Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1998-05-13
2003-12-30
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S007100, C435S007210, C435S007240, C435S007940, C435S810000, C435S975000, C436S063000, C436S069000, C436S501000, C436S527000, C436S533000, C436S822000, C422S051000, C422S051000, C422S068100, C530S416000, C530S807000
Reexamination Certificate
active
06670196
ABSTRACT:
BACKGROUND
The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be or describe prior art to the invention.
Existing methods for determining ratios of biological molecules involve multiple steps and often require a large amount of time to perform. These methods often utilize two or more components, usually antibodies, specific for each of the biological molecules. Thus, two or more discrete assays need to be conducted to determine the ratio. Hence, these systems prolong the time required to determine the ratio and also accumulate reagent costs.
In addition, many of the existing methods for determining the concentrations of biological molecules utilize several components, usually antibodies or labeled antigens, at concentrations in excess of the concentration of the biological molecules in a sample. Non-competitive or sandwich assays function by the use of antibodies in excess of the biological molecules. Competitive immunoassays function through a competition of binding of a biological molecule and a labeled biological molecule for a limited concentration of antibody. Because some biological molecules, such as hemoglobin or cell receptors, occur at high concentrations in biological fluids, existing methods that require components to be in excess of the biological molecules are of limited application. In addition, samples generally require a dilution prior to assay.
Determining the ratio of biological molecules has proved to be an important indicator for many medical conditions and procedures. In particular, the determination of the ratio of related biological molecules is useful. Related biological molecules are formed in an organism when a biological molecule becomes modified. Biological molecules can become modified, for example, by covalent chemical alteration or by the reversible binding of molecules.
Biological molecules can become chemically modified in an organism in an intermolecular fashion. For example, hemoglobin, a blood-borne oxygen carrier in organisms, can become modified by glucose moieties when the blood stream contains high levels of glucose. In the blood stream, the aldehyde group of glucose condenses with valine of hemoglobin to form a Schiff base. This reversible reaction is followed by a virtually irreversible rearrangement in which the double bond shifts to C-2 of the sugar to give a stable fructose derivative of hemoglobin. Stryer,
Biochemistry,
3rd Ed., W. H. Freeman and Co., New York 1988. Hemoglobin that is modified in this manner is referred to as hemoglobin A1-C.
In addition, biological molecules can be modified in an intramolecular fashion. For example, troponin I, which normally exists in a reduced form in muscle cells, is oxidized when it is released into the blood stream of organisms suffering from a myocardial infarction. In particular, cysteine moieties within a discrete troponin I molecule can oxidize to form an intramolecular disulfide linkage. Methods of detecting related forms of troponin I that are released from muscle cells after a myocardial infarction are disclosed in PCT publication WO 96/33415.
Biological molecules can also become reversibly modified when high-affinity ligands bind to them. Cell receptors, for example, which are presented on the surface of a cell, can bind natural ligands or synthetic ligands with equilibrium dissociation constants in the micromolar to picomolar range.
SUMMARY
The invention relates in part to novel methods of rapidly determining the ratio of biological molecules. The invention also relates in part to a kit for determining the ratio of related biological molecules.
The invention increases the rate for determining ratios of biological molecules as compared to the rates of determining these ratios using existing methods. The invention increases the rate for determining ratios of biological molecules by reducing the number of steps required for measuring the ratio.
Applicant has discovered that the ratio of biological molecules can be rapidly detected without measuring the absolute concentrations of the biological molecules by using a binding molecule, preferably an antibody, that recognizes each of the biological molecules but binds only one of the biological molecules at a time.
FIG. 1
, which depicts one embodiment of the invention, serves as an illustrative example for the rapid determination of the ratio of biological molecules. The number of steps are reduced by probing a sample with a first component that binds a fraction of each of the biological molecules of interest. When the concentration of the first component is less than the concentrations of the biological molecules, the first component binds the biological molecules in a ratio related to the ratio at which the biological molecules exist in solution.
In one embodiment, the binding of one of the biological molecules to the first component excludes the binding of the other, even though the first component has the capacity of binding each of the molecules independently. The distribution of the biological molecules bound to the first compound is a statistical distribution that is directly related to the distribution of biological molecules in the sample.
These two features of the first component, the multiple binding feature and the exclusive binding feature, allow the first component to bind the biological molecules in a ratio related to the ratio of the biological molecules in the sample. For example, if the first component can bind each of molecules A and B, and A and B exist in the sample at a 3 to 1 ratio, the bound first component will have bound A and B in a 3 to 1 ratio or nearly this ratio.
Biological molecules A and B bind to the first component in a ratio related to their ratio in the sample, the relative on rates of the A and B binding to the first component determining the final ratio of A and B bound to the first component. Thus, the ratio of A to B can be bound to the first component in a ratio that is proportional to the ratio of A to B existing in a sample.
The second component of the invention detects the complex formed between the first component and one of the biological molecules. This complex may be detected when the second component binds to only one of the biological molecules, e.g., A or B, or if the second component binds to the complex formed between one of the biological molecules and the first component. The latter instance may provide an advantage if the biological molecules exist at high concentrations in the sample with respect to the concentration of the second component, since the second component will bind the complex comprising one biological molecule and the first component and not the unbound biological molecule.
Once the second component binds the complex comprising the first component and a biological molecule, a signal can be measured from a reporter molecule linked to one of the components of the invention. This signal can be applied to a standard curve that relates the signal to a ratio of the biological molecules. The standard curve can be prepared by measuring the signal, by the methods described herein, for samples prepared with known ratios of the biological molecules.
When biological molecules do not bind to the first component with equal affinity, standard curves relating the ratio to a signal generated by one of the components, preferably the first component, can be utilized to determine the ratio of A to B in the sample. In addition, normalization factors can be utilized to determine the ratio of A to B in a sample.
The ratio of the biological molecules is determined most rapidly when the components and the sample are mixed together at the same time and in the same vessel. This approach minimizes the number of steps required to determine the ratio of biological molecules, and thereby represents an advantage over existing techniques for determining the ratio of biological molecules. In particular, applications of the methods and kits described herein relate in part to increasing the efficiency of monitoring dru
Biosite, Inc.
Cheu Changhwa J.
Foley & Lardner
Le Long V.
Warburg Richard J.
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