Generic signalling mechanism for detection of analytes

Details Generic signalling mechanism for detection of analytes Generic signalling mechanism for detection of analytes

1999-06-21

2002-03-26

Nguyen, Bao-thuy L. (Department: 1641)

Chemistry: analytical and immunological testing

Involving diffusion or migration of antigen or antibody

C435S007100, C435S007920, C435S007930, C435S091500, C435S007950, C435S810000, C435S975000, C436S518000, C436S528000, C436S539000

Reexamination Certificate

active

06362008

ABSTRACT:

BACKGROUND OF THE INVENTION
The general class of small analytes which are to be assayed includes small molecular weight (about 10,000 daltons or less, preferably about 6,000 daltons or less, which is less than the size usable in a sandwich assay format, and which is capable of being recognized and bound by a ligand) pesticides, drugs, toxins, mycotoxins and drug metabolites, and further includes trichothecenes, fumonisins, antibiotics, and fragments of microorganisms, such as bacteria, parasites, viruses, yeast and mold, and fragments of mammalian cells. In particular, non-invasive screening procedures for assessing the exposure of humans to these substances require the ability to quantify both the target analyte and in some cases, metabolites, such as hydroxylated derivatives which include Aflatoxin M
1
, derived by hydroxylation from Aflatoxin B
1
, and/or other metabolites in body fluids such as milk, serum and urine.
Both the parent and the present inventions relate to a small analyte detecting assay which is novel because, in one test, it incorporates the best aspects of Enzyme Linked Immunosorbent Assay (ELISA) and Immunoaffinity chromatography (IA), but is not one or the other, since in contrast to ELISA, neither the parent nor the present invention use an enzyme signaling method, and in contrast to IA, the parent and present inventions depend on a competition and, therefore, do not result in isolation solely of purified target analyte, nor in isolation of target analyte at high efficiency. In addition, the inventions can be applied as general signal generation methods for a variety of target analytes.
Previously, with each new target analyte, a visualization approach specific to that target analyte was needed, which requires great expenditure of Research and Development resources. In the majority of cases this problem is solved by the parent invention which is drawn to a generic signaling assay method that is readily adaptable to most target analytes. However, some small analytes are hydrophobic and hard to chemically modify or difficult to work with within the context of the parent invention. The present invention overcomes the problems associated with this sub-set of small analytes while conserving the approach of the parent invention, resulting in greater efficiency of non-invasive immunological screening procedures and reduced Research and Development costs.
Finally, the method of the present invention is highly sensitive, i.e. has a very low detection limit, and yet is more rugged than other methods (such as enzyme-based methods) because the method of the invention has a longer shelf-life and is more forgiving in the hands of a user.
SUMMARY OF THE INVENTION
The invention uses an affinity matrix material for detecting a small analyte target comprising a solid phase sorbent material and a first ligand such as a monoclonal or polyclonal antibody, or a non-antibody ligand specific for the target analyte as well as a conjugated version of the analyte, in which the first ligand is bound to the sorbent material. This aspect of the invention provides a novel and widely useful method of testing for the presence of a small analyte by use of a ligand that binds to both the small analyte and conjugated small analyte, in which the ligand is bound to a solid phase sorbent material. Examples of solid phase sorbent materials are; sepharose and other agarose gel compositions, dextrans, magnetic beads or particles, charged nylon membranes, carbon and silicon granular preparations and the like, including glass beads and plates.
The invention can be summarized as follows. Any small target analyte and any conjugate decorated with multiple target analytes which are capable of competitively binding a first antibody or specific ligand that is linked to a solid support matrix can be employed. A second antibody or specific ligand which recognizes both the analyte and the multiple-analyte-decorated conjugate, and that has a fluorescent label, or any chemical compound that serves as a direct label, including colorimetric and isotopic labels, but specifically excluding all enzymes and indirect signal generators, is used. A sample that is spiked with the multiple-analyte-decorated conjugate is exposed simultaneously, or sequentially, in either order, to the first ligand and the second, tagged ligand. The amount of the label detected indicates the concentration of the endogenous amount of the analyte in the sample in inverse relationship as described below.
A test sample is mixed or may be incubated with a pre-determined amount of the multiple-analyte-decorated conjugate. When the test sample containing the multiple-analyte-decorated conjugate is passed over the immunoaffinity matrix, the first antibodies can bind competitively to two species: free analyte and multiple-analyte-decorated conjugate. The matrix is then exposed to a second ligand, such as a mono- or polyclonal antibody that is tagged or fluorescently labeled. Either within the matrix or upon elution, high label presence is seen in a clean sample. Conversely, only a small amount of the tag or label is detected in the matrix or eluant of a test sample that is highly contaminated with the free analyte.
It is an object of this invention to provide a kit for rapidly and accurately determining the presence or absence of small analytes in a sample quantitatively or non-quantitatively as desired. Each component of the kit(s) may be individually packaged in its own suitable container. The individual containers may also be labeled in a manner which identifies the contents. Moreover, the individually packaged components may be placed in a larger container capable of holding all desired components. Associated with the kit may be instructions that explain how to use the kit. These instructions may be written on or attached to the kit.
The present invention is drawn to a small analyte that is less than 10,000 Da, which is tagged with a non-enzyme label.
The present invention is further drawn to an affinity matrix for the detection of a small analyte comprising:
a solid phase sorbent material; and
a first ligand which is specific for both said small analyte and the small analyte which is conjugated or tagged with a non-enzyme label, wherein said first ligand is immobilized on said sorbent material.
The present invention is further drawn to a method for detecting small analytes in a test sample which comprises the steps of:
exposing a sample believed to contain a small analyte in combination with a predetermined amount of said analyte that decorates a non-enzyme conjugate, to a solid phase sorbent material that has immobilized thereon a first ligand that is specific for both said analyte and said multiple-analyte-decorated conjugate;
washing said solid phase sorbent material to remove non-specifically associated sample material;
exposing said solid phase sorbent material to a second ligand which is specific for said analyte and said multiple-analyte-decorated conjugate, and which is tagged or labeled;
washing said solid phase sorbent material to remove non-specifically associated tagged second ligand;
detecting the presence and amount of said tagged second ligand, for example, by subjecting said resin to UV light to induce fluorescence.
In addition, the present invention is further drawn to a method for detecting small analytes in a test sample which comprises the steps of:
exposing a sample believed to contain a small analyte in combination with a predetermined amount of said analyte that decorates a non-enzyme conjugate, to a solid phase sorbent material that has immobilized thereon a first ligand that is specific for both said analyte and said multiple-analyte-decorated conjugate;
washing said solid phase sorbent material to remove non-specifically associated sample material;
exposing said solid phase sorbent material to a second ligand which is specific for said analyte and said analyte-decorated conjugate, and which is tagged or labeled;
washing said solid phase sorbent material to remove non-specifically associated tagged second ligand;
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