15
Dependent Claim
77 words
A method as claimed in claim 1 in which at least one of the mediator and enzyme is chemically linked to a nucleic acid probe sequence whereby specific binding of the probe sequence to the target sequence in a single-strand nucleic acid material to be investigated affects the electrochemical availability of the chemically linked species as detected by a sensor electrode in presence of the enzyme substrate, whereby the presence of the target sequence can be detected
Claim 16
Independent Claim
196 words
A method of detecting a target sequence in a nucleic acid material which comprises: (a) providing a single strand nucleic acid material to be investigated for a given target sequence; (b)selecting a probe material with a sequence of nucleic acids complementary to the target sequence; (c) choosing a procedure from among (i) chemically linking the probe with an enzyme and adding the enzyme-linked probe to a solution containing both a substrate for the enzyme and a mediator, (ii) chemically linking the probe with a mediator and adding the mediator-linked probe to a solution containing both a substrate and an enzyme for the said substrate; (iii) chemically linking the probe with a mediator enzyme combination and adding the so-modified probe to a solution containing a substrate for the enzyme; (d) contacting the solution containing the chemically linked probe sequence with a sensor electrode whereby charge is transferred by the mediator to the electrode from the enzyme-catalysed substrate reaction; and (e) contacting the solution with the single stranded material, whereby alteration in the amount of charge transferred is an indication of a specific binding reaction between the probe and target affecting the availability of enzyme, mediator or combination.
Claim 17
Dependent Claim
56 words
A method as claimed in claim 15 wherein the mediator is linked indirectly to the probe sequence by a linker group and a material reactive to the linker group is present on the electrode, whereby the whole complex is present on the electrode and binds to the probe sequence to cause an alteration in electrode current.
Claim 18
Dependent Claim
17 words
A method as claimed in claim 15, 16 or 17 in which the mediator is a ferrocene.
Claim 19
Dependent Claim
17 words
A method as claimed in claim 15, 16 or 17 in which the mediator is a carboxyferrocene
Claim 20
Dependent Claim
26 words
A method as claimed in claim 15, 16 or 17 in which the substrate/enzyme system is chosen from a glucose/glucose oxidase and a glucose/glucose dehydrogenase system.
Claim 21
Dependent Claim
76 words
A method as claimed in claim 1 which comprises immobilising a ligand to be assayed on a suitable surface, thereafter carrying out a specific binding reaction with excess of a suitable antiligand itself chemically linked to an enzyme, prior to remixing the excess and adding a substrate for the immobilised enzyme; and contacting with a mediator and a sensor electrode, so that the charge transferred to the electrode is proportional to the amount of enzyme present.
Claim 22
Independent Claim
118 words
A method of heterogeneous enzyme-linked immunoassay comprising: (a) immobilising at a suitable surface a ligand to be assayed; (b) contacting with the ligand excess of an enzymatically active species consisting of an antiligand chemically linked with an enzyme to bring about a specific binding reaction with the immobilised ligand and give an immobilised enzymatically active species in an amount corresponding to that of the ligand to be assayed; (c) removing excess non-immobilised chemically-linked enzyme/antiligand, and (d) contacting the immobilised enzyme with a substrate therefor and with charge-transferring mediator compound whereby an electrode in contact therewith signals a charge corresponding to the amount of immobilised enzyme, from which the original amount of ligand to be assayed may be derived.
Claim 23
Dependent Claim
14 words
A method as claimed in claim 22 in which the mediator is a ferrocene.
Claim 24
Dependent Claim
14 words
A method as claimed in claim 23 in which the mediator is a carboxyferrocene.
Claim 25
Dependent Claim
22 words
A method as claimed in claim 21, 22 or 23 in which the substrate/enzyme is chosen from glucose/glucose oxidase and glucose/glucose dehydrogenase.
Claim 26
Dependent Claim
60 words
A method as claimed in claim 1 in which the specific binding reaction takes place at the surface of a sensor electrode to block or alter the characteristics of the surface at least in part whereby the existence or amount of a decrease in detected electrical charge is a measure of the existence or extent of the specific binding reaction.
Claim 27
Independent Claim
137 words
A method of immunoassay comprising the steps of: (a) taking a liquid medium containing a ligand to be assayed; (b) adding thereto a known amount of a substrate capable of undergoing enzyme-catalysed reaction with an enzyme; (c) contacting the liquid medium with a sensor electrode comprising at its surface a combination of (i) antiligand capable of undergoing a specific binding reaction with the ligand to be assayed (ii) the said enzyme and (iii) a mediator compound to transfer charge from the enzyme to the electrode when the enzyme is catalytically active, so as to provide a signal; and (d) comparing the signal with a signal received in the absence of the ligand, and deriving the amount of ligand present as a function of blockage or conformational change at the electrode surface caused by the specific binding reaction.
Claim 28
Dependent Claim
14 words
A method as claimed in claim 26 in which the mediator is a ferrocene.
Claim 29
Dependent Claim
14 words
A method as claimed in claim 27 in which the mediator is a carboxyferrocene.
Claim 30
Dependent Claim
23 words
A method as claimed in claim 26, 27 or 28 in which the substrate/enzyme system is chosen from glucose/glucose oxidase and glucose/glucose dehydrogenase.
Claim 31
Dependent Claim
20 words
A method as claimed in claim 1, 2 or 3 in which the enzyme and mediator are chemically linked together.
Claim 32
Independent Claim
97 words
A method for determining the presence of a selected ligand species present in a mixture of compounds, comprising reacting the ligand species with an anti-ligand to form a ligand/anti-ligand complex, performing an enzyme catalyzed reaction whose rate is representative of the amount of said complex formed, and sensing said enzyme catalyzed rate electrochemically by exposing a mixture comprising said enzyme and an electron-transfer mediator to an electrode having an electrically conductive surface, whereby said electron-transfer mediator is capable of transferring electrons between said electrode surface and said enzyme at a rate representative of said enzyme-catalyzed reaction rate.
Claim 33
Dependent Claim
21 words
A method as claimed in claim 10 in which the chemically linked mediator-analyte conjugate is F c - CH 2 -theophylline
Claim 34
Dependent Claim
59 words
An electrode configuration supporting at its surface (a) a conjugate between a ligand species and a mediator, (b) an antiligand therefor (c) an enzyme substrate and (d) an enzyme catalytically active on the substrate, wherein at least the conjugate is separate from the antiligand and the enzyme is separate from its substrate either by spatial location or physical state.
Claim 35
Dependent Claim
52 words
A method of assay in which an electrode as claimed in claim 34 is contacted with a medium containing an unknown amount of the ligand species to be assayed, and the signal or rate of change of signal received fromthe electrode is observed as a measure of the said unknown amount .
References Cited (Backward Citations)
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