Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-16
2004-09-28
Ponnaluri, Padmashri (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007900, C435S007920, C436S518000, C530S387100, C530S388900, C530S389100, C549S223000
Reexamination Certificate
active
06797479
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the quantification of vancomycin in a test sample. In particular, the present invention relates to immunogens, antibodies prepared from such immunogens, and labeled reagents for the specific quantification of vancomycin in a test sample, preferably for use in fluorescence polarization immunoassays.
BACKGROUND OF THE INVENTION
For the past 30 years, vancomycin has been the drug of choice for the treatment of Gram-positive infections caused by methicillin resistant
Staphylococcus aureus
. It is also the treatment in bacterial infections in patients allergic to &bgr;-lactam antibiotics. Vancomycin is produced by
Amycolatopsis orientalis
(previously designed
Nocardia orientalis
and
Streptomyces orientalis
). Vancomycin is resistant to Gram-negative organisms. Cross resistance with other antibiotics is unknown and in spite of its long usage, there have been few reports of the emergence of resistant organisms during therapy. Vancomycin is not absorbed from the gastrointestinal tract, and hence the antibiotic is used to treat enterocolitis caused especially by
Clostridium difficile
in the gut. Nagarajan, R., J.
Antibiotics,
46:1181 (1993). Vancomycin exerts its antibacterial action by binding preferentially to peptide intermediates involved in the biosynthesis of bacterial cell wall peptidoglycan.
Vancomycin is eliminated via the kidneys. The half life of the drug, 5-11 hours in normal patients, is extended to 2-5 days in patients with renal insufficiency, and is even longer in dialysis patients. While vancomycin is a relatively safe drug; adverse effects which have been observed include nephrotoxicity and autotoxicity.
For safe administration of vancomycin, it is customary to quantify its levels in patient blood. It has been suggested that because the drug stays longer in the body of a renally impaired patient, exposure to internal body temperature for longer periods results in the accumulation of degradation products which are known as Crystalline Degradation Products I and II (CDP-I and CDP-II). CDP-I and CDP-II are rotational isomers which can be separately isolated. Vancomycin and its two major degradation products CDP-I & CDP-II are shown in
FIGS. 1-3
, respectively.
It is known that vancomycin is unstable in an aqueous environment. U.S. Pat. No. 4,670,258 to Harris, et al. discloses a composition of vancomycin and a tripeptide which is said to stabilize the drug in an aqueous solution. However, such tripeptides can interfere with immunoassay techniques. For example, such interference may occur where an antibody competes with a stabilizing peptide for the same binding site of the analyte.
Historically, vancomycin concentrations in biological fluids have been determined by fluorescence immunoassay (FIA), high performance liquid chromatography (HPLC), radio immunoassay (RIA), the enzyme multiplied immunoassay technique (EMIT) or microbiological techniques. While HPLC is considered by those skilled in the art as the most accurate of all methods for quantification of vancomycin, it is a slow and labor intensive method which requires highly trained personnel and specialized equipment which is not always available in every clinical setting.
More recently, fluorescent polarization techniques have been used to assay for vancomycin. Fluorescent polarization techniques are based on competitive binding immunoassay principles. The principle behind fluorescent polarization is that a fluorescent labeled compound, when excited by a linearly polarized light, will emit fluorescence having a degree of polarization inversely proportional to its rate of rotation. Therefore, when a fluorescent labeled tracer-antibody complex is excited by a linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and emitted. When a “free” tracer compound (i.e., unbound to an antibody) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate produced in a competitive binding immunoassay.
Fluorescent polarization techniques and compounds suitable for use as fluorescent labels have been described in the art. For instance, U.S. Pat. Nos. 4,510,251 and 4,614,823, to Kirkemo et al., disclose fluorescent polarization assay for ligands using aminomethyl fluorescein derivatives, respectively. U.S. Pat. No. 4,476,229, to Fino et al., discloses substituted carboxyfluoresceins, including those containing a vancomycin analog, for use in fluorescence polarization immunoassay. U.S. Pat. Nos. 4,420,568 and 5,097,097 to Wang et al., disclose fluorescent polarization immunoassay utilizing substituted triazinylaminofluoresceins as tracers. Wang in U.S. Pat. No. 4,420,568 discloses reaction of vancomycin and dichlorotriazinylaminofluorescein (DTAF). However, this patent does not describe the structure of the product of such a reaction or its application in the heterogeneous system. Griffin et al., (JACS 115, 6482 (1993)) describe a selective method for the synthesis of vancomycin derivatives bearing alkyl, imidazole and amine functional groups attached to the C-terminus and indicated usefulness of this method for preparation of derivatives bearing different functional groups. However, there is no description of the synthesis of immunogenic material or immunocomponents and their use for quantification of vancomycin.
Commercially available fluorescent polarization assays (FPIA) for vancomycin are available. For instance, commercially available assays (Abbott TDX®, TDXFLX® assays, (hereinafter referred to as the “commercially available Abbott Vancomycin assays(s)”)) include reagents for the quantitative measurement of vancomycin in serum or plasma samples. These assays use a vancomycin derivative labeled with a dichlorotriazinylaminofluorescein (DTAF) (hereinafter referred to as the “commercially available tracer”), and sheep polyclonal antibodies against vancomycin (hereinafter referred to as “commercially available antibodies”).
FPIAs have an advantage over radioimmunoassays (RIA) in that there are no radioactive substances to dispose of and they are homogeneous assays that can be easily and rapidly performed. However, it has been reported that the commercially available vancomycin assays show an occasional increase in measured vancomycin values which do not conform with HPLC measurements. These increases have been attributed to increased cross-reactivity with CDP-I and CDP-II. As noted above, the isomers CDP-I and CDP-II can be separately isolated. As expected, any solution made from CDP-I will always contain an equilibrium mixture of both isomers. Thus, measures of CDP-I cross-reactivity reported herein measure the cross-reactivity of the equilibrium mixture.
Thus there exists a continuing need for improved assays which can quickly and accurately determine the concentration of vancomycin in the presence of cross-reactive degradation products in biological fluid. Accordingly, the present invention provides unique antibody reagents and labeled reagents for the quantification of vancomycin in a test sample. The invention also provides immunoassay methods which utilize these unique reagents. Also provided are synthetic procedures for preparing immunogens which are employed for the production of such antibody reagents, as well as procedures for preparing such labeled reagents.
Also provided are antibody reagents which can be used, and in many instances are critical, for constructing stable vancomycin calibrators and controls which can be used in assays to measure vancomycin concentration.
According to the present invention, the labeled reagents and the antibody reagents offer an advance in the art beyond previously known procedures when used in an immunoassay for the quantification of vancomycin in a test sample. Specifically, it was discovered that the antibody reagents of the present invention have essentially no cross-reactivity with the metabolites CDP-I and CDP-II. Moreover, the antibody reagents of the prese
Adamczyk Maciej
Brate Elaine M.
Perkowitz Mary M.
Rege Sushil D.
Abbott Laboratories
Anderson Regina M.
Ponnaluri Padmashri
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