Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-28
2002-02-26
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007210, C435S007400, C435S007500, C435S007700, C435S007900, C435S007910, C435S962000, C435S968000, C436S537000, C436S544000, C436S545000, C436S546000, C436S800000, C436S815000, C436S817000, C436S814000
Reexamination Certificate
active
06350579
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns compositions and methods for the detection of analytes which may be present in a wide range of possible concentrations through the use of two or more labeled binding partners. In a particular embodiment, two or more separately distinguishable labels are each joined to binding partners designed to bind two mutually exclusive target regions of the same analyte. In this preferred embodiment, the label joined to each said binding partner is present at a different specific activity. In other embodiments, the labels are present on the binding partners at the same specific activity, or are not separately distinguishable. The present invention provides for the extension of the dynamic range of any assay to be used for detection of any analyte, using any label type or combination of label types. The binding partners must be able to bind the analyte in a target region-specific manner; otherwise the binding partners may be of any type. The invention is useful in any application in which the detection of analyte is desired: these include, without limitation, nucleic acid hybridization assays, antibody binding assays, enzyme-substrate interactions, cytokine-receptor interactions, hapten-ligand interactions, and the like.
BACKGROUND OF THE INVENTION
Assays for the detection and/or quantification of analytes exist in many different forms and formats. In many cases, the amount of the analyte sought to be detected in a sample is not great enough for direct detection or quantification. In such cases, a secondary molecule able to bind to or interact with the analyte must be used to indicate the presence or amount of analyte in a given sample. Unless the secondary molecule is directly detectable, the secondary molecule must be conjugated with a label which will be detected when the secondary molecule binds the analyte. For the purposes of the present application, such secondary molecules able to bind or interact with analytes will be referred to as “binding partners” or “probes”. A non-exclusive list of analytes sought to be detected are antibodies, proteins, cell-surface receptors, cytokines, hormones, antigens, nucleic acids, metals, molecular complexes such as polymeric arrangements of proteins or other macromolecules, and the like. Likewise, binding partners for the detection of such analytes may include, without limitation: antibodies, proteins, antigens, haptens, nucleic acid probes, chelating agents, enzymes, enzyme substrates, and analogs of these.
One commonly used assay format is the enzyme-linked immuno-absorption assay (ELISA). In this assay format the analyte is contacted with a primary antibody able to bind at least one domain or “target region” thereon. After the excess antibody is washed free of the resulting analyte: antibody complex, the primary antibody is contacted with an enzyme-labeled secondary antibody to which it will specifically bind. The sample is then given a chromogenic enzyme substrate, and incubated under conditions favoring enzyme-mediated reaction of the substrate. The resulting colored product and its intensity after a given reaction time are indications of the presence and amount, respectively, of the analyte originally present in the sample. Illustrative examples of enzymes used in such assay methods are &bgr;-galactosidase, acid phosphatase, and alkaline phosphatase; a non-exhaustive list of enzyme substrates for use with such enzymes include x-gal (5-bromo-4-chloro-3-indolyl-&bgr;-D-galacto-pyranoside) and p-nitrophenyl phosphate. Other such enzymes and substrates are well known by those of skill in the art. Variations of this assay method exist; for example, the primary antibody may be linked to an enzyme thus eliminating the secondary antibody step. Nevertheless, these assay methods feature common steps involving contacting of the analyte with a labeled binding partner and subsequent detection of the analyte-bound label as an indication of the presence or amount of analyte.
While ELISA utilizes an enzyme label which is indirectly detected, other methods exist for the direct detection of labeled binding partner. Thus, Campbell, et al., U.S. Pat. No. 4,496,958 describes chemiluminescent acridinium labeling compounds for use in labeling binding partners in multiple assay formats; this patent is incorporated by reference as part of the present application. Additionally, other labeling compounds such as radionuclides, fluorescent, bioluminescent, phosphorescent, luminescent, chemiluminescent, or electrochemiluminescent compounds, chromophores, and dyes are known in the art and are commonly used as labeling agents in a variety of assay formats, both direct and indirect, including immunoassay and nucleic acid hybridization assays.
In addition to distinctions between assays based on the type of analytes to be detected, the type of binding partner with which the analyte binds, and the type of label used, assays can also be classified according to whether the method involves the immobilization of the analyte, or the analyte: binding partner complex. In the most common assay format, known as a “heterogeneous” or biphasic assay system, a probe is allowed to bind its analyte—usually under conditions of probe excess. Either the analyte molecule or the probe molecule may be immobilized to a solid support, thus causing the resulting probe: analyte complex to become immobilized—alternatively, and preferably, the complex may be immobilized following its formation in the liquid phase. After probe: analyte complexes have been immobilized, the excess uncomplexed probe molecules are washed away. If the probe molecules are directly labeled, the label may now be detected as an indication of the presence of analyte. In a variation of this format, probe: analyte complexes may also be separated from free probe, by means such as gel filtration chromatography, electrophoresis, electrofocusing, and other separation methods based on size or charge of the probe: analyte complex.
Alternatively, and more rarely, an assay may be designed to take place wholly in a single phase without a step resulting in the physical separation of probe: analyte complex from free probe. Such assay methods are termed “homogeneous” assays. In such methods, usually either the analyte: probe complex, or the free probe is altered after formation of the complex to permit the separate detection of analyte in the presence of the free probe. One such way of differentiating free probe from probe-bound analyte involves alteration or selective inactivation of the label joined to the probe rather than the free probe molecule itself. Arnold, et al., U.S. Pat. No. 5,283,174, describes homogeneous methods employing a oligonucleotide probe joined to a label which is capable of selective inactivation or alteration based on whether the labeled probe is bound to its target or not. These methods may be used in a single tube without the need for washing or decanting. This patent enjoys common ownership with the present application, and is incorporated by reference herein.
Assay methods exist which may utilize aspects of both homogeneous and heterogeneous assays. These methods may, for example, employ a single phase selective alteration of the probe or the probe: analyte complex followed by a physical separation step to further decrease the level of background in the assay. Such a “hybrid” assay format is described as one aspect of the multiple analyte assay described in Nelson et al., U.S. Pat. No. 5,658,737 which is incorporated by reference herein.
Regardless of the assay format used, a number of factors exist in all assays which can limit their sensitivity and the range of possible analyte concentrations that can be accurately detected or measured. One such factor is the level of background present in the assay. “Background” is a term used to describe probe or label in the assay which is not bound specifically to analyte and which may mask positive results at low analyte levels. Thus, for example, in a heterogeneous assay, background may be provided by a small amount of probe whi
Ceperley Mary E.
Fisher Carlos
Gen-Probe Incorporated
Gritzmacher Christine
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