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  Determination of concentration of fk778 by competitive immunoassayUSPTO Application #: 20070269841Title: Determination of concentration of fk778 by competitive immunoassay Abstract: Methods and kits for measurement of concentration of FK778 in a biological sample by means of an immunoassay, preferably a competitive immunoassay. In one aspect, the method and kit involve the use of (a) an antibody to FK778 conjugated to a label, e.g., an acridinium label, (b) an antibody to FK778 not conjugated to a label, (c) a solid phase containing an antibody to a first hapten, e.g., a fluorescein hapten, and (d) a bihapten comprising a first hapten and FK778 or an analogue of FK778, e.g., a bihapten comprising a fluorescein hapten and a FK778 hapten. In another aspect, the method and kit involve the use of (a) antibody to FK778, (b) a bihapten comprising FK778 or an analogue of FK778 and a first hapten, e.g., a bihapten comprising the fluorescein hapten and the hapten of FK778 or an analogue of FK778, and (c) a pretreatment reagent. (end of abstract)
Agent: Robert Deberardine Abbott Laboratories - Abbott Park, IL, US Inventors: Thomas G. Spring, Elaine M. Brate, Shelley Holets-McCormack, Rajarathnam E. Reddy, Donald D. Johnson, Yon-Yih Chen, You Pan USPTO Applicaton #: 20070269841 - Class: 435007930 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Assay In Which An Enzyme Present Is A Label, Heterogeneous Or Solid Phase Assay System (e.g., Elisa, Etc.), Competitive Assay The Patent Description & Claims data below is from USPTO Patent Application 20070269841. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a division of U.S. application Ser. No. 11/327,711, filed Jan. 6, 2006. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates to detection and measurement of FK778 or analogues of FK778 in biological samples, and, more particularly, detection and measurement of FK778 or analogues of FK778 in biological samples by means of competitive immunoassay. [0004] 2. Discussion of the Art [0005] FK778 (previously known as HMR 1715, X92 0715, or MNA 715) is structurally similar to A77 1726, the active metabolite of leflunomide. Like A77 1726, FK778 is a malononitrilamide; these compounds are effective immunosuppressants in experimental models of autoimmune diseases and in allo- or xeno-transplantation. While leflunomide (Arava.TM.) has been released for clinical use in rheumatoid arthritis, the long plasma half-life of A77 1726 in humans (15-18 days) makes the drug undesirable for use in clinical transplantation. FK778 has a shorter plasma half-life in humans and thus holds promise that it might be useful in clinical transplantation. The structures of FK778 and A77 1726 are set forth below. [0006] FK778 is described in greater detail in the following articles, all of which are incorporated herein by reference: [0007] Bilolo et al., "SYNERGISTIC EFFECTS OF MALONONITRILIAMIDES (FK778, FK779) WITH TACROLIMUS (FK506) IN PREVENTION OF ACUTE HEART AND KIDNEY ALLOGRAFT REJECTION AND REVERSAL OF ONGOING HEART ALLOGRAFT REJECTION IN THE RAT", Transplantation, Vol. 75, 1881-1887, No. 11, Jun. 15, 2003. [0008] Birsan et al., "In vivo pharmacokinetic and pharmacodynamic evaluation of malononitrilamide FK778 in non-human primates", Transpl. Int. (2003) 16: 354-360. [0009] Birsan et al., "Effects of the malononitrilamide FK778 on immune functions in vitro in whole blood from non-human primates and healthy human volunteers", Transplant Immunology 11 (2003) 163-167. [0010] Fawcett et al., "FK778: A Powerful Immunosuppressive, But Will It Really Be Good for You?", Transplantation, Volume 78, Number 1, Jul. 15, 2004. [0011] Evers et al., "Inhibition of human cytomegalovirus signaling and replication by the immunosuppressant FK778", Antiviral Research xxx (2004) xxx-xxx. [0012] First et al., "NEW DRUGS TO IMPROVE TRANSPLANT OUTCOMES", Transplantation, Vol. 77, S88-S92, No. 9, May 15, 2004 Supplement. [0013] Jin et al. "A novel leflunomide derivative, FK778, for immunosuppression after kidney transplantation in dogs", Surgery, Volume 132, Number 1, 72-79, July 2002. [0014] Savikko et al., "Leflunomide Analogue FK778 Is Vasculoprotective independent of its Immunosuppressive effect: Potential Application for Restenosis and Chronic Rejection", Transplantation 2003: 76: 455, Transplantation, Vol. 76, 471-473, No. 3, Aug. 15, 2003. [0015] Savikko et al., "LEFLUNOMIDE ANALOGUE FK778 IS VASCULOPROTECTIVE INDEPENDENT OF ITS IMMUNOSUPPRESSIVE EFFECT: POTENTIAL APPLICATIONS FOR RESTENOSIS AND CHRONIC REJECTION", Transplantation, Vol. 76, 455-458, No. 3, Aug. 15, 2003. [0016] Slauson et al., "Flow cytometric analysis of the molecular mechanisms of immunosuppressive action of the active metabolite of leflunomide and its malononitrilamide analogues in a novel whole blood assay", Immunology letters 67 (1999) 179-183. [0017] Vanrenterghem et al., "The Effects of FK778 in Combination With Tacrolimus and Steroids: A Phase II Multicenter Study in Renal Transplant Patients". Transplantation, Volume 78, Number 1, Jul. 15, 2004. [0018] Detection and measurement of FK778 in biological samples is important for monitoring therapeutic drugs, as an aid in adjusting drug dosage. The concentration of drug in plasma correlates to the degree of immunosuppression. [0019] FK778 can be determined by LC/Tandem Mass Spectrometry. Methods for LC/Tandem Mass Spectrometry are described in the following reference, which is incorporated herein by reference: [0020] Therapeutic Drug Monitoring of Immunosuppressant Drugs by High-performance Liquid Chromatography-Mass Spectrometry. Taylor, Paul J. Therapeutic Drug Monitoring 26(2):215-219, April 2004 Determination of the presence and amount of FK778 or analogues of FK778 in a biological sample can be determined by a competitive diagnostic assay. Small molecule, competitive diagnostic assays usually require a labeled component that can compete with the analyte for available antibody sites. The labeled component is typically referred to as a tracer. Examples of the labeled component include radioactive tracers, fluorescent tracers, chemiluminescent tracers, and enzyme tracers. Typically, the labeled component consists of the analyte or an analogue of the analyte coupled to a label. [0021] The probability that a particular reagent comprising an antibody to FK778 and a labeled component will be useful in a sensitive assay for FK778 can be assessed by knowledge of the dose response curve. The dose response curve for a FK778 assay is a plot of the ratio of the response in the presence of FK778 analyte to the response in the absence of FK778 analyte as a function of the concentration of the FK778 analyte. The dose response curve for a given FK778 assay is unique for each reagent comprising an antibody to FK778 and a tracer and is modulated by the competition between the tracer and the analyte for sites on the antibody to the analyte. [0022] The problem with a typical FK778 competitive immunoassay on an automated chemiluminescent analyzer is that the tracer comprising FK778 and an acridinium label has a very potent signal. Consequently, the tracer must be diluted to a very low concentration to be measured by the analyzer. FK778 analyte is present at a very high concentration in biological samples. Accordingly, the sample must be diluted more than 1000-fold to compete effectively with the tracer. This degree of sample dilution is typically not available on an automated analyzer. Failure to provide such a dilution results in an assay in which the concentration of FK778 exceeds the concentration of the tracer by so much that the dose response curve is too steep in the dynamic range for a reliable assay. It is desired to develop a competitive assay that allows effective competition between a tracer and the analyte but contains a labeled component that is not as potent as the tracer comprising FK778 and an acridinium label. It would be desirable to provide a competitive immunoassay format capable of detecting levels of FK778 above 10 .mu.g/mL and below 250 .mu.g/mL, concentrations that are clinically useful but difficult to measure. [0023] The company that developed the FK778 drug for clinical use (formerly Fujisawa, now Astellas) also developed and evaluated a series of monoclonal antibodies to FK778 using an ELISA procedure. It was required that these antibodies have sufficient affinity for FK778 in order to be used in an immunoassay. In addition, even when antibodies that demonstrated an appropriate affinity for the FK778 analyte were developed, many of these antibodies demonstrated the undesirable property of high cross-reactivity to structurally similar analogues of FK778, such as metabolites. These antibodies were further screened by Fujisawa for degree of cross-reactivity to metabolites and an antibody having low cross-reactivity was selected. SUMMARY OF THE INVENTION [0024] This invention provides methods and kits for measurement of concentration of FK778 in a biological sample by means of an immunoassay, preferably a competitive immunoassay. [0025] In one aspect, the method and kit involve the use of (a) an antibody to FK778 conjugated to a label, e.g., an acridinium label, (b) an antibody to FK778 not conjugated to a label, (c) a solid phase containing an antibody to a first hapten, e.g., a fluorescein hapten, and (d) a bihapten comprising a first hapten and FK778 or an analogue of FK778. e.g., a bihapten comprising a fluorescein hapten and a FK778 hapten. The bihapten (d) provides a bridge between (a) the antibody to FK778 conjugated to a label and (b) the solid phase containing an antibody to the first hapten. The concentration of the bihapten (d) can be optimized within a concentration range to compete effectively with the analyte, FK778, and provide a good dose response curve without excessive predilution of the FK778 sample. The concentration of the conjugate (a) can be modulated downward by addition of unlabeled antibody, so as not to exceed the detection capability of a commercially available chemiluminescent reader. The ratio of the labeled antibody (a) to the unlabeled antibody (b) typically ranges from about 1:135 to about 1:225, and preferably is about 1:175. [0026] In this aspect, the invention involves identifying a bihapten having the appropriate affinity for both an antibody to the first hapten and an antibody to FK778 or an analogue thereof in the bridging. i.e., bihapten, format. The compounds that are amenable to detection by the method and kit of the present invention include FK778, a slightly cross-reactive FK778 metabolite known as M3, and the active metabolite of leflunomide, known as A77,1726. [0027] The method comprises the steps of: [0028] (a) incubating a mixture comprising (1) a test sample suspected of containing FK778, (2) a solid phase coupled to an antibody specific for a first hapten, (3) a bihapten comprising the first hapten and FK778 or an analogue of FK778, and (4) a reagent mixture comprising an antibody to FK778 conjugated to a label and an antibody to FK778 not conjugated to a label to form a detectable complex comprising (i) the antibody to FK778 conjugated to the label, (ii) a bihapten comprising the first hapten and FK778 or an analogue of FK778, and (iii) the solid phase coupled to the antibody specific for the first hapten; [0029] (b) separating the solid phase coupled to the antibody specific for a first hapten from the mixture; [0030] (c) measuring the amount of label coupled to an antibody specific for FK778, which is bridged by a bihapten bound to an antibody specific for the first hapten that is bound to the solid phase; and [0031] (d) determining the amount of FK778 in the test sample from the amount of label measured. [0032] A kit containing the reagents for carrying out the above-described assay comprises (a) a mixture of an antibody to FK778 conjugated to a label and an antibody to FK778 not conjugated to a label, (c) a bihapten comprising a first hapten and FK778 or an analogue of FK778, (b) a solid phase coupled to an antibody specific for the first hapten. [0033] In another aspect, the method and kit involve the use of (a) antibody to FK778, (b) a bihapten comprising FK778 or an analogue of FK778 and a first hapten, e.g., a bihapten comprising the fluorescein hapten and the hapten of FK778 or an analogue of FK778, and (c) a pretreatment reagent. The method of this aspect comprises the steps of: Continue reading... 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