REAGENTS AND METHODS FOR THE DETECTION AND QUANTIFICATION OF VANCOMYCIN IN BIOLOGICAL FLUIDS

Abstract: Immunoassay reagents, methods and test kits for the specific quantification of vancomycin in a test sample are disclosed. The reagent comprises antibodies prepared with immunogens of Figure 6 wherein P is an immunogenic carrier material and X is a linking moiety. Also described is the synthesis of labeled reagents of Figure 8 wherein Q is a detectable moiety, preferably fluorescein or a fluorescein derivative, and X is a linking moiety.

Description: REAGENTS AND METHODS FOR THE DETECTION AND QUANTIFICATION OF VANCOMYCIN IN BIOLOGICAL FLUIDS 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 a 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 the patients allergic to b- lactam antibiotics. Vancomycin is produced by Amycolatopsis orientalis (previously designated 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 the antibiotic is used to treat enterocolitis caused especially by Clostridium difficile in the gut. 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 & 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 Figures 1-3, respectively. It is known that vancomycin is unstable in an aqueous environment. U.S. Patent 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. 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 the 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 the 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 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. Patent Nos. 4,510,251 and 4,614,823, to Kirkemo et al., disclose fluorescent polarization assay for ligands using aminomethyl fluorescein derivatives as tracers, and the aminomethyl fluorescein derivatives, respectively. U.S. Patent 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. Patent 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. Patent No. 4,420,568 discloses reaction of vancomycin with dichlorotriazinylamino- fluorescein (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 fluorescence polarization assays (FPIA) for vancomycin are available. For instance, commercially available assays (Abbott TDX ® . TDXFLX ® assays, Abbott Laboratories, Abbott Park, II.; hereinafter referred to as the "commercially available Abbott Vancomycin assay(s)") include reagents for the quantitative measurement of vancomycin in serum or plasma samples. These assays use a vancomycin derivative labeled with a dichlorotriazinylamino- fluorescein (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 radio immunoassays (RIA) in that there are no radioactive substances to dispose off 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 a 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. 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. Summary of the Invention The present invention provides a method for the quantification of vancomycin in a test sample, wherein: (a) the test sample is contacted with an antibody reagent having antibodies which are capable of specifically binding to vancomycin and are produced with an immunogen of Figure 6 wherein P is an immunogenic carrier material, and X is a linking moiety of from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain -0-0 linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms, and a labeled reagent of Figure 8 wherein Q is a detectable moiety and X is from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain -0-0 linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms, to form a reaction solution; and (b) measuring the amount of the labeled reagent in the reaction solution which either is or is not bound with an antibody as a function of the amount of vancomycin in the test sample. The invention further provides the above method wherein fluorescence polarization is employed. In a preferred method of the invention the antibody is produced with an immunogen of Figure 6 and the labeled reagent is as shown in Figure 8. This invention further provides novel immunogens of Figure 6 which are useful to produce antibodies which specifically bind vancomycin. This invention also provides the hybridoma cell line designated as HB 11834 and monoclonal antibodies produced thereby. Such monoclonal antibodies are most preferred for the quantification of vancomycin, most preferably by fluorescence polarization. Also provided are kits useful for the quantification of vancomycin in a test sample having antibody reagents of Figure 6 and labeled reagents of Figure 8. Preferred kits have antibodies produced from an immunogen of Figure 6; most preferred are monoclonal IgG antibodies produced from an immunogen of Figure 5. The present invention also provides synthetic procedures for preparing immunogens which are employed for the production of such antibody reagents, and for preparing such labeled reagents. Brief Description of the Figures FIGURE 1 shows the structure of vancomycin. FIGURE 2 shows the structure of one of the major metabolites of vancomycin, CDP-I. FIGURE 3 shows the structure of another of the major metabolites of vancomycin, CDP-II. FIGURES 4a through 4c illustrate a representative synthetic pathway for coupling vancomycin to carrier protein. FIGURES 5a through 5d illustrate the synthetic pathway for coupling vancomycin to thyroglobulin according to the method of the present invention. FIGURE 6 shows the structure of the immunogen of the invention. FIGURE 7 shows the structure of the most preferred immunogen of the invention. FIGURE 8 shows the structure of the labeled reagent of the invention. FIGURE 9 shows the general structure of the most preferred labeled reagent of the invention. FIGURES 10a and 10b illustrate the synthetic pathway for coupling vancomycin to fluorescein according to the method of the present invention. FIGURE 11 shows the results of a correlation of an existing commercial assay with an assay of the present invention utilizing the most preferred antibody of the invention. FIGURE 12 shows the correlation of an assay of the present invention with HPLC. FIGURE 13 shows the results of a fluorescence polarization immunoassay of the present invention. Detailed Description of the Invention As used in this specification and the attached claims, the following words shall have these respective meanings: "Heteroatom" means nitrogen, oxygen, sulfur and phosphorous. "CHCI3" means chloroform, "CDCI3" means deutero chloroform, "MeOH" means methanol, "DMF" means dimethylformamide, "CH2CI2" means methylene chloride, "Et2θ" means diethyl ether and "DMSO" means dimethylsufoxide. "Linking moiety", "tether", "spacer", "spacer arm", and "linker" are used interchangeably and are meant to define any covalentiy bound chemical entity that separates one defined substance (such as a hapten) from a second defined substance (such as a immunogenic carrier or detectable moiety). The present invention provides immunogens, antibodies prepared from such immunogens, and labeled reagents which are suitable for use for the quantification of vancomycin. The specific quantification of vancomycin is accomplished by first contacting a test sample with a labeled reagent (also referred to as a tracer) of the present invention and an antibody reagent of the present invention, either simultaneously or sequentially in either order, and then measuring the amount of the labeled reagent which either has or has not participated in a binding reaction with the antibody reagent as a function of the amount of vancomycin in the test sample. The antibodies and labeled reagents of the present invention are especially useful in fluorescence polarization immunoassays (FPIA) for the specific quantification of vancomycin. According to a preferred embodiment of the present invention, the labeled reagent and the antibody reagents are used in a fluorescence polarization immunoassay which combines specificity with the speed and convenience of homogeneous methods to provide a reliable quantification of vancomycin in a test sample and avoidance of interference from the major metabolites of vancomycin, i.e., CDP-I and CDP-II. The test sample can be any naturally occurring bodily liquid, or an extract or dilution thereof, and includes, but is not intended to be limited to whole blood, serum, plasma, urine, feces, saliva, cerebrospinal fluid, brain tissue, and the li ke . As is known to one of ordinary skill in the art, when preparing specific antibodies and complementary labeled haptens one needs to consider the chemical structure of both the immunogen used to elicit the antibody response and the labeled hapten. Traditionally, one attaches the hapten to the carrier protein through a site on the hapten that is remote from the unique features of the hapten that are critical for achieving selective antibodies. Likewise, when preparing a labeled hapten able to bind to such antibodies, it is customary to attach the label through the same site on the hapten as employed for linking the carrier protein to the hapten. One reason behind such an approach is that the carrier protein may sterically block access of the immune system to that part of the hapten. The complementary labeled hapten is synthesized by attaching its label to the same site on the hapten as the immunogen uses for attachment of its carrier protein, so as not to interfere with antibody binding to the critical features of the hapten. Therefore, it was surprisingly and unexpectedly discovered that the vancomycin immunogen and labeled vancomycin of the present invention, which are derived from different sites of attachment on vancomycin, lead to development of antibodies specific for vancomycin and an assay displaying an excellent cross-reactivity profile for the major metabolites of vancomycin. Among the most surprising discoveries is that, based on the limits of the sensitivity of the assay, the monoclonal antibody secreted by HB 11834 displays no detectable cross-reactivity with CDP-I. This results in an improved assay for the quantification of vancomycin. Synthesis of Immunogens Antibodies of the present invention, both polyclonal and monoclonal, are produced with immunogens prepared from a vancomycin molecule which is conjugated to the carrier protein via the carboxylic acid terminal of vancomycin as shown in the general formula of Figure 6 wherein P is an immunogenic carrier material and X is a linking moiety. In the immunogens of the present invention, X is preferably a linking moiety consisting of from 0 to 50 carbon and hetero atoms, including not more than ten heteroatoms, arranged in a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked, that cyclic moieties contain six or fewer members, and that branching may occur only on carbon atoms. As would be understood by one skilled in the art, the immunogenic carrier material P, can be selected from any of those conventionally known. In most instances, P will be a protein or polypeptide, although other materials such as carbohydrates, polysaccharides, lipopolysaccharides, poly(amino) acids, nucleic acids, and the like, of sufficient size and immunogenicity can also be employed. Preferably, the immunogenic carrier material is a protein such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), thyroglobulin, and the like. In the preferred immunogen, P is thyroglobulin and X is -NH(CH2)3 C(=0)-. The most preferred immunogen is shown in Figure 7. However, the compound of Figure 7 is not limited to a single conjugate of vancomycin and the immunogenic carrier, as one skilled in the art would realize. Rather, the ratio of vancomycin derivative to immunogenic carrier is defined by the number of chemically available functional groups on the immunogenic carrier P and controlled by the ratio of the two materials in the synthesis. The degree of substitution on P by the vancomycin derivative can vary between 1 to 100% of the available functional groups on the immunogenic carrier. The level of substitution is preferably between 10% to 95%; and more preferably, between 15% to 85%. As stated above, the immunogenic conjugate of the present invention is prepared by coupling vancomycin to a carrier material via the carboxylic acid terminal of vancomycin. As shown in Figures 4a through 4c, vancomycin is coupled according to methods known to those skilled in the art with a bifunctional compound designated V-X-Y wherein X is a linking moiety and V- and -Y are functional groups one of which can react with the carboxylate of vancomycin (I) and the other with chemically available functional groups on P. Many bifunctional linkers are known in the art. For example, heterobifunctional linkers are described in, e.g. U.S. Patent 5,002,883 to Bieniarz, et al. Heterobifunctional linkers may be preferred in some cases due to the specificity of their ends for one functional group or another. Likewise, for convenience in the synthesis of the immunogen, the functional groups V- and -Y may be protected, and deprotected at the desired time, following techniques well known to, or easily aquired by, those skilled in the art (see, e.g., T. W. Greene and P. G. M. Wutts, "Protective Groups in Organic Synthesis, 2nd Ed." 1991 , John Wiley and Sons). Generally, in the preparation of the immunogens of the present invention, V is selected from the group consisting of -OH, -halo (-CI, -Br, -I), -SH, or -NHR'-, where R' is selected from H, alkyl, aryl, substituted alkyl or substituted aryl. Y may be selected from the group consisting of carboxy (-C(=0)OH), amino (-NH2), aldehyde (-CH(=0)), or azido (-N3). As stated above, X is a linking moiety from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged in a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked, that the sequence V- X-Y cannot contain -O-O- linkages, that cyclic moieties contain six or fewer members, and that branching may occur only on carbon atoms. Referring now to the representative synthetic scheme shown in Figures 4a - 4c, reaction of vancomycin (Fig. 4a) with V-X-Y produces tethered intermediate compound (Fig. 4b) having linking moiety X with a functional group Y. The functional group -Y, can be reacted in any of several ways, known to those skilled in the art, with the functional groups on an immunogenic carrier. It is preferable to form amide bonds, which typically are quite stable. Amide bonds are formed by first activating the carboxylic acid moiety [Y=(- C(=0)OH)] of the spacer arm by reaction with an activating reagent such as 1 ,3-dicyclohexylcarbodiimide and an additive such as N-hydroxysuccinimide. The activated form (Figure 4b) is then reacted with a buffered solution containing the immunogen ic carrier materials. Alternatively, the carboxylic acid group may be converted, with or without isolation, into a highly reactive mixed anhydride, acyl halide, acyl imidazolide, or mixed carbonate and then combined with the immunogenic carrier materials. As is readily apparent to one with ordinary skill in the art, there are many reagents that can be used to form amide bonds other than those listed above and such reagents require no special mention. Alternatively, a spacer arm with a terminal amine functionality (Y=-NH2) can be transformed into a highly reactive N-hydroxysuccinimide urethane by reaction with N,N'- disuccinimidyl carbonate in a suitable solvent, such as acetonitrile or dimethylformamide. The resultant urethane is then reacted with the immunogenic carrier materials in a buffered, aqueous solution to provide an immunogen. Additionally, a spacer arm with a terminal aldehyde functionality [Y = -CH(=0)] can be coupled to the immunogenic carrier materials in a buffered, aqueous solution and in the presence of sodium cyanoborohydride, by reductive amination according to methods known to those skilled in the art. Alternatively, spacer arms containing an alcohol group [Y =-OH] can be coupled to the immunogenic carrier materials by first reacting it with phosgene or phosgene equivalent, such as di- or triphosgene or carbonyldiimidazole, resulting in the formation of a highly reactive chloroformate or imidazoloformate derivative (usually without isolation). The resultant active formate ester is then reacted with the immunogenic carrier materials in a buffered, aqueous solution to provide an immunogen. Alternatively, when Y = -N3, the tethered intermediate can be coupled to the immunogenic carrier by photolysis in aqueous buffered solution. The preferred immunogen of Figure 6 is thus prepared according to the scheme of Figures 5a through 5d. The carboxyl group of vancomycin (Fig. 5a) is activated with dicyclohexylcarbodiimide and N-hydroxy benzotriazole (HOBT). Further reaction with the linker, 4-amino butyric acid methyl ester [V=-NH2, X=-(CH 2 ) 3 -, Y = -CO2H] which after hydrolysis gives the tethered intermediate [X=-(CH 2 )3-, Y= -CO2H]. Y is then activated with 1 -(3-Dimethylaminopropyl)-3- ethyl carbodiimide (EDAC) and coupled to P. Those skilled in the art will recognize that other methods for peptide bond formation could be employed with equal success. Thus, in the manner just described, vancomycin can, via this and other reactive sites on the molecule such as amines or alcohols, be coupled to immunogenic carrier materials by various conventional techniques known in the art where P is an immunogenic carrier material as described previously. Furthermore, in a manner analogous to linking haptens to carrier materials, spacer arms can be conjugated to solid supports having functional groups such as amino, hydroxyl or carboxyl groups that are reactive in a complementary sense with reactive groups on the spacer arm. The result is a solid phase which can be used to separate or purify antibodies against the hapten. Such coupling techniques are also well known in the art. Production of Antibodies The immunogens according to the present invention may be used to prepare antibodies, both polyclonal and monoclonal, according to methods well known in the art. Generally, a host animal, such as a rabbit, goat, mouse, guinea pig, or horse is injected at one or more of a variety of sites with the immunogen, normally in a mixture with an adjuvant. Further injections are made at the same site or different sites at regular or irregular intervals thereafter with bleedings being taken to assess antibody titer until it is determined that optimal titer has been reached. The antibodies are obtained by either bleeding the host animal to yield a volume of antiserum, or by somatic cell hybridization techniques or other techniques known in the art to obtain monoclonal antibodies, and can be stored, for example, at -20°C. Besides intact immunoglobulins, the term antibodies as used herein includes antigen binding fragments of the immunoglobulins which may be produced by known methods, e.g., Fab, F(ab')2 and Fv. It is to be noted that the replacement of the commercially available antibody with the preferred antibody of the present invention alone improves the performance of the vancomycin assay. It shall also be noted that the preferred method of the invention utilizes antibodies which do not bind metabolites that are not intended to be detected, to the extent such binding interferes with the accuracy of the assay, e.g., CDP-I AND CDP-II. Preparation of the Labeled Reagent As noted above, the labeled vancomycin reagent of the present invention is prepared by attachment of the label at the secondary amino terminal of vancomycin, that is, a position which differs from the position at which the carrier protein is attached. Labeled reagents of the present invention for vancomycin have the general formula shown in Figure 8 wherein Q is a detectable moiety, preferably a fluorescent moiety; and X is a linking moiety. In the preferred labeled reagent, Q is a fluorescein derivative chosen from the group consisting of 4'- aminomethylfluorescein, 5-aminomethylfluorescein, 6-amino- methylfluorescein, 6-carboxyfluorescein, 5-carboxy- fluorescein, 5 and 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein; and X is preferably a linking moiety consisting of from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged in a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms. In the more preferred labeled reagent, Q is chlorotriazinylaminofluorescein and X=0, i.e., the vancomycin derivative is directly attached to the fluorescein derivative. The preferred labeled reagent of the invention has the structure shown in Figure 9. In a manner analagous to the synthesis of the immunogenic conjugate, the labeled reagents of the invention are synthesized from vancomycin by first differentially protecting the primary amino group (see, T. W. Greene and P. G. M. Wutts, "Protective Groups in Organic Synthesis, 2nd Ed." 1991 , John Wiley and Sons) followed by selectively reacting the secondary amino group with the detectable moiety. More specifically, the preferred labeled reagent can be synthesized as shown in Figures 10a and 10b by: (i) reacting vancomycin base with dilute HCI at pH 6.0 to protect the primary amino group as a quaternized nitrogen followed by (ii) reacting it with dichlorotriazinylaminofluorescein (DTAF) to give the labeled reagent. In its most preferred aspect, the above synthetic methods are used to produce the labeled reagents of Figure 9. Vancomycin Assay utilizing Fluorescence Polarization Immunoassay By following a fluorescence polarization immunoassay (FPIA) format employing the reagents of the present invention, the concentration, or level, of vancomycin in a test sample can be accurately quantified. To perform a FPIA for the specific quantification of vancomycin, calibration curves are generated from calibrators having known concentration vancomycin. Generally, fluorescent polarization techniques are based on the principle that a fluorescent tracer, when excited by plane polarized light of a characteristic wavelength, will emit light at another characteristic wavelength (i.e., fluorescence) that retains a degree of the polarization relative to the incident stimulating light that is inversely related to the rate of rotation of the tracer in a given medium. As a consequence of this property, a tracer substance with constrained rotation, such as in a viscous solution phase or when bound to another solution component with a relatively lower rate of rotation, will retain a relatively greater degree of polarization of emitted light than if in free solution. Therefore, within the time frame in which the ligand and tracer compete for binding to the antibody, the tracer and ligand binding rates should yield an appropriate proportion of free and bound tracer with the preservation of important performance parameters such as selectivity, sensitivity, and precision. When performing a fluorescent polarization immunoassay for the specific quantification of vancomycin according to the present invention, a test sample suspected of containing vancomycin is contacted with an antiserum or monoclonal antibodies prepared with immunogens according to the present invention, in the presence of labeled reagent of the present invention. Plane polarized light is then passed through the solution to obtain a fluorescent polarization response and the response is detected as a measure of amount of vancomycin present in the test sample. The fluorescence polarization assays can be conducted in commercially available automated instruments (e.g., AxSYM®, TDx®, and TDxFLx®, Abbott Laboratories). According to the present invention, it has been unexpectedly and surprisingly found that superior fluorescence polarization immunoassay assay results for the quantification of vancomycin are obtained when employing an antibody derived from the immunogen shown in FIG. 6 with the fluorescent labeled reagent shown in FIG 8. In particular, it was unexpectedly and surprisingly found that the use of the labeled reagent of FIG 9 in combination with a monoclonal antibody produced in response to the immunogen of FIG 7, resulted in an assay which shows very low, essentially zero, (that is, below the limits of the sensitivity of the assay) cross-reactivity to the major metabolites of vancomycin, CDP-I and CDP-II. Most preferred is the fluorescence polarization method which employs monoclonal IgG antibody produced by the hybridoma designated ATCC HB 11834. The amount of tracer bound to the antibody varies inversely to the amount of vancomycin present in the test sample. Accordingly, the relative binding affinities of vancomycin and the tracer to the antibody binding site, are important parameters of the assay system. Other Assay Formats In addition to fluorescence polarization immunoassays, various other immunoassay formats can be followed for the quantification of vancomycin according to the present invention. Generally, such immunoassay systems depend upon the ability of an immunoglobulin, i.e., a whole antibody or fragment thereof, to bind to a specific analyte from a test sample wherein a labeled or detectable reagent is employed to determine the extent of binding. Such detectable labels include, but are not intended to be limited to, enzymes, radiolabels, biotin, toxins, drugs, haptens, DNA, RNA, liposomes, chromophores, chemiluminescens, colored particles and colored microparticles, and fluorescent compounds such as those described earlier. Typically, the extent of binding in such immunoassay system formats is determined by the amount of the detectable moiety present in the labeled reagent which either has or has not participated in a binding reaction with the analyte and requires that the amount of the detectable moiety detected and measured can be correlated to the amount of analyte present in the test sample. For example, in a competitive immunoassay system, the substance being measured (often referred to as a ligand) competes with a substance of close structural similarity to that of the ligand and which is coupled to a detectable moiety (often referred to as a tracer) for a limited number of binding sites on antibodies specific to the portion or portions of the ligand and tracer with structural similarity. Test Kits A test kit according to the present invention includes reagents necessary to perform a desired immunoassay for the quantification of vancomycin in a test sample. The test kit may be presented in a commercially packaged form as a combination of one or more containers holding the necessary reagents, and/or as a composition or admixture where the compatibility of the reagents will allow. Preferably, a test kit includes all reagents, standards, buffers, diluents, etc. which are necessary to perform the assay. Particularly preferred is a test kit for the fluorescent polarization immunoassay quantification of vancomycin in a test sample, which includes fluorescent tracer compounds and antibodies as described above for the quantification of vancomycin. It is to be understood that the test kit can, of course, include other materials as are known in the art and which may be desirable from a user standpoint, such as sample pretreatment solutions, buffers, diluents, standards, and the like. The present invention will now be illustrated, but is not intended to be limited to, the following examples. EXAMPLE 1. Synthesis of Vancomycin Immunogen a) Synthesis of methyl-4-amino butyrate 4-Amino butyric acid (5.00g, 48.5mmol) is taken in a 200 mL round bottom flask. Dimethoxy propane (80mL, 65 mmol) is added to the flask with stirring. Concentrated hydrochloric acid (15mL) is added to the reaction and stirred at room temperature overnight. Solvents are removed under reduced pressure on a rotary evaporator without heating. The solid is dissolved in a minimum amount of MeOH and is reprecipitated with ether. The precipitated solid is filtered under suction and washed with Et 2 0 (2X50 mL). The solid (yield: 6.9 g (96%)) is then dried under vacuum. 1 H NMR of the free amine(CDCI 3 ): 2.1 (quintet, 2H), 2.5 (triplet, 2H), 3.2 (broad triplet, 2H), 3.7 (singlet, 3H), 8.1 (singlet, 2H) b) Synthesis of vancomycin-aminobutyrate derivative Vancomycin (500mg, 0.34 mmol) base is taken in a 25mL round bottom flask. DMSO (4mL) is added and stirred, with warming as necessary, until a clear solution results. Methyl-4- amino butyrate .HCI from Example 1 (a) (506mg, 3.4 mmol) is added to the reaction followed by hydroxybenzotriazole (105mg, 0.69 mmol) and triethylamine (0.0976 mL, 0.69 mmol). Reaction is stirred at room temperature for 3 to 7 days under N 2 . Reaction is followed by HPLC. After the starting material had been consumed, the precipitated solid is removed by filtration and the filtrate is purified by reverse phase HPLC using a C-18 column as described below. The collected fractions (yield: 310 mg) are lyophilized. Analytical HPLC conditions are as follows: The column is 7.8mmX300mm C-18 (Bondapak C-18, Waters, Marlborough, MA) with a continuous gradient mobile phase of acetonitrile: ammonium acetate (50mM) (10% acetonitrile to 50% acetonitrile developed over 15 min.) at a flow rate of 3.0 ml/min. Detection is at 254nm. Preparative HPLC conditions are as follows: The column is 19mmX250mm C-18 (Dynamax 60A C-18, Rainin, Woburn, MA) with a continuous gradient mobile phase of acetonitrile: ammonium acetate (50mM) (10% acetonitrile to 50% acetonitrile developed over 15 min.) at a flow rate of 8.0 ml/min. Detection is at 254nm. Mass Spectrometry (MS): Electron Spray lonization (ESI) MH + 1547, (M2H) + 774. c) Synthesis of vancomycin hapten The vancomycin-aminobutyrate derivative from Example 1 (b) (165mg, 0.1 mmol) is dissolved in DMF/Water (2mL:3mL) in a 25mL round bottom flask. The flask is cooled to 0°C and lithium hydroxide (56mg, 1.3mmol) is added. Reaction is stirred at 0°C for 2hrs and warmed to room temperature and followed by HPLC. After the starting material has been consumed the reaction is directly purified by preparative HPLC using a reverse phase column. The solvent is lyophilized to give the product (yield: 160 mg). Both analytical and preparative HPLC are as stated above. Mass Spectrometry (MS): ESI MS gives (MH)+ at 1533 indicating the correct molecular weight of the hydrolyzed linker attached to vancomycin. d) Synthesis of Immunogen Thyroglobulin (100mg, 0.0002mmol) is dissolved in sodium phosphate monobasic buffer (5mL, pH adjusted to 6.7 with dilute NaOH). Vancomycin hapten from Example 1 (c) (50mg, 0.0321 mmol) is added followed by EDAC (9.2mg, 0.0482mmol). The resultant reaction is stirred for 2 days at room temperature. The contents are transferred to a membrane and dialyzed with 0.1M Na HP0 4 buffer (monobasic, pH7.8 adjusted with NaOH) for 2 days changing the solvent every 4 hrs. The contents in the dialysis bag are lyophilized to yield 130mg of the desired immunogen. EXAMPLE 2. Production of Anti-vancomycin Antibody 15-109- 592 A female, 6-8 weeks old, RBF/DnJ mouse (Jackson Laboratories, Bar Harbor, Maine) is immunized with the vancomycin immunogen of Example 1 emulsified with Freund's adjuvant (Difco, Detroit, Ml). The primary immunization is administered with Freund's Complete Adjuvant and subsequent boosts with Freund's Incomplete Adjuvant. The animal boosting interval for this long term immunized animal is at weeks 1 , 3, 5, 12, 17 and 24 with the respective dosage level at 25, 12.5, 12.5, 10, 10 and 10 μg per animal at one subcutaneous site and one intraperitoneal site for the first three boosts and at two subcutaneous sites each for the last three boosts. Periodically, bleeds are made to confirm that an antibody response is developing. The animal was allowed a 7 week rest period before a 10μg boost was administered to the spleen 3 days prior to fusion. On the day of the fusion, the mouse is euthenized by a quick cervical dislocation and the spleen is removed. The splenocytes are washed one time in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco, Grand Island, New York) and centrifuged at 1000 RPM for 10 minutes. The pelleted splenocytes are combined with SP2/0 myeloma cells (Dr. Milstein, Cambridge, UK) at 1 :1 ratio, washed in IMDM, and centrifuged. The supematent is removed and 1 ml of 50% polyethylene glycol (PEG; American Type Tissue Culture Collection, Rockville, MD) is added to the pellet for 1 minute as the pellet is resuspended in IMDM containing hypoxanthine, aminopterin, thymidine (HAT Gibco) and 15% Fetal bovine serum (FBS; Hyclone Labs, Logan, Utah). To enhance the fusion frequency, 0.5% Salmonella typhimurium mitogen v/v (STM; RIBI immunochem Research, Inc., Hamilton, Montana) and 1 % v/v ORIGEN (Igen, Rockville, MD) are added to the fusion cell suspension and plated into 24 well tissue culture plates. The primary screening of the fusion occurred on day 10 confluent cultures. A commercial assay (TDx®; Abbott Laboratories) is used to detect anti-vancomycin reactivity in supernate samples. The tissue culture supernate is loaded in duplicate into the sample well and 10μl of either the A calibrator (O μg/mL vancomycin) or the F calibrator (100 μg/mL vancomycin) from the commercial calibrator kit is loaded into the pre-dil well. Diluent buffer is placed in the S and P pots of the reagent pack and the commercial tracer is used in the T pot. Because hybridoma culture supernatents are very dilute, sample volume is increased to 90μl. The polarization of the samples is measured and only one hybrid, 15-109, is identified as specific to vancomycin as measured by a decrease in polarization in the presence of the F calibrator (See Table 1 ). This is due to the free vancomycin binding to the antibody and blocking the tracer from binding, therefore causing a decrease in the signal. Table 1 a μg/ml Vancomycin b Negative Control is an irrelevant antibody tissue culture supernate. c Positive Control is Vanco clone 3-266-279 tissue culture supernate. Hybrid 15-109 is cloned by limiting dilutions from 1 - 100 to 1 -100,000. The cloning media is IMDA with 10% v/v FBS and 1% v/v HT Supplement (Gibco). A 200μl cell suspension is added to each well of a 96-well tissue culture plate. The hybrid, now designated 15-109-133, is selected for further evaluation based on additional screening of the clone supernate of confluent cultures. The monoclonal antibody hybrid 15-109-133 is first concentrated 10-fold using an Amicon filtration system. Then, the raw antibody (the antibody is still in fetal bovine serum) is cut using saturated ammonium sulfate (50%). The solution is then centrifuged at 4000 RPM (revolution per minute) and the supernate is discarded. The pellet is resuspended into PBS (pH 7.4) at a volume 1/10th the original volume after concentration. This antibody solution is then dialyzed in PBS (pH 7.4) and after dialysis is diluted using the commercial buffer (phosphate, azide, and bovine gamma globulin buffer) as follows: straight, 1 :2, 1 :4, 1 :8, 1 :16, and 1 :32. The samples are run in duplicates in the sample well using a sample volume of 10 (100μL) instead of 2 (20μL) using the same Mode 1 vancomycin assay previously discussed. The instrument calculates the mP (millipolarization values) as described previously and the dilution of antibody generating the highest mP is chosen as the dilution of antibody to use in the S pot (antibody pot) in the reagent kit. The antibody is diluted into phosphate buffer including 10% glycerol and 5% BSA). The tracer pot contains the existing market tracer re-purified by HPLC and diluted in a Tris buffer (Plus 0.7% SDS and 0.5% LDS). This purified tracer is diluted to 1.7μg/mL in the tracer pot. The popper consists of 20mM copper sulfate + 2.5% 5-SSA. This reagent pack is loaded into the instrument with vancomycin (analyte) at 0, 5, 10, 25, 50, and 100μg/mL as the calibrators run in duplicates along with controls at 7, 35, and 75μg/mL. Assay 16 with Mode 1 pipetting is used on the instrument and a standard curve is calibrated and stored. CDP-1 samples at various concentrations are run to ensure no CDP-1 cross reactivity exists. Based on the further screenings, a clone, now designated 15-109-592, is selected for deposit. The isotype of the monoclonal antibody secreted from the cell line identified as 15-109-592 was determined on a antibody isotyping kit (Mouse Monoclonal, Southern Biotech, #5080-05, Birmingham, Alabama). The assay is performed according to the vendor recommendations and the results indicate an isotype of lgG1 , kappa light chain. Cell Line Deposit In accordance with the Budapest Treaty, the hybridoma cell line, designated as hybrid 15-109-592, is deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, United States of America. The deposit date is 16 February 1995 and the ATCC number assigned to the cell line is HB 11834. EXAMPLE 3. Synthesis of Vancomycinchlorotriazinylamino- fluorescein Tracer Vancomycin base (576mg, 0.4mmol, 1.5 eq) is dissolved in DMF (8mL) (with warming if necessary to 40°C) in a 50mL round bottom flask with a wide mouth. A small pH electrode is inserted to monitor the pH of the reaction. A solution of dichlorotriazinyl- aminofluorescein.HCI (DTAF; 132mg., 0.26 mmol, 1 eq.) in DMF (2mL) is added to the flask. The reaction turns orange-yellow and the pH dropped to 6.0 ± 0.5 and is stirred overnight while maintaining this pH. Reaction products are monitored by analytical HPLC. After all the DTAF had been consumed DMF is removed under vacuum to about 2-3ml. The reaminder is purified by HPLC. Appropriate fractions are collected and the solvent is lyophilized to give an orange yellow powder (yield: 350 mg). Analytical HPLC conditions are as follows. The column is a 3.9mmX300mm C-18 (DYNAMAX C-18, Rainin) with a continuous gradient mobile phase (A = ammonium acetate; B = acetonitrile (50mM); % B = 10, % B = 50 developed over 15 min.) at a flow rate of 1.5 ml/min. Dectection is at either 254 or 486 nm. Preparative HPLC utilizes the same conditions as above with a 19mmX250mm C-18 column (DYNAMAX C-18, Rainin). MS: ESMS gives MH+ at 1908, (M2H) + at 953 EXAMPLE 4. Fluorescence Polarization Immunoassay For Vancomycin The tracer (Example 3) and monoclonal antibody #15-109- 592 (Example 2) are optimized to perform similar to or better than the TDX®/TDXFLX® Abbott Vancomycin assay with the advantage of no CDP-1 cross reactivity in the presence of vancomycin. As discussed previously, by adding a constant concentration of antibody and tracer to a test sample, the ratio of vancomycin-antibody complexes to tracer-antibody complexes that are formed is directly proportional to the amount of vancomycin in the sample. When the mixture is excited with linearly polarized light and the polarization of fluorescence emitted by unbound tracer and tracer-antibody complexes is measured, one is able to quantitate or qualitate the prescence of vancomycin in a test sample. The results can be quantified by net millipolarization units (mP) and span. Net millipolarization indicates the polarization detected when a maximum amount of tracer is bound to the antibody (i.e., in the abscence of vancomycin). The higher the net mP units, the better the binding of the tracer to the antibody. Assay span is the difference between the net millipolarization values obtained when the maximum tracer is bound in the abscence of any vancomycin and the net millipolarization obtained when a specific amount of vancomycin is present in the test sample. The millipolarization units are automatically interpolated from a stored standard curve and expressed as the amount of vancomycin (microgram) per mL of sample. The purified (ammonium sulfate cut) vancoymcin antibody 15-109-133 is diluted in phosphate buffer with 2.5% Bovine Serum Albumin and 10% glycerol to a concentration of 20μg/mL which composes the S pot. The tracer pot (T pot) is a Vancomycin-DTAF tracer diluted in Tris buffer with 0.7% sodium lauryl sulfate and 0.5% lithium lauryl sulfate to a concentration of 0.275μg/mL. Together these two components along with the pretreatment pot (P pot) yield a 96.94 mP span with intensity values ranging from 3500 to 4500 units. (Intensity values are a measure of the effect of the antibody and tracer reacting together. As either the antibody or tracer concentration is increased in the assay, the intensity value gets larger.) The accuracy of the vancomycin assay of the present invention is shown by comparing it with the commercially available vancomycin assay using 56 patient samples. Both assays correlated to give an R=0.996 and y=0.92x-0.008 (FIGURE 11 ). Furthermore, the assay of the present invention was compared to HPLC quantification. The new assay correlation against HPLC gave an R=0.998 and y=1.007+1.053 (FIGURE 12), while the commercially available vancomycin assay correlated against HPLC gave an R=0.996 and y=1.088x+1.252 (Data not shown). Both the assay of the present invention and the commercially available vancomycin assays have a sensitivity of less than 2.00μg/mL and can detect between 0 and 100μg/mL of vancomycin in a sample. Samples containing greater than 100μg/mL of vancomycin can be automatically diluted twofold or fourfold by reducing the sample volume to 1.0 or 0.5 as instructed in the assay manual. The precision of both the assay of the present invention and the commercially available vancomycin assay are the same. All between run, within run, and total coefficient of variation (CV) are less than 6% (See Table 2). For cross reactivities, vancomycin is tested at levels 0, 40, and 80μg/mL with various cross reactants present at levels of 0, 1 , 10, 50, and 100μg/mL. Surprisingly, all cross reactants, including CDP-1 , show less than 2% cross reactivity with vancomycin which means all readings are below the sensitivity of the assay (2 μg/ml) . In contrast, the commercially available vancomycin assay has elevated levels of CDP-1 cross reactivity ranging from 39.58% to 65% with or without vancomycin present. Surprisingly, the antibody of the present invention shows no detectable cross-reactivity to CDP-1 at the highest concentrations tested. These results are a significant improvement over the existing commercial assay. (Refer to Table 3 for CDP-1 cross reactivity data.) Figure 13 is representative of the data showing mP at 0-100 μg/mL vancomycin utilizing the method of Example 4. Table 2 PRECISION PRESENT INVENTION Target Value (μg/mL) 7.00 35.00 75.00 Mean (N=80) 7.13 35.96 74.48 Within Run SD 0.39 0.55 1.37 Within Run %CV 5.6% 1.6% 1.9% Between Run SD 0.00 0.60 0.81 Between Run %CV 0% 1.7% 1.1 % Total Precision SD 0.42% 1.05 2.38 Total Precision %CV 5.9% 3.0% 3.3% COMMERCIALLY AVAILABLE VANCOMYCIN ASSAY Target Value (μg/mL) 7.00 35.00 75.00 Mean (N=80) 6.78 34.39 72.76 Within Run SD 0.17 0.64 1.43 Within Run %CV 2.5% 1.9% 2.0% Between Run SD 0.20 0.84 1.15 Between Run %CV 3.0% 2.5% 1.6% Total Precision SD 0.40% 1.12 2.53 Total Precision %CV 5.9% 3.3% 3.5% TABLE 3 CDP-1 CROSS REACTIVITY ALL VALUES ARE IN μg/mL 3a. 0 μg/mL VANCOMYCIN CDP- l (μg/mL) PRESENT INVENTION VALUES COMMERCIAL ASSAY VALUES 1 0.22 N.D. 0.92 N.D. 2.5 0.15 N.D. 1.61 N.D. 5 0.26 N.D. 3.25 65% 7 LOW N.D. 4.54 64.86% 10 0.13 N.D. 6.42 64.2% 15 LOW N.D. 8.87 59.13% 20 0.46 N.D. 1 1.04 55.20% 25 0.18 N.D. 12.94 51.76% 50 0.67 N.D. 19.79 39.58% N.D. = Less than sensitivity at 2.00μg/mL vancomycin 3b. 40μg/mL VANCOMYCIN PRESENT INVENTION COMMERCIAL ASSAY Sample μg/mL Diff/Sample-Control %C.R. μg/mL Diff/Sample-Control %C.R. Control 41.06 38.94 1 μg/mL CDP- l 40.86 -0.20 N.D. 39.99 1.05 N.D. Control 41.06 38.94 lOμg/mL CDP- l 40.52 -0.54 N.D. 43.74 4.80 48% Control 39.27 37.16 50uε/mL CDP-l 39.13 -0.14 N.D. 55.49 18.33 36.66% Control 36.98 36.50 l OOμg/mL CDP-l 37.22 0.24 N.D. 67.35 30.85 30.85% The vancomycin antibody (the S pot) 15-109-592 of the present invention can be stored at 45° C for 14 days and, it was unexpectedly discovered that the monoclonal is highly resistant to change due to freeze/thaw cycles. The monoclonal antibody can undergo three freeze / thaw cycles with minimal changes in span and intensity values. Additionally, since the antibody is a monoclonal, assay parameters such as span, cross reactivity, and stability are essentially the same from lot to lot. Furthermore, manufacturability is improved as the hybridoma may be cultured using hollow fiber tissue culture systems. The antibody also can survive at two airset fluctuations (approximately 1 .5° C) in a clinical analyzer; thus the kinetics of the assay are also stable in the analyzer environment (about 34 +/- 0.5° C). Finally, use of a pretreatment solution (10% 5-sulfosalicylate, 0.1 M Tris, 20mM copper sulfate) allows bilirubin interference (up to 30mg/dL) to be less than 5%, and it reduces carryover (of a 250μg/mL vancomycin sample) to less than the sensitivity of the assay, i.e. 2%. The pretreatment solution removes the protein from any protein bound vancomycin in order to release the vancomycin for assaying.

Claims: We Claim: 1 . A method for the quantification of vancomycin in a test sample, the method comprising the steps of: (a) contacting the test sample with (i) an antibody reagent comprising antibodies which are capable of specifically binding to vancomycin produced with an immunogen of the formula: wherein P is an immunogenic carrier material, and X is a linking moiety of from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain -O-O linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms, and (ii) a labeled reagent of the formula: wherein Q is a detectable moiety and X is from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain -O-O linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms, to form a reaction solution; and (b) measuring the amount of the labeled reagent in the reaction solution which either is or is not bound with the antibody as a function of the amount of vancomycin in the test sample. 2. The method of claim 1 wherein the detectable moiety is selected from the group consisting of enzymes, chromophores, fluorescent molecules, chemiluminescent molecules, phosphorescent molecules, and luminescent molecules. The method of claim 1 , wherein the labeled reagent is and the antibody is produced with an immunogen of the formula 4. The method of claim 3 wherein the antibody is secreted by a hybridoma cell line designated A.T.C.C. HB 11834. 5. An antibody specific for vancomycin produced with an immunogen of the formula: wherein P is an immunogenic carrier material and X is from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain -0-0 linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms. 6. A continuous hybridoma cell line which secretes IgG antibody which specifically binds to vancomycin. 7. The hybridoma cell line designated as A.T.C.C. HB 1 1 834. 8. A monoclonal antibody capable of specifically binding vancomycin and having no cross-reactivity with CPD- 1. 9. A test kit for the quantification of vancomycin in a test sample, the test kit comprising: (a) an antibody reagent comprising antibodies which are capable of specifically binding vancomycin in a test sample, the antibody produced with an immunogen of the formula: wherein P is an immunogenic carrier material and X is a linking moiety of from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain -0-0 linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms; and (b) a labeled reagent which is capable of displacing the binding of the antibody to the vancomycin. 1 0. The kit of claim 9 wherein the labeled reagent is of the formula: wherein Q is a detectable moiety and X is of from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain -0-0 linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms.

Inventors: ADAMCZYK MACIEJ, BRATE ELAINE M, PERKOWITZ MARY M, REGE SUSHIL D, ADAMCZYK, MACIEJ, BRATE, ELAINE, M., PERKOWITZ, MARY, M., REGE, SUSHIL, D.
Applicants: ABBOTT LAB, ABBOTT LABORATORIES
Application year: 1996
Main classification: G01N33
IPC codes: C07K16, C12N5, C12N15, C12P21, G01N33