Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-08-20
2003-10-21
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S004000, C435S005000, C435S007100, C436S501000, C436S506000, C436S507000, C436S536000, C436S538000, C436S543000
Reexamination Certificate
active
06635469
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the general field of analyte detection, assays, and methods for the separation of particular compounds from a mixture.
BACKGROUND OF THE INVENTION
Assays of Biomolecules: Assays can be used to determine whether, and how much of, an analyte is present in a sample. In some cases, such assays rely on selective binding or complexation (specific or nonspecific) of the analyte in the sample with an exogenously supplied capture reagent or binding partner.
Effects of endogenous binding partners on assays: Often such samples contain an endogenous component that forms a complex with the analyte, and the resulting endogenous complex may interfere with detection of the analyte. For example, detection by absorbance, fluorescence, molecular weight, or other analyte characteristics may be adversely affected by endogenous complexes. Where detection itself depends on formation of a complex with an exogenously supplied reagent, analyte present in endogenous complexes may be unable to effectively complex with the exogenous binding partner (as is required for detection in such assays). In that way, the endogenous complex interferes with the assay's reliability. For example, the analyte goes undetected or is incompletely detected—i.e., it provides a false negative or non-quantitative result.
This problem can be illustrated with standard enzyme-linked immunosorbent assays (ELISAs), in which sample antigen is detected only if it is recognized and bindable by immobilized antibody. Endogenous sample antibodies that react with the analyte may prevent at least some portion of the analyte from complexing with one or both of the exogenous assay reagents (antibodies), thereby reducing the effectiveness of the assay.
Assays for antigens or antibodies that are characteristic of a pathogen are particularly susceptible to problems caused by endogenous binding partners. If the patient being assayed has developed an immune response to the analyte antigen, a significant portion of sample antigen may be present in undetectable endogenous antigen/antibody complexes. Similarly, in serology assays where the antibody is the analyte to be measured, some of the sample antibody that the assay is designed to measure may be complexed with endogenous pathogen antigen.
In addition to endogenous antibody/antigen complexes, other endogenous complexes can interfere with assays, for example, various serum globulins can interfere with immunoassays for thyroxine, estradiol, cortisol, and testosterone. See, Thorell, J. I., and Larson, S. M, in “Radioimmunoassay and Related Techniques,” C. V. Mosby, St. Louis, 1978. Vitamin B
12
assays are perturbed by the binding of transcobalamin. See, Laue et al. Blood 26:202 (1965). Immunoassays for prostate-specific antigen (PSA) are perturbed by endogenous complexes with a serine protease inhibitor, &agr;
1
-antichymotrypsin. See, Lilja et al.
Clin. Chem
. 37:1618-1625 (1991).
Another area in which endogenous complexes may seriously affect assay results is the use of tumor antigens to mark the tumor's presence, e.g. in an immunoassay. Frequently, these tumor markers may be masked by endogenous complexes. For example, serum thyroglobulin autoantibody interferes with detection of differentiated thyroid carcinoma. Another example of the difficulty of obtaining accurate quantitation of a serum tumor antigen is the epithelial mucin MUC-1. Gorevitch et al.,
Br. J. Cancer
, 72:934-938 (1995); and Hilgers et al. Scand.
J. Clin. Ob. Invest. Suppl
., 221:81-86 (1995).
A particular problem which may be related to endogenous complex formation has surfaced in HIV assays. Tsiquaye et al.,
AIDS
, 2:41-45 (1988); McHugh et al.,
J. Infect. Dis
., 158:1088-1091 (1988); Nishanian et al.,
J. Infect. Dis
., 162:21-28 (1988); and Carini et al.,
Scand. J. Immuno
., 26:1 (1987). Other assays in which this problem can arise include: epithelial mucin (MUC-1 and PEM) assays (Gorevitch et al.,
Br. J. Cancer
, 72:934-938 (1995); and Hilgers et al. Scand.
J. Clin. Ob. Invest. Suppl
., 221:81-86, 1995); Hi histones in assays for systemic lupus (Wesierska-Gadek et al.
Arthritis Rheum
, 33:1273-1278, 1990); assays for the tuberculosis pathogen (Dlugovitzky et al.
Braz. J. Med. Biol. Res
. 28:331-335, 1995); alpha-fetoprotein assays to detect hepatocellular carcinoma (Tsai et al.,
Br. J. Cancer
, 72:442-446, 1995); assays for Yersinia enterocolitca and Yersinia pseudotuberculosis (Didenko et al.
J. Basic Microbiol
. 35:163-170, 1995); and assays for the leprosy pathogen (Sinha et al.
Int. J. Lepr. Other Mycobact. Dis
., 60:396-403, 1992).
Various methods have been described to dissociate endogenous antibody/antigen complexes and thereby to improve assay sensitivity, including solvent extraction, heating, protein precipitation, use of competitive inhibitors, and pH changes. For example, Mosier, U.S. Pat. No. 4,656,251, and Weil et al.,
J. Immunology
, 134:1185-1191 (1985), disclose pretreating a canine sample to break up immune complexes before assaying for heartworm antigens. Mosier '251 discloses a process that includes acidification to dissociate the complex, followed by heating to denature dissociated antibodies. Weil discloses (p. 1186, right column) a process including the addition of EDTA followed by heating.
Accelerating High Sensitivity Assays: While sensitivity may be improved by lengthening incubation time (e.g., overnight), high throughput and automation are also important goals that may be inconsistent with lengthy incubation. As high throughput automated instruments have become widely utilized, assay results are needed more quickly (i.e., within a few minutes). The need to accelerate analyte/binding partner interactions may be addressed by adding a large excess of the exogenous binding partner, or by using temperature conditions above optimum to drive the binding reaction as far as possible in an acceptable assay time.
Separation of Biomolecules: A widely accepted method for purification of bioactive compounds is affinity chromatography. This method is based on the premise that many bioactive compounds bind to other molecules with extraordinary specificity. These other molecules are commonly referred to as “ligands.” For example, ligands that-have been identified for binding specific compounds include, but are not limited to, nucleic acids, vitamins, carbohydrates, fats, and proteins (e.g., enzymes, antibodies, and receptors).
The first step in affinity chromatography typically is identification of a ligand that binds specifically to the compound of interest. Such ligands are already known for many enzymes and other compounds. Once a ligand has been identified and obtained, the ligand can be attached to a solid support. The solid support can, for example, be trapped within a porous sack or, more commonly, immobilized in a porous column. A solution known to contain, inter alia, the compound of interest is generally flushed through the column so that the solution comes into binding contact with the immobilized ligand.
The quantity of immobilized ligand required depends on the amount of the desired compound expected to be present. Typically, each ligand can bind to a limited number of (e.g., often one) molecules of the compound. Numerous complications render this generalization less valid in practice, however. For example, steric constraints can limit the number of molecules of the compound that can exist within a given volume, especially if the compound is, for example, a relatively large molecular weight, multi-domain protein. Also, there can be other, undesirable compounds capable of weakly binding to the same ligand that the compound of interest binds tightly to. The latter problem can become especially acute if the undesired weakly binding compound is present in excess (i.e., relative to the desired compound). Therefore, it is desirable to promote high affinity, high specificity interactions.
In a typical preparative application of affinity chromatography, an impure solution containing the desired compound is passed through a porous mater
Green David J.
Laugharn James A.
Litt Gerald J.
BBI Bioseq, Inc.
Chin Christopher L.
Fish & Richardson P.C.
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