Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-09-08
2002-02-05
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C422S051000, C422S051000, C422S051000, C422S082010, C422S082020, C435S007900, C435S007930, C435S288200, C435S291400, C435S817000, C436S518000, C436S524000, C436S525000, C436S528000, C436S544000, C436S806000, C436S807000, C204S400000, C204S403060, C204S415000
Reexamination Certificate
active
06344333
ABSTRACT:
The present invention relates to assays that employ an enzyme label or tag that acts on a substrate by obtaining electrons from an electrode (electrocatalysis) and an apparatus for use in such assays.
BACKGROUND OF THE INVENTION
Immunoassay techniques are based on the ability of antibodies to form complexes with the corresponding antigens or haptens. This property of highly specific molecular recognition of antigens by antibodies leads to high selectivity of assays based on immune principles. The high affinity of antigen-antibody interactions results in great sensitivity of immunoassay methods. The use of a label or indicator to verify that an antigen/antibody interaction has occurred is the basis for immunoassay methods.
Immunoassay techniques have been used mainly in clinical analyses and medical diagnostics. However, immunoassay applications in other areas such as environmental control, food quality control, etc. are expanding. Certain limitations in assaying techniques due to existing procedures have limited somewhat the expansion into such other areas.
In this respect, during the last few years a significant number of publications have dealt with non-conventional (alternative) immunoassay techniques designed to expand the accuracy or applicability of immunoassays. In most cases the development of alternative immunoassay techniques aims at improvements in performance of conventional immunoanalysis. Often such improvement attempts are directed to decreasing analysis times, increasing assay sensitivity, and simplifying and automating assay procedures.
For example, the utilization of enzymes able to catalyze electrochemical reactions by direct (mediatorless) mechanism (bioelectrocatalysis) would allow for the detection of immuno-interactions in real time. Such applications of bioelectrocatalysis in the development of immunosensors are based on the self-assembling or displacement of molecule/label complexes or “molecular transducers” on the surface of an electrode that has been modified by immunospecies that bind the complex. Ordinarily these immunospecies would be complimentary to the immunoconjugate which includes the electrocatalytically active enzyme-label.
Antigen immobilized on the electrode surface interacts with the enzyme-labeled antibody which results in the attachment of the enzyme to the electrode surface. Attachment of the electrocatalytic active enzyme on the electrode surface initiates, in the presence of a substrate, an electrocatalytic reaction. Therefore, the formation of an antigen-labeled antibody complex on the electrode surface is accompanied by an assembling of the molecular transducing layer. The rate of electron transfer can be limited by the efficiency of electrical connection between the enzyme-label and the electrode surface, which is already modified by the immobilized immunospecies.
A potentiometric immunosensor based on mediatorless bioelectrocatalysis has been utilized which employed laccase enzyme as an electrocatalyst-label. The electrocatalytic property of the enzyme in the reaction of oxygen electroreduction (reaction 1) allowed the detection of the biospecific interaction of a laccase-labeled receptor, or antibody, with a ligand modified electrode. Formation of a complex between the laccase labeled antibody and antigen on the electrode surface results in a considerable shift in electrode potential due to the catalytic reduction of over voltage. Analysis was performed in a competitive scheme, and a single measurement was made with 20 minutes. Such a potentiometric immunoassay does not require an electrochemically active mediator. The reaction substrates were atmospheric oxygen and electrons that were transferred directly from the electrode to the oxygen molecule via the active site of the enzyme. Insulin was used as a model analyte.
In the above immunoassay sensor, the electron which is the “second substrate” of enzymatic reaction can be captured by the enzyme-label only from the electrode surface. Therefore, only molecules intimately attached to the electrode surface generate electrochemical signal. The rate of attachment of electrocatalyst molecules to the electrode surface is proportional to the rate of formation of the immuno-complex on the electrode surface. The rate of attachment of electrocatalyst molecules to the electrode surface is proportional to the rate of formation of the immuno-complex on the electrode surface. The rate of immunointeraction on the electrode surface can be directly monitored by amperometric or potentiometric mode.
However, assays based on mediatorless bioelectrocatalysis are limited in that primarily one of the two assay procedures set forth below are utilized and only a single assay measurement may be taken before the electrode is regenerated or replaced by a new electrode. The two competitive assay procedures are:
(a) Competitive Immunoassay With an Initial Label-free Electrode—An electrode having no attached analyte/enzyme label is utilized as a starting point and a measured amount of analyte media along with a measured quantity of analyte/enzyme label are assayed by a competitive binding assay procedure. After maximum association with the electrode has occurred the amperometric or potentiometric measure result is compared to that of an electrode having 100% analyte/enzyme label associated. The difference in measurements corresponds proportionally to the amount of analyte in the media being assayed. and
(b) Displacement Immunoassay With an Initial Label-loaded Electrode—An electrode having the maximum amount of attached analyte/enzyme label (a filly loaded electrode) is utilized as a starting point and a measured amount of analyte media is assayed by a competitive binding assay procedure. After maximum displacement of the analyte/enzyme label from the electrode by the analyte of the media has occurred the amperometric or potentiometric measure result is compared to that of the initial fully loaded electrode (having 100% analyte/enzyme label associated). The difference in measurements corresponds proportionally to the amount of analyte in the media being assayed.
In addition to the laccase enzyme label, the potentiometric immunosensor employing peroxidase as an electrocatalyst-label has also been developed. The basic principle is the same as for the laccase based immunosensor. The electrode surface is modified by an immobilized antigen (rabbit IgG). The peroxidase-antibody conjugate associates with the antigen on the electrode surface. Once added to the media, and on reaching the electrode surface, the antibody-conjugated peroxidase starts to catalyze the electro-reduction of hydrogen peroxide. This results in an increase (anodic shift) in the electrode potential.
Both the laccase and peroxidase label immunosensors based on bioelectrocatalytic detection (as discussed above) allow direct detection of immunointeraction in real time. However, these sensors must be regenerated or replaced (e.g., disposable sensors) after each measurement. Accordingly, such immunoassay procedures do not allow continuous monitoring of the analyte. In addition, such procedures are a multi-stage process that result in a general complexity of analysis and require a highly qualified technician to conduct the assay.
Accordingly, there is a need for immunoassay procedures that can be continuous, particularly automatable procedures or procedures that do not require highly qualified technicians to conduct the assay.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an improved bioelectrocatalysis immunoassay apparatus for detecting an analyte, the apparatus comprising a sensing device with an electrode, wherein the sensing device has the ability to monitor changes in the amount of analyte in a liquid without requiring regeneration or replacement of the electrode or of the reagents used in the assay. Preferably, the sensing device of the apparatus is capable of multiple intermittent and/or continuous immunoassay measurements of the same analyte without a requirement for regeneration or replacement of the electrode o
Chin Christopher L.
Grant Alan J.
Olstein Elliot M.
Synectig Corporation
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