Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is organic
Reexamination Certificate
2001-03-02
2003-09-16
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is organic
C436S525000, C436S535000, C436S544000, C436S805000, C436S815000, C436S826000
Reexamination Certificate
active
06620628
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the analysis of analytes having a net charge or their derivatives of analytes having a net charge.
Immunoassays can be used to determine the presence and quantity of a range of analytes including antigens, antibodies, therapeutic drugs, narcotics, enzymes, hormones, and proteins. Their specificity gives these assays particular utility in clinical chemistry. A variety of media are available for performing such assays including, for example, dry immunoassay elements such as multicomponent slides; microtiter plates; “dip and read” test strips; bead and tube tests; and microparticles. The different configurations of the immunoassay kits in which the assay is performed are referred to herein as analytical elements. They can comprise, among many others, a bead onto which an antibody is adhered (“immobilized”) to serve as a receptor, a cup having a surface for similar use, or a polymer layer within a slide onto which sample and reagent are applied, spread, and reacted.
In these assays, conjugate pairs are formed from either analyte (“ligand”) and receptor combinations or labeled analyte (“labeled ligand”) and receptor combinations. In competitive binding immunoassays, labeled ligand competes with unlabeled ligand for reaction with a fixed amount of a particular receptor. Signal measurements such as light absorbance or reflection density are taken of either the bound or unbound labeled ligand after appropriate treatment with various reagents such as chromophores or flourometrically sensitive materials. Unknown ligand concentration is then determined from the measured signal of the labeled ligand after removing (e.g., by washing away) the other species that are not useful for calculating ligand concentration. Thus, it is important to have a reliable method to separate properly bound and unbound forms of ligands or labeled ligands. Failure to do so can lead to inaccurate or imprecise results.
In actual practice, signal measurements can be obtained from labeled ligand, a labeled derivative or analog of the ligand, or a labeled receptor which binds in a specific manner to a ligand as in the case of a sandwich assay.
In general, ligands that are capable of binding non-specifically to, for example, hydrophobic sites and/or ligands that possess a net charge and are capable, therefore, of binding non-specifically to oppositely charged centers are particularly problematic in analytical systems. In such cases, a step may be required to separate free from bound species. That is, they tend to bind to components of assay elements while their labeled counterparts bind to receptors. This makes separation of bound and unbound species incomplete. The same can be said of the labeled ligand when it is the unlabeled ligand that is sought to be bound to the receptor. Aminoglycosides such as gentamicin and tobramycin are two amine rich analytes that possess a net positive charge under conditions in which one or more of the amine groups is protonated—generally below about pH 11. These analytes are particularly important and yet immunochemical methods of quantitatively measuring them are vulnerable to the inaccuracy and imprecision described above. It is particularly desirable to improve methods for measuring these analytes and others like them.
U.S. Pat. No. 4,547,460 proposes the use of quarternary ammonium compounds as additives for elements of an immunoassay. The ammonium compounds are used to reduce interference from bilirubin and proteins. Presumably, such interference is reduced as a result of complexing bilirubin and/or proteins with the quarternary ammonium compounds. Of course, this can only occur where the bilirubin or protein have a different charge than the ammonium compound.
U.S. Pat. No. 5,279,940 proposes the use of cationic surfactants as signal enhancers in chemiluminescene-based analytical elements. The surfactant is part of the mix of components that provides the enhanced signal. It is not involved in removing substances whose presence would otherwise generate a signal leading to an inaccurate result.
U.S. Pat. No. 4,153,668 proposes using positively charged polymers in an analytical element to more uniformly disperse a liquid containing a negatively charged analyte (a protein bound or proteinaceous substance). The patent discloses only the use of polymers having a net charge that is opposite that of the analyte.
Immunoassay accuracy and precision can still be improved where the analyte has a net charge.
SUMMARY OF THE INVENTION
The invention is an assay element for analyzing a charged analyte. The assay element employs an immobilized receptor and a material having a net charge which is the same as that of the analyte.
In one aspect of the invention the analyte is amine rich and the material is a polymer having a net positive charge and the analysis in which the element is used is a competitive binding immunoassay.
In yet another aspect of the invention, the element is a polymer having a net charge such as poly(acrlyamide-co-N-(3-methacrylamidopropyl)-N,N,N-trimethylammonium chloride.
DETAILED DESCRIPTION OF THE INVENTION
Immunoassays for analytes having a net charge can be improved by the incorporation of a suitable substance possessing the same net charge into analytical element for determining the presence or amount of the analyte. By the same net charge, it is meant that if an analyte possesses a net positive (negative) charge then the substance possesses a net positive (negative) charge, but they are not required to possess the same quantity of charge. By way of illustration, if an analyte possesses a net charge of +2, the substance must possess a net positive charge, but it is not required to be a net charge of +2. It can be any positive charge. Such an element acts to prevent ligand and/or labeled-ligand from adhering nonspecifically. This enhances accuracy and precision in, for example, competitive binding assays resulting in the production of a signal more truly representative of the true concentration of analyte in a sample.
In the assays of this invention, a receptor such as an antibody is generally immobilized in or on a component of the analytical element comprising a material having a net charge that is the same as that of the analyte. The element can be a layer of film; the surface of a cup; a fibrous layer used, for example, in “dip and read” format; a bead; a tube surface; or other medium. The receptor is specific for the analyte having the net charge. Either before the receptor is bound to the element or after the receptor is immobilized on an analytical element, sample containing the analyte is added to it. The analyte may have been previously mixed with labeled-analyte. Alternatively, the labeled-analyte could be added later. In any case, analyte and labeled analyte will then compete with each other for sites on the receptor. Because the component of the assay element to which the receptor is bound and/or some other component or surfaces nearby or in contact with the component of the element of the analytical device also has a net charge that is the same as the ligand or labeled ligand, the species which is not meant to be preferentially bound to the receptor will be repelled by the element to which it is affixed. In a subsequent step in which the species not meant to be bound is to be removed (e.g., by washing), it is not able to nonspecifically bind to the element. Accordingly, it is more readily removed from the assay. Thus, in a yet further step in which the bound ligand or bound labeled ligand is exposed to further reagents such as enzyme substrate and then measured for the generation of a signal, there is a reduced level of nonspecifically bound ligand or labeled ligand to generate such an undesirable signal. This results in an analyte measurement that is more accurate and more precise. Alternatively, the label that has been separated from that which is specifically bound to the receptor can be measured with greater accuracy and precision. This assay could also be performed in an inverse format wher
Danielson Susan
Mauck Linda
Sutton Richard
Swartz Jerome
Ceperley Mary E.
Ortho-Clinical Diagnostics, Inc.
Volyn Todd F.
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