Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-23
2001-11-20
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S534000
Reexamination Certificate
active
06319670
ABSTRACT:
FIELD OF THE INVENTION
This application relates generally to methods and compositions for conducting binding assays, more particularly to those which measure the presence of an analyte of interest by measuring electrochemiluminescence emitted by one or more labeled components of the assay system. More specifically, the invention relates to precise, reproducible, accurate homogeneous or heterogeneous specific binding assays of improved sensitivity using electrochemiluminescent components.
BACKGROUND OF THE INVENTION
Numerous methods and systems have been developed for the detection and quantitation of analytes of interest in biochemical and biological substances. Methods and systems which are capable of measuring trace amounts of microorganisms, pharmaceuticals, hormones, viruses, antibodies, nucleic acids and other proteins are of great value to researchers and clinicians.
A very substantial body of art has been developed based upon binding reactions, e.g., antigen-antibody reactions, nucleic acid hybridization techniques, and protein-ligand systems. The high degree of specificity in many biochemical and biological binding systems has led to many assay methods and systems of value in research and diagnostics. Typically, the existence of an analyte of interest is indicated by the presence or absence of an observable “label” attached to one or more of the binding materials.
Electrochemiluminescent (ECL) assays provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescence is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of such ECL techniques, reference is made to U.S. Pat. No. 5,221,605, U.S. Pat. No. 5,591,581, U.S. pat. No. 5,597,910, PCT published application WO90/05296, PCT published application WO92/14139, PCT published application WO90/05301; PCT published application WO96/24690, PCT published application US95/03190, PCT application US97/16942, PCT published application US96/06763, PCT published application WO95/08644, PCT published application WO96/06946, PCT published application WO96/33411, PCT published application WO87/06706, PCT published application WO96/39534, PCT published application WO96/41175, PCT published application WO96/40978, PCT/US97/03653 and U.S. application 08/437,348 (U.S. Pat. No. 5,679,519). The disclosures of the aforesaid applications are incorporated by reference. Reference is also made to a 1994 review of the analytical applications of ECL by Knight, et al. (Analyst, 1994, 119: 879-890) and the references cited therein. The disclosure of the aforesaid articles are also incorporated by reference.
While electrochemiluminescence assays are significantly improved over chemiluminescence, fluorescence, ELISA and radioisotope-based assays, as well as other assay techniques, there is always a desire to improve assays by increasing the signal or modulation signal obtained from a binding event. By doing so one can improve the ratio of signal to background noise and, therefore, the sensitivity of the assay. Increasing the signal of an ECL assay also has several instrumental advantages including the following: i) less sensitive (and less expensive) light detection systems are required; ii) smaller samples are required; iii) electrodes and instrumentation may be miniaturized so as to allow for instruments that are smaller and/or devices that run many assays concurrently in a small area.
OBJECTS OF THE INVENTION
1. It is therefore a primary object of this invention to provide methods, reagents and compositions, for conducting of electrochemiluminescence binding assays which improve one or more characteristics of the assay or the instruments used to conduct the assay.
2. It is a further and related object of this invention to increase the number of photons emitted per binding event in an electrochemiluminescence assay.
3. It is a further and related object of the invention to improve the sensitivity of electrochemiluminescence assay by increasing the signal and thereby increasing the ratio of signal to background.
4. It is still a further and related object of the invention to reduce the sensitivity requirements for the light detection system used in electrochemiluminescence instruments.
5. It is still a further and related object of the invention to miniaturize ECL electrodes and instrumentation and thereby decrease the size the instruments themselves.
6. It is still a further and related object of the invention to miniaturize ECL electrodes and instrumentation and thereby increase the number of assays that can be run concurrently in one device.
SUMMARY OF THE INVENTION
These and other objects of the invention are achieved using microparticles comprised of an electrically conductive material having (a) one or more copies of an assay ligand immobilized on its outer surface, and (b) a plurality of electrochemiluminescent moieties immobilized on its outer surface. The assay ligand may be linked to the electrochemiluminescent moiety. More specifically, it has now been found that colloidal gold is a highly advantageous conductive material with which to form microparticles. Colloidal gold particles having one or more assay ligands immobilized on its outer surface and a plurality of ECL moieties immobilized on its outer surface can be used in a wide range of assay formats, including those based on detecting the ECL from moieties immobilized on the particle and those based on the modulation by the particles of the ECL from free ECL moieties in solution. The objects of the present invention may also be achieved using microparticles that do not comprise electrically conductive material.
Assays for an analyte of interest present in a sample are conducted by (a) forming a composition comprising (i) the sample, and one or more microparticles of the invention; (b) incubating said composition to form a complex; (c) causing the complex to bind to an assay-ligand immobilized on an electrode; and (d) conducting an electrochemiluminescence measurement in the presence of electrochemiluminescence reactants.
Assays employing bound ECL moieties can be carried out in a similar sequence of steps. The complex formed includes an assay ligand, the microparticle of the invention (wherein said microparticle contains ECL moieties) and the assay-ligand immobilized on the electrode so as to furnish a plurality of ECL moieties at the electrode. The electrochemiluminescence measurement is conducted there in the presence of reactants.
DETAILED DESCRIPTION OF THE INVENTION
Definition of Terms
“Assay-ligand” means a binding substance which may be an analyte or an analog thereof; a binding partner of the analyte or an analog thereof; additional binding partners of the binding partner or analog thereof; or a reactive component capable of reacting with the analyte, an analog thereof or a binding partner or analog thereof. These species can be linked to a combination of one or more binding partners and/or one or more reactive components and/or an analyte or its analog or a combination thereof. It is also within the scope of the invention for a plurality of the aforementioned species to be bound directly, or through other molecules to an analyte or its analog.
The term assay-ligand, therefore, includes analytes that can be measured by a binding assay, e.g., proteins (including oligopeptides, polypeptides, glycoproteins, lipoproteins and peptide analogs), nucleic acids (including mononucleotides, olignucleotides, polynucleotides, ribonucleic acids, deoxyribonucleic acids, and nucleic acid analogs), lipids, steroids, carbohydrates (including sugars and polysaccharides), porphyrins, alkaloids, nucleotides, nucleosides, amino acids, fatty acids, viruses, microorganisms, and biological cells (incl
Gudibande Satyanarayana
Martin Mark T.
Sigal George B.
Wilbur James L.
Wohlstadter Jacob N.
Evans, Esq. Barry
Houtteman Scott W.
Kramer Levin Naftalis & Frankel LLP
Meso Scale Technology LLP
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