Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-06-22
2004-10-05
Tate, Christopher R. (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C435S007500, C436S533000, C530S322000
Reexamination Certificate
active
06800608
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to reagents and methods for rapidly and quantitatively assaying the concentration of analytes in biological samples. More particularly, this invention includes stabilized vancomycin bidentate conjugates and other glycopeptide antibiotic bidentate conjugates and uses thereof in immunoassay formats for assaying the concentration of vancomycin in a test sample.
BACKGROUND OF THE INVENTION
The ability to determine the concentration of therapeutic agents in a biological sample is of broad importance in medicine. For example, glycopeptide antibiotics such as vancomycin, eremomycin, ristocetin A, etc., are clinically important in the treatment of post-surgical staphylococcal infections. However, even these otherwise beneficial drugs can induce life-threatening symptoms if abused or mis-dosed. Indeed, the adverse side-effects of these antibiotics, such as nephrotoxicity and ototoxicity, have been well-documented (Costa Silva, V. L. et al.,
Renal Physiol.
10:327-337 (1987); Fee, W. E. et al.,
Rev Infect. Dis.
5 (Suppl. 2):S304 (1983); Lane, A. Z. et al.,
Amer. J. Med.
62:911 (1977)). Thus, a narrow margin exists between the therapeutic dosage and toxicity-inducing overdosages (Witchitz, J. L. et al.,
Nour. Presse Med.
11:489-491 (1982); Damien, J. M. et al.,
Ann. Biol. Clin.
48:217-220 (1984). In view of the wide use of these therapeutic agents, and the importance of accurately assaying the concentration of antibiotics in patient samples, a variety of methods have been developed to permit the screening of large numbers of patients.
Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular analyte or “antigen.” An antigen is a substance which is capable of inducing an immune response, i.e., antibody production, when introduced into an animal or human body. The region of an antigen that is recognized by an antibody and to which the antibody binds is referred to as an “epitope.”Although large molecules such as proteins or other “antigens” possess multiple epitopes, low molecular weight molecules such as most pharmacological agents possess only a single epitope. Such low molecular weight molecules are referred to herein as “haptens.”
The simplest immunoassay involves merely incubating an antibody that is capable of binding to a predetermined molecule (i.e., the “analyte”) with a sample that is suspected to contain the analyte. The presence of the target molecule is determined by the presence, and is proportional to the concentration, of any immune complexes that form through the binding of antibody and the analyte. In order to facilitate the separation of such immune complexes from the unbound antibody initially present, a solid phase is typically employed. For example, in particle enhanced immunoassays, either the antibody or the antigen is immobilized on latex particles. The presence of the target molecule is then determined by incubating the immobilized antibody or antigen in the presence of the analyte-containing sample.
Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways. For example, the support can be incubated in the presence of a labeled, second antibody (i.e., a “sandwich” immunoassay) that is capable of binding to a second epitope of the target molecule. Immobilization of the labeled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the targets in the sample. In an alternative assay, the sample is incubated with a known amount of labeled targets and antibody binding sites. The presence of any target molecules in the sample competes with the labeled target molecules for the antibody binding sites. Thus, the amount of labeled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecules in the sample. This is known as a competitive immunoassay.
The various immunoassay formats can be further divided into two main classes depending upon whether the assay requires the separation of bound species from unbound species. Heterogeneous immunoassays require such purification and, hence, entail a separation or isolation step. In contrast, homogeneous assays are designed such that the removal of bound species from unbound species is unnecessary. Because homogeneous assays lack a separation step, and are more easily automated, they are more desirable than heterogeneous assays in applications that entail the screening of large numbers of patients.
If the immune complex is large enough, it will become capable of scattering light, or of spontaneously precipitating. In such cases, agglutination, nephelometric, or turbidimetric immunoassay methods may be employed. Nephelometric methods measure the light scattered by a suspension of particles or reflected toward a detector that is not in the direct path of light (Sternberg, J. C.,
Clin. Chem.
23:1456-1464 (1977)). In contrast, turbidimetric methods measure the reduction of light transmitted through the suspension of particles or aggregates. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. In both nephelometry and turbidimetry, the rate of change in light scatter may also be measured, and provides an indication of the amount of antigen present. Agglutination assays measure the precipitation of antibody-antigen complexes. Such assays can be extremely sensitive and are amenable to automation. Because nephelometric and turbidimetric methods do not require the separation of the initially present antibody from the immune complexes formed in the assay, such assays are homogenous immunoassays.
The requirement of producing large immune complexes has limited the applicability of nephelometric, turbidometric, or agglutination immunoassays to high molecular weight molecules, such as proteins, that possess several epitopes (i.e. antibody binding sites). In particular, many haptens such as therapeutic agents have only a single epitope and, as such, are incapable of forming the large immune complexes needed for such immunoassays.
Two approaches have been exploited to define agglutination assays for haptens. One approach is a particle enhanced immunoassay involving the agglutination of antibody-coated particles with a polyepitopic species or a developer antigen containing at least two covalently coupled hapten analogs (e.g., a protein carrier, such as BSA) (Mongkolsirichaikul, D. et al.,
J. Immunol. Meth.
157:189-195 (1993)). The agglutination reaction requires the use of a developer antigen or a polyepitopic species because a molecule that has only one epitopic site cannot bind two antibodies, and hence cannot cross-link two antibodies together. Such cross-linking is, however, an essential step in the formation of large immune complexes. The second particle enhanced approach involves the agglutination of hapten-coated particles and antibody for the agglutination reaction.
With either method, the hapten or drug in the sample competitively binds to the antibody binding sites and results in inhibition or reduction of the immunoagglutination. Particle agglutination assays for therapeutic drugs and drugs of abuse which use hapten-coated particles are commercially available. Examples of such assays are PETINIA (Du Pont) and AbuScreen (Roche), Advisor (Abbott) and that of Mitsubishi.
A third solution to this problem has recently been described by Yan, et al. in U.S. Pat. No. 5,747,352, which is incorporated herein by reference. Yan et al. disclose a particle-enhanced homogeneous assay for aminoglycoside antibiotics, including vancomycin. The method is based on a latex-avidin bidentate assay for vancomycin using a biotinylated vancomycin bidentate conjugated to an avidin-latex particle. In the bidentate immunoassay method described by Yan et al., the biotinylated vancomycin/avidin latex particle conjugate is incubated with an anti-vancomycin antibody and a test sample. The inhibition of agglutination between the conjugate and the
Cheng Anthony K.
Kim Julie S.
Beckman Coulter Inc.
Chism B. Dell
Hill D. David
Hogan & Hartson LLP
May William H.
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