Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-02-17
1996-06-18
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435188, 435975, 436518, 436525, 436534, 436815, 5462784, G01N 33535
Patent
active
055276865
DESCRIPTION:
BRIEF SUMMARY
1. FIELD OF THE INVENTION
The present invention relates to methods for determining the presence of an analyte in a sample. More particularly, homogeneous liquid-phase and heterogeneous liquid-phase/solid-phase release assays that are highly specific and sensitive are provided.
2. BACKGROUND OF THE INVENTION
Immunoassays utilize the specific binding capabilities of antibodies to detect the presence of target molecules in solution. Although the general principle is applicable to a broad range of problems, major commercial interest has centered on medical diagnostic applications for a wide variety of analytes in biological fluids such as blood, saliva, and urine.
Several types of immunoassays, useful for distinct applications, already exist. Each such assay type requires a way of distinguishing whether binding sites on an antibody are occupied or free. Typically this is accomplished by means of label such as an atom, molecule, enzyme or particle attached permanently to either the antibody or to an analog of the analyte.
Sensitivity and specificity are key parameters of an immunoassay. Specificity relates primarily to the antigen binding site of the antibody, which is inherent to selection of variable region gene segments and is independent of the assay configuration. Sensitivity relates primarily to the affinity of the antibody for its ligand(s) and to the inherent detectability of the label. For example, radioisotopes, used for radioimmunoassay, can be detected at significantly lower concentrations than fluorescent molecules. Enzyme labels are detectable at concentrations similar to fluorescent labels. When substrates that produce fluorescent or chemiluminescent products are used with enzyme labels, the sensitivity of resulting immunoassays is comparable or greater than with radioisotope labels.
Many conventional assay techniques are considered competitive in that the analyte and labeled component have comparable affinities of the antibody binding site. One example of such a competitive method is found in U.S. Pat. No. 3,817,837 by Rubenstein and Ullman which describes a technique in which ligand and enzyme-bound-ligand compete for antibody binding sites. Since binding of the antibody to the enzyme-bound-ligand alters its enzymatic activity, the concentration of ligand present can be estimated by measuring the rate at which such a mixture converts substrate to product.
Immunoassays can be further characterized as homogeneous and heterogeneous. In a heterogeneous method, the label is equally detectable in bound and unbound states. To obtain any meaningful assay results physical separation of the bound versus unbound antibody is required. A common strategy for accomplishing this separation entails associating the label to a solid phase which can be physically separated from the liquid phase prior to the detection step. A typical heterogeneous assay is the Tandem EIA from Hybritech, Inc.
In a homogeneous method, the detectable property of the label is inherently different depending on whether bound or unbound. In its bound state, the label will haver greater or lesser signal intensity. Usually, binding of antibody to the labeled ligand causes a decrease in signal intensity, e.g., when the label is an enzyme. Typical products in this category include the EMIT line of enzyme immunoassays from Syva Company and the TDX line of fluorescence polarization immunoassays from Abbott Diagnostics.
Two further characteristics of immunoassays are particularly noteworthy. These are the minimal concentration of analyte that can be detected, and the dynamic range of detection. The dynamic range is the range of analyte concentrations over which signal from a label changes from zero to maximum. The order in which the sample, the antibody, and a labeled component are combined can significantly affect both of these key parameters by affecting the degree of binding of the labeled component, which in turn affects detection of the label.
In certain known assay methods, the antibody and the analyte are combined prior to addition of
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Fitzpatrick Judith
Lenda Regina
Scheiner Toni R.
Serex, Inc.
Wolski Susan C.
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