Europaisches Patentamt J European Patent Office © Publication number: 0 457 21 3 A2 Office europeen des brevets EUROPEAN PATENT APPLICATION © Application number: 91107624.8 © intCiAGOIN 33/532, G01N 33/533, G01N 33/94, C07D 405/12 © Date of filing: 10.05.91 Priority: 16.05.90 US 524195 Inventor: Dubler, Robert Edward 5041 Adele Drive Date of publication of application: Gurnee, Illinois 60031 (US) 21.11.91 Bulletin 91/47 Inventor: Jonas, Patrick J. 1608 Alexander Court Designated Contracting States: Waukegan, Illinois 60085(US) AT BE CH DE ES FR GB IT LI NL Inventor: Grote, Jonathan 753 Durham Lane Applicant: ABBOTT LABORATORIES Grayslake, Illinois 60030(US) CHAD-0377, AP6D/2, One Abbott Park Road Inventor: Nelson, Jane Ann Abbott Park, Illinois 60064-3500(US) 623 Constitution Drive No.3 Palatine, Illinois 60074(US) Inventor: Adamczyk, Maciej 174 Quail Haven Court Gurnee, Illinois 60031 (US) Representative: Modiano, Guido et al Inventor: Cantarero, Luis A. MODIANO, JOSIF, PISANTY & STAUB 1319 Dunleer Drive Modiano & Associati Via Meravigli, 16 Mundelein, Illinois 60060(US) 1-20123 Milano(IT) © Barbiturate assay, tracers, immunogens, antibodies and kit. © The present invention is directed to a fluorescence polarization immunoassay for barbiturates, to the various components needed for preparing and carrying out such an assay, and to methods of making these compo- nents. Specifically, tracers, immunogens and antibodies are disclosed, as well as methods for preparing them and a reagent kit containing them. The tracers and the immunogens are made from substituted barbiturate compounds. A fluorescein moiety is included in the tracer, while a poly(amino acid) forms a part of the immunogen. The assay is conducted by measuring the degree of polarization retention of plane-polarized light that has been passed through a sample containing antiserum and tracer. — </-nh CM — ' Y-N" < d b CO FIG.1-1 FIG. 1-2 FIG.1-3 rs m > o > o X ^° // o UJ FIG.1-4 FIG. 1-5 FIG.1-6 Rank Xerox (UK) Business Services EP 0 457 213 A2 BACKGROUND OF THE INVENTION Technical Field 5 This application is a continuation-in-part of copending application U.S. Serial No. 284,781, filed on December 12, 1988, which is a continuation-in-part of application U.S. Serial No. 870,671, filed on June 4, 1986, now abandoned. The present invention relates to a method and reagents for a fluorescence polarization immunoassay procedure for determining the amount of barbiturate in fluids, especially biological fluids such as serum, w plasma or urine, and to a method of making the reagents. More specifically, the invention relates to (1) reagents (tracers and antibodies, and a kit containing the same) for determining the amount of barbiturate in a sample; (2) immunogen compounds used to raise antibodies; (3) synthetic methods (for making the tracer and immunogen compounds); and (4) analytical methods for conducting the assay. 75 Background Art Barbiturates are central nervous system depressants. Therapeutically, they are used as sedatives, hypnotics and anticonvulsants. Although the legal availability of barbiturates has declined, they are frequently abused sedative or hypnotic drugs and are commonly used to commit suicide. 20 The physiological absorption, action and toxicity of barbiturates vary widely and are dependent on the nature of the 5-substituted groups and imino-hydrogens. Approximately 35% of the barbiturate in blood is plasma protein bound. Barbiturates are distributed in various tissues and organs. Barbiturates are primarily metabolized in the liver and, with a few exceptions, are generally excreted in urine mainly as nonactive metabolites. 25 The most commonly abused barbiturates are the short to medium acting: secobarbital, pentobarbital, amobarbital, etc. These are widely used to reduce excitation states due to the use of stimulants. Tolerance to these drugs can develop from chronic use, and death may occur from either overdose or abrupt withdrawal of the drug. In the past, barbiturate levels in urine have typically been measured by high performance liquid 30 chromatography (HPLG), gas chromatography (GC), enzyme immunoassay (EIA), substrate-linked fluores- cence immunoassay (SLFIA) and radioimmunoassay (RIA). These methods are reasonably specific for detecting drug levels; however, they are not without drawbacks. HPLC and GC methods require sample extraction procedures and the assay time is lengthy. Both EIA and SLFIA involve enzyme reactions and have the following disadvantages: 1) the reagents are relatively unstable; 2) any components in the 35 biological samples which may influence the enzyme reaction in EIA or SLFIA (such as enzyme inhibitors or enzymes which catalyze similar reactions) will affect the assay results; and 3) EIA and SLFIA measure either absorbance or fluorescence, and any compounds in the biological samples which may affect absorbance or fluorescence (such as lipid, hemoglobin, bilirubin or other chromophores or fluorophores) will affect the accuracy of the results obtained from these assays. RIA reagents have the following shor- 40 tcomings: 1) short shelf-life; 2) radiation hazards; and 3) problems associated with the storage and disposal of radioactive materials. Typically, competitive binding immunoassays are used for measuring ligands in a test sample. (For purposes of this disclosure, a "ligand" is a substance of biological interest to be determined quantitatively by a competitive binding immunoassay technique.) The ligands compete with a labeled reagent, or "ligand 45 analog," or "tracer," for a limited number of binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody: the amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations. so Fluorescence polarization provides a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane-polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of molecular rotation. Accordingly, when a tracer-antibody conjugate having a fluorescent label is excited with plane-polarized light, the emitted 55 light remains highly polarized because the fluorophore is constrained by the antibody from rotating between time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane-polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate. As a result, the light emitted from the unbound tracer molecules is depolarized. EP 0 457 213 A2 A problem that heretofore has prevented the accurate determination of barbiturates and other "drugs of abuse" in urine by fluorescence polarization techniques is that of riboflavin interference. Riboflavin, or vitamin B2, is a common constituent of many foods and of commercially available vitamin supplements. Riboflavin is excreted primarily in the urine and has a fluorescence spectrum quite similar to that of 5 fluorescein. As a result, the presence of riboflavin in even moderate amounts in urine samples creates an interference which can produce erroneous results. While ordinary consumption of riboflavin is unlikely to produce more than trace amounts of riboflavin in the urine, test results can readily be distorted by the consumption of excessive quantities of vitamin supplements by persons wishing to prevent detection of barbiturate use. io The present invention is characterized by a more uniform cross-reactivity for the commonly used barbiturates. Further, this invention offers an advance in the art, in that tracers, a method for making the tracers, and an assay using the tracers, are provided specifically for the determination of barbiturates without riboflavin interference. 75 SUMMARY OF THE INVENTION The present invention is directed to a fluorescence polarization assay for barbiturates; to tracers, immunogens and antibodies for use in the assay; to a reagent kit; and to methods for making the tracers, immunogens and antibodies. 20 A first aspect of the invention relates to the discovery of unique tracers and immunogens having novel structures. According to the first aspect of the invention, the tracers and the immunogens are represented by the structural formulas shown in Figure 27 and Figure 2, respectively, wherein, for the tracers: (1 ) W is oxygen or sulfur; (2) R1 is an alkyl, alkenyl, aryl, or alkynyl group having a total of from 1 to 12 carbon atoms, and 25 including from 0 to 1 halogen atoms, arranged in a straight or branched chain, and including up to one aliphatic or aromatic ring structure; (3) R3 is R4-FI; (4) Fl is fluorescein or a fluorescein derivative; and (5) R4 is a linking group which: 30 (a) has a total of from 0 to 15 carbon atoms and heteroatoms, arranged in a straight or branched chain, and includes up to one aliphatic or aromatic ring structure, said heteroatoms being 0, N, S, P or F; and (b) has a total of from 0 to 5 heteroatoms; and, for the immunogens: 35 (1) W is oxygen or sulfur; (2) R1 is an alkyl, alkenyl, aryl, or alkynyl group having a total of from 1 to 12 carbon atoms, including from 0 to 1 halogen atoms, and including up to one aliphatic or aromatic ring structure; (3) R2 is R-Q; (4) Q is a poly(amino acid), a poly(amino acid) derivative or another immunogenically active carrier; and 40 (5) R is a linking group which: (a) has a -CH2-, a -CH = , a 0 or a -NH- at that end of said linking group which is linked to Q; (b) has a total of from 0 to 20 carbon atoms and heteroatoms, arranged in a straight or branched chain, and includes up to one aliphatic or aromatic ring structure, said heteroatoms being 0, N, S, P so or F; and (c) has a total of from 0 to 7 heteroatoms.