WO1993020094A1 - Haptenes, traceurs, immunogenes et anticorps destines a la quinoline - Google Patents

Haptenes, traceurs, immunogenes et anticorps destines a la quinoline Download PDF

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Publication number
WO1993020094A1
WO1993020094A1 PCT/US1993/002899 US9302899W WO9320094A1 WO 1993020094 A1 WO1993020094 A1 WO 1993020094A1 US 9302899 W US9302899 W US 9302899W WO 9320094 A1 WO9320094 A1 WO 9320094A1
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group
conjugate
oligonucleotide
hapten
compound according
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PCT/US1993/002899
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James R. Fino
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Abbott Laboratories
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/20—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/36—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/60—Quinoline or hydrogenated quinoline ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to novel quinoline hapten compounds, to tethered intermediates, to immunogens useful for preparing antibodies, to tracer compounds useful for assaying the haptens, to oligonucleotides labeled with the haptens and to kits containing these reagents.
  • the invention also relates to various methods for making and/or using the novel haptens and the derivatives specified above.
  • the hapten label or "hook” may be used either to isolate a desired target sequence (i.e.. by hybridization with a haptenated oligonucleotide and collection of the haptens with a specific binding partner); or to attach a detectable signaling moiety to a target sequence (e.g. by probing target with a haptenated oligonucleotide and using an anti-hapten conjugate with a detectable signal generating compound such as a fluorophore, chemilumiphore, colloidal particle or enzyme).
  • a detectable signaling moiety e.g. by probing target with a haptenated oligonucleotide and using an anti-hapten conjugate with a detectable signal generating compound such as a fluorophore, chemilumiphore, colloidal particle or enzyme.
  • a label- phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis.
  • a label- phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis.
  • DNA synthesis reaction conditions are quite severe (e.g. iodine oxidation and ammonium hydroxide cleavage) and many haptens (e.g.
  • a linker having a protected terminal amine is attached to the desired end of the oligonucleotide.
  • the amine can be deprotected and, under milder conditions, reacted with a label.
  • the invention has a further advantage in that successfully labeled oligonucleotides can easily be isolated from unlabeled oligonucleotides by an affinity separation method using a specific binding partner, e.g. an antibody, for the hapten.
  • a specific binding partner e.g. an antibody
  • fluorescence polarization assays require tracers comprising an analyte-hapten coupled to a fluorescent molecule.
  • the analyte-hapten and a known amount of tracer are allowed to compete for a limited amount of a specific binding member for the hapten, and the labeled tracer is thereby partitioned between a bound and free form.
  • the signal from the bound form is differentiable from the signal from the free form, so that the amount of analyte-hapten can be estimated.
  • FPIA fluorescence polarization immunoassay
  • Japanese patent publication 1169357 discloses (according to the WPI abstract) antibodies to carcinogenic heterocyclic amines such as 2- amino-3-methylimidazo (4,5-f) quinoline.
  • the antibodies are raised against immunogen conjugates made by common methods. They are said to cross react with 3-amino-l,4-dimethyl-5H pyrido (4,3-b) indole, and to be specific for mutagenic and carcinogenic heterocyclic amines.
  • French patent publication 2601 141 (Alberici, et al.) describes a conjugate prepared by coupling a 4-amino-quinoline compound to a carrier protein.
  • Various compounds are given in Table 1. All have an exocyclic amino group at the 4 position, except the last two, 7-methylquinoline (7-MQ) and
  • the present invention is derived from the class of compounds which are based on the quinoline core structure:
  • a is selected from the group consisting of hydrogen (H), hydroxy (OH), protected hydroxy, mercapto (-SH), protected mercapto, nitro (-NO2), sulfo (-SO3-), and 3-nitrobenzyloxy (-O-CH2C6H4-NO2) and Z is -0-, or -S
  • the compound of formula I has a at the 5 position.
  • the invention relates to a tethered intermediate quinoline compound having the following structure:
  • a is selected from the group consisting of hydrogen (H), hydroxy (OH), protected hydroxy, mercapto (-SH), protected mercapto, nitro (- N0 2 ), sulfo (-SO3-), and 3-nitrobenzyloxy (-O-CH2C.5H4-NO2);
  • Z is -0-, or -S (O2)- ; and A is a linking moiety of the formula -L-y, wherein y is a functional group that can react directly or after activation with functional groups in a second molecule and L is a spacer group consisting of from 1 to 50 atoms.
  • L will include 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 in the sequence -L-y, that the sequence -L-y cannot contain -O-O- linkages, that cyclic moieties contain 6 or fewer members, and that branchings may occur only on carbon atoms.
  • protecting group may be the same or different, although some protecting groups are preferred for a particular function being protected.
  • Protecting groups may be selected from among the many known protecting groups.
  • A may ideally comprise a lower alkyl substituent having a reactive y functional group.
  • a ZA substituent is logicaly referred to generally as an alkoxy quinoline.
  • A When Z is -S(O2)-, A ideally comprises a linker bonded to the S via nitrogen atom to form a sulfonamide quinoline. Generally, the remainder of the linker between the N and the y function comprises lower alkyl. In either case, especially useful y groups include hydroxy, carboxy and phosphoramidite, or protected forms of these groups.
  • the present invention relates to conjugates of the following structure:
  • a conjugation partner may be selected from the group consisting of an immunogenicity conferring carrier molecule (to form an immunogen), a detectable label molecule (to form a tracer), an oligonucleotide (to form a separable or detectable probe), and a solid phase (to form an affinity support).
  • the invention relates to antibodies, either polyclonal or monoclonal, which are reactive with the above compounds (I), (II) or (HI). Such antibodies may be prepared by the process of injecting an immunogen (HI) into an animal and recovering the antibodies.
  • the invention relates to the following methods of using the above compounds:
  • an antibody reactive with the compounds said antibody being attached to or adapted for attachment to either solid supports or detectable labels.
  • the oligonucleotide probe may be hybridized with a target and the antibody may be used to separate or detect it.
  • the phosphoramidite may be used to label one's own oligonucleotide during its synthesis, while the anitbody is used as before.
  • Antigen is defined in its usual sense, to refer to a molecule or compound which is capable of eliciting an immune or antibody response in a challenged animal.
  • Compounds which are not antigenic by themselves can sometimes by made to elicit the immune response by coupling the compound (a "hapten") to an "immunogenicity conferring carrier” molecule to form an "immunogen". While such haptens are not “antigenic” in the strict sense, they are capable of imitating antigens and have many properties in common with antigens. Thus, the terms antigen and hapten are often used interchangeably.
  • both haptens and antigens have at least one "determinant” which, as used herein, refers to a region of the antigen or hapten which is involved in specific binding reactions between the antigen or hapten and an antibody.
  • Some haptens and antigens have more than one determinant region or site and thus are “polyvalent”. In essence, it is the determinants which differentiate antigens, and therefore, antibodies from one another on the basis of immunological specificity.
  • hapten is defined as any compound having the quinoline core structure shown below:
  • a and Z are defined as above. Note that the core structure excludes the A group so that other tethers or linkers may be connected to the carbon.
  • immunogenicity conferring carriers include, for example, naturally occurring poly(amino-acids), albumins and serum proteins such as bovine thyroglobulin (BTG), globulins, lipoproteins, ocular lens proteins, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovine gamma globulin (BGG), thyroxine binding globulin (TBG), and the like.
  • BSG bovine thyroglobulin
  • KLH keyhole limpet hemocyanin
  • BGG bovine gamma globulin
  • TBG thyroxine binding globulin
  • synthetic poly(amino-acids) can be utilized such as polylysine, etc.
  • any molecule which is capable of conferring antigenicity to a hapten is an "immunogenicity conferring carrier.”
  • hapten-specific binding member refers to a member, such as an antibody or receptor, that specifically binds to the hapten.
  • the determinants on the hapten are responsible for the specific binding of the binding member to the hapten.
  • the most common and usual specific binding member is an antibody, either polyclonal or monoclonal.
  • alkyl refers to monovalent straight or branched aliphatic radicals which may be derived from alkanes by the removal of one hydrogen, and have the general formula CnH2n+l- Alkyl substituents may have from 1 to about 30 carbons, more practically 1 to about 20.
  • Lower alkyl refers to alkyls having from 1 to about 6 carbons. Examples of lower alkyl include CH3-, CH3CH2-, CH3CH(CH3)-, and CH3(CH2)4-.
  • Alkenyl refers to monovalent straight or branched aliphatic radicals which may be derived from alkenes by the removal of one hydrogen, and have the general formula C n H2n-l- Alkenyl substituents may have from 1 to about 30 carbons, more practically 1 to about 20.
  • Lower alkenyl refers to alkenyls having from 1 to about 6 carbons.
  • Olefinic is a synonym for alkenyl.
  • Aryl refers to a monovalent radical derived from aromatic hydrocarbons or heteroaromatic compounds by the removal of one hydrogen. Aryl substituents have ring structures, such as those of phenyl, naphthyl and 2- thienyl. Typically, aryl substituents are planar with the ⁇ electron clouds of each carbon remaining on opposite sides of the plane. Aryl substituents satisfy the Huckel (4n+2) ⁇ electrons rule.
  • Protecting groups are defined as groups that can be removed under specific conditions, but which shelter or hide a reactive atom or functionality temporarily during intermediate reactions under other conditions.
  • Protecting groups for hydroxyl, amino and thiol functionalities are well known in the art (T. W. Greene, Protective Groups in Organic Synthesis, John Wiley and Sons, NY, 1981).
  • halides are the most useful leaving groups in nucleophilic reactions
  • the hydroxyl group in carboxylic acids may be converted to a halide.
  • an acid is formed by the reaction of a carboxylic acid with thionyl chloride or oxalyl chloride/DMF.
  • the hydroxyl group in carboxylic acids may be converted to a leaving group by first activating the carboxylic acid moiety by reaction with an activating reagent such as 1,3-dicyclohexylcarbodiimide and an additive such as N-hydroxysuccinimide or conversion into a highly reactive mixed anhydride, acyl imidazolide, or mixed carbonate. It should be recognized that one of normal skill in the art may determine other leaving groups which would also work.
  • linking moiety A is also referred to as a "tether”. These terms refer to the spacer molecule having the formula:
  • y is a reactive functional group that can react directly or after activation with the functional groups in a second molecule, e.g. the conjugation partner, Q; and L is a spacer group consisting of from 1 to 50 carbon and heteroatoms.
  • L will include 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 direcdy linked in the sequence -L-y, that the sequence -L-y cannot contain -O-O- linkages, that cyclic moieties contain 6 or fewer members, and that branchings may occur only on carbon atoms.
  • A is already linked to Q
  • y is chosen from the group consisting of hydroxy (-OH), thiol
  • the linking moiety often comprises a bifunctional compound designated x-L-y wherein x is also a functional group (selected from the same group as y) which can react with functional groups on the hapten or -R.
  • x is also a functional group (selected from the same group as y) which can react with functional groups on the hapten or -R.
  • Many bifunctional linkers are known to one skilled in this art. For example, heterobifunctional linkers are described in, e.g. U.S. Patent 5,002,883 (Bieniarz). These are preferred in some cases due to the specificity of their ends for one functional group or another.
  • a “Michael acceptor” is defined in the art as follows: "The nucleophilic addition of enolate ( or analogous) anions to the carbon- carbon double bond of ⁇ , ⁇ -unsaturated ketones, aldehydes nitriles or carboxylic acid derivatives, [is] a process known as the Michael reaction
  • the unsaturated compounds in the reaction often called Michael acceptors, may include any unsaturated system having a functional group capable of stabilizing the carbanionic intermeditate
  • the Michael acceptors may also add a variety of nucleophiles such as alcohols, thiols, and amines.” H. O. House, Modern Synthetic Reactions, W.A. Benjamin, Inc., Menlo Park CA, 1972, pp. 595-96.
  • Common functional groups which can activate a double bond to this kind of nucleophilic addition (thereby forming Michael acceptors) include, -
  • Rt can be alkyl or aryl.
  • exemplary Michael acceptors include:
  • Solid supports refer to a wide variety of support materials.
  • Polymeric plastics such as polystyrene, polypropylene, and polytetrafluoroethylene, are exemplary.
  • Glass is also a useful support.
  • Supports may take any size or shape, including beads, microparticles, tubes, rods, plates, wells and cuvettes.
  • Supports may include functional groups for conjugation, or may be derivatized prior to conjugation. Alternatively, supports may be coated or adsorbed in some cases. Supports should be physically separable from reagent solutions, based on size, weight, shape, charge, magnetic properties or some other physical property. It will be realized that two distinct uses for solid supports are described herein.
  • antibodies can be purified using supports conjugated to tethered intermediates; and secondly, supports to which anti-hapten antibodies are attached are useful for separating and/or detecting oligonucleotides labeled with haptens, and for competitive hapten-analog assays.
  • Antibodies are prepared by developing an immune response in animals to the immunogens described hereinafter. The immunogen is administered to animals such as rabbits, mice, rats, sheep or cows by a series of injections according to techniques generally known in the art. An antibody, according to the present invention, is raised in response to an immunogen of the invention which is derived from the haptens described above.
  • polyclonal and monoclonal antibodies recognize specific epitopes on an immunogen, and, while typically polyclonal antibodies have been utilized in the present invention, both may be suitable.
  • Polyclonal antibodies consist of a mixture of multiple antibodies, each recognizing a specific epitope, some of which may be present on the carrier molecule. Techniques for preparing polyclonal antibodies generally are well known in the art. It is well within the skill of the ordinary practitioner to isolate antibodies which are specific for the hapten portion of the immunogen. Affinity chromatography is but one method.
  • Monoclonal antibodies specific for just one determinant or epitope, may be prepared eliciting an immune response as before.
  • B-lymphocyte cells are removed from the spleens of the animals by standard procedures, and the B-lymphocyte cells are then fused with myeloma fusion partners according to standard procedures, such as those described in Kohler and Milstein, "Continuous Culture of Fused Cells Secreting Antibody of Predefined Specificity," Nature.256, 495 (1975).
  • Label refers to labels capable of providing a directly detectable signal, as well as to molecules like haptens, which can indirectly be detected. In this way, “label” is interchangeable with “reporter” or “hook”. However, at times is is necessary to distinguish “label” from a moiety which is capable of generating a measurable detectable signal, usually an electromagnetic radiation signal.
  • detectable label or “signalling” label or moiety is used when the intent is to differentiate from hapten-type labels or "hooks”.
  • traceer refers to a conjugate of hapten with a detectable signalling label. The tracer permits a determination or assay of the amount of hapten present in an unknown solution.
  • the tracer signalling label is a fluorescent molecule as described hereinafter, although the tracer signalling label may encompass other detectable labels, including by way of example and not limitation, radioisotopes, chemilumiphores and colloidal particles.
  • the choice of the fluorescent molecule for forming the tracer is advantageously flexible and is largely up to the preferences of the practitioner. It will be readily appreciated that the fluorescent labels are ideally chosen in accordance with their size, that is, the smaller the molecule, the more rapidly it can rotate, and the more effective it is as an FPIA tracer component.
  • the preferred fluorescent labels are fluorescein and fluorescein derivatives.
  • any of the following fluorescein derivatives can be used: dansyl chloride, fluorescein amine, carboxyfluorescein, a-iodoacetamidofluorescein, ⁇ -aminomethylfluorescein, 4'-N- alkylaminomethylfluorescein, 5-aminomethylfluorescein, 2,4-dichloro-l,3,5- triazin-2-yl-aminofluorescein (DTAF), 4-chloro-6-methoxy-l,3,5-triazin-2-yl- aminofluorescein, fluorescein isothiocyanate.
  • Especially preferred derivatives are aminomethylfluorescein and 5-carboxyfluorescein.
  • Other tracer detectable labels are also known in the literature, particularly associated with other detection techniques.
  • oligonucleotide refers to short segments of nucleic acid having a minimum of about 5 nucleotides and a maximum of several hundred nucleotides. Although, oligonucleotides longer than about 30 nucleotides are often called polynucleotides, the term oligonucleotide is used herein to encompass the longer chains as well.
  • the nucleic acid may be RNA or DNA, although DNA is generally preferred.
  • the DNA may be natural or synthetic, although the invention excels in the automated synthesis of DNA.
  • Haptens which are structurally similar to quinoline have the following general structure: wherein a is hydrogen (H) or nitro (-NO2) and Z is -0-, or -S (O 2 )- .
  • a is hydrogen (H) or nitro (-NO2) and Z is -0-, or -S (O 2 )- .
  • the available bond to Z is occupied by a linker or a second molecule as described below.
  • Hapten starting materials may be purchased (in some cases) or synthesized as described in the example section below, c. Tethered intermediates
  • linker molecules x-L-y containing reactive functional groups x and y capable of coupling to complementary reactive groups on another molecule or macromolecule
  • A -L-y.
  • the remainder of the hapten molecule retains a structure substantially similar to those of the desired determinant(s).
  • Many methods for linking a hapten to another molecule are known in the art. Preferred methods involve activating a functional group on the hapten or linker, attaching a linker or tether to the hapten via the activated group.
  • the free end of the tether, y is available for coupling to the desired molecule, Q.
  • Q desired molecule
  • hapten (I) Reaction of the hapten (I) with the tether or linker produces tethered intermediate compounds (II) having a tether with a functional group y .
  • the reaction conditions for each of these reaction steps can be obtained in the examples or the literature.
  • Immunogens can be produced from a wide variety of tethered intermediates.
  • the immunogens of the present invention have the following general structure:
  • the tether functionality, y, of tethered intermediates ( ⁇ ) can be reacted in any of several ways known to those skilled in the art with the amino groups on a protein carrier. It is frequently preferable to form amide bonds, which typically are quite stable. Amide bonds are formed by first activating the carboxylic acid moiety y of the tethered intermediate by reaction with an activating reagent such as 1,3- dicyclohexylcarbodiimide and an additive such as N-hydroxysuccinimide. The activated form of the hapten is then reacted with a buffered solution containing the immunogenicity conferring carrier.
  • an activating reagent such as 1,3- dicyclohexylcarbodiimide and an additive such as N-hydroxysuccinimide.
  • the carboxylic acid hapten 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 immunogenicity conferring carrier.
  • a highly reactive mixed anhydride, acyl halide, acyl imidazolide, or mixed carbonate and then combined with the immunogenicity conferring carrier.
  • a suitable solvent such as acetonitrile or dimethylformamide.
  • the resultant urethane is then reacted with the immunogenicity conferring carrier in a buffered, aqueous solution to provide an immunogen.
  • a tethered intermediate with a terminal aldehyde functionality [II, y -
  • phosgene or a phosgene equivalent such as di or triphosgene or carbonyldiimidazole
  • tethered intermediates 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, y on the linker of the intermediate.
  • the result is a solid phase which can be used to separate or purify antibodies against the hapten.
  • Tracers of the present invention look very much like the immunogens described above, except that Q is a signalling moiety. Tracers have the general structure:
  • A is a spacer/linker as defined above except that the reactive function y has already been reacted with, Q, a detectable label.
  • detectable labels which can be detected in homogeneous systems are preferred. Particularly preferred are fluorescein and fluorescein derivatives.
  • Tracers of the invention find use in assays for quinoline derivatives, including oligonucleotides derivatized with this hapten.
  • A consist of 1 to 12 carbon and heteroatoms. Longer chains reduce the differential polarization effects by distancing the label from the high molecular weight molecule that modulates its polarization properties.
  • Tethered intermediates (H) containing an amino group, a carboxyl group or an alcohol group in the tether can be coupled to fluorescein or a fluorescein derivative to prepare the tracers of the present invention.
  • Tethered intermediates with a terminal amine functionality 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.
  • a suitable solvent such as acetonitrile or dimethylformamide.
  • an amine- terminated tethered intermediate can be activated to an isocyanate. The resultant product is then reacted with an amino fluorescein derivative to form a urea tracer.
  • An amino-group-containing hapten can also be coupled to a carboxyfluorescein derivative which has been activated with N- hydroxysuccinimide in a suitable solvent.
  • Tethered intermediates with a terminal carboxyl group on the linker can be coupled to an amino-terminal fluorescein derivative by first activating the carboxylic acid moiety of the tether by reaction with an activating reagent such as 1,3-dicyclohexylcarbodiimide and an additive such as N-hydroxysuccinimide. The activated intermediate is then reacted with a solution of the fluorescein derivative, resulting in the formation of a tracer.
  • the carboxylic acid hapten 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 fluorescein derivative.
  • tethered intermediates containing an alcohol group can be coupled to the fluorescein by first reacting the tethered intermediate with phosgene or a 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 an amino-terminal fluorescein derivative resulting the formation of a tracer. 4 Oligonucleotides a. Structure of Oligonucleotides Oligonucleotides (oligonucleotides) can be produced from a wide variety of tethered intermediates. The oligonucleotides of the present invention have the following general structure:
  • oligonucleotides labeled with haptens find uses in nucleic acid hybridization assays, including amplification assays. Haptenated oligonucleotide probes are well adapted for separation and/or detection of PCR products (see e.g. EP-A-357011) and or LCR products (see e.g. EP-A-439 182). In combination with other haptens, (e.g.
  • the tether functionality, y can be the same as defined above for tracers and immunogens.
  • Oligonucleotides can be labeled by reacting the y functionality with an amino or hydroxyl function of the oligonucleotide, or by direct reaction with the phosphorous via oxidative amination of an H-phosphonate reagent.
  • Amino functionalities are present in the purine and pyrimidine bases, but these sites are less preferred for labeling because of their importance in hybridization. Amino functionalities can be introduced to the 5' and/or 3' ends of oligonucleotides using reagents such as Aminomodifier® (Clontech, Palo Alto). Hydroxyl functions are typically formed during automated synthesis.
  • a preferred method for adding a hapten to the 3' end of an oligonucleotide is disclosed in co-pending, co-owned U.S. patent application Serial No. 630,908, filed December 20, 1990, the disclosure of which has already been incorporated.
  • a preferred method for adding a hapten to the 5' end is through the use of a phosphoramidite reagent as described in Thuong, et al. or Cohen, et al. cited above in the Background Section.
  • Scheme I begins with the synthesis of the tether.
  • the starting compound is an amino-protected carboxylate 1 wherein W is a spacer group of from 1 to about 50 atoms arranged in a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that (a) not more than two heteroatoms are directly linked, (b) cyclic moieties contain 6 or fewer members, and (c) branching occurs only on carbon atoms; R* and R***" are independently hydrogen, alkyl of from 1-10 carbon atoms, an amino-protecting group or aryl; alternatively R* or R*0 when taken together with W and the nitrogen atom to which they are attached may form a cyclic amine.
  • Compound 1 is carboxyl- activated (step 1) via a nucleophilic acyl substitution reaction, followed by reaction with Meldrum's acid (2,2-dimethyl-l,3-dioxane-4,6-dione, 2) and R ⁇ OH (R2 is alkyl of from 1-6 carbon atoms) to yield the diketoester 3 which is then reduced (step 4) with sodium borohydride under reflux conditions to the diol 4. The primary hydroxyl of the diol is then protected by dimethoxytritylation (step 5).
  • the amino is then N-deprotected deprotected (step 6) to 5 which is reacted with hapten (6, Z is nitro or sulfonyl) to form the tethered hapten 7.
  • the tethered hapten is then phosphoramidated at the secondary alcohol (step 7 ) to the phosporamidite-linked hapten 8 .
  • the phosphoramidite-linked hapten may then be used directly to introduce the hapten into a synthetic oligonucleotide at any position.
  • oligonucleotide-labeled oligonucleotides are prepared by reacting an quinoline phosphoramidite with the 5' hydroxyl of a nucleotide attached to a growing oligonucleotide chain.
  • the labeled oligonucleotides are purified by standard procedures, e.g., Gait, Oligonucleotide Sythesis: A Practical Approach (IRL Press, Washington, D.C.: 1984).
  • Scheme 1 Preparation of Phosphoramidite-linked Hapten
  • a tethered hapten (II) is reacted in the presence of carbon tetrachloride with a phosphonate group already incorporated in to the oligonucleotide, via oxidative amidation.
  • the tracers and antibodies raised against immunogens of the present invention produce excellent results in a fluorescence polarization assay of the present invention for the semi-quantitative detection of hapten derivatives.
  • the assay is performed in accordance with the following general procedure: 1) a measured volume of standard or extracted test sample containing or suspected of containing hapten derivatives is delivered to a test tube;
  • the amount of tracer-antibody complex is measured by fluorescence polarization techniques known per se to determine the presence or amount of the analyte in the test sample.
  • the preferred procedure was designed to be conducted on the TDx® Therapeutic Drug Monitoring System or the ADxTM Abused Drug System, IMx® Fluorescence Polarization and Microparticle Enzyme Immunoassay (MEIA) Analyzer all of which are available from Abbott Laboratories, Abbott Park, Illinois.
  • TDx Therapeutic Drug Monitoring System
  • ADx Abused Drug System
  • IMx Fluorescence Polarization and Microparticle Enzyme Immunoassay
  • millipolarization units The results can be quantified in terms of "millipolarization units", “span” (in millipolarization units) and “relative intensity”.
  • the measurement of millipolarization units indicates the maximum polarization when a maximum amount of the tracer is bound to the antibody in the absence of any analyte in the test sample. The higher the net rnillipolarization units, the better the binding of the tracer to the antibody.
  • the span is an indication of the difference between the net millipolarization and the minimum amount of tracer bound to the antibody. A larger span provides for a better numerical analysis of the data. For the purposes of the present invention, a span of at least 15 millipolarization units is preferred.
  • the intensity is a measure of the strength of the fluorescence signal above the background fluorescence. Thus, a higher intensity will give a more accurate measurement.
  • the intensity is determined as the sum of the vertically polarized intensity plus twice the horizontally polarized intensity.
  • the intensity can range from a signal of about three times to about thirty times the background noise, depending upon the concentration of the tracer and other assay variables. For the purposes of the present invention, an intensity of about three to about twenty times that of background noise is preferred, although it is within the skill of the routineer to optimize the signal for each particular system.
  • the pH at which the method of the present invention is practiced must be sufficient to allow the fluorescein moiety to exist in its open form.
  • the pH can range from about four to nine, preferably from about six to eight, and most preferably from about 7 to 7.5.
  • Various buffers can be used to achieve and maintain the pH during the assay procedure.
  • Representative buffers include borate, phosphate, carbonate, Tris, barbital and the like.
  • the particular buffer used is not critical to be present invention, but the Tris and phosphate buffers are preferred.
  • the preferred FPIA procedure is especially designed to be used in conjunction with the Abbott TDx® Clinical Analyzer, the Abbott TDxFLxTM or the Abbott ADx® Drugs of Abuse System, all three of which are available from Abbott Laboratories, Abbott Park, Illinois.
  • the calibrators, controls, or unknown samples are pipetted directly into the sample well of the TDx® sample cartridge.
  • One of the advantages of this procedure is that the sample does not require any special preparation.
  • the assay procedure from this point is fully automated.
  • the sample is mixed with the pretreatment solution in dilution buffer and a background reading is taken.
  • the fluorescence tracer is then mixed with the assay.
  • the antibody is then finally mixed into the test solution. After incubation, a fluorescence polarization reading is taken.
  • the fluorescence polarization value of each calibrator, control or sample is determined and is printed on the output tape of an instrument, such as the Abbott TDx® Analyzer, TDxFLxTM or ADx® System.
  • An instrument such as the Abbott TDx® Analyzer, TDxFLxTM or ADx® System.
  • a standard curve is generated in the instrument by plotting the polarization of each calibrator versus its concentration using a nonlinear regression analysis.
  • the concentration of each control or sample is read off of the stored calibration curve and printed on the output tape.
  • the tracer, antibody, pretreatment solution, wash solution, calibrators and controls should be stored between about 2 degrees C and about 8 degrees C while the dilution buffer should be stored at ambient temperature.
  • a standard curve and controls should be run every two weeks, with each calibrator and control run in duplicate. All samples can be run in duplicate.
  • the preferred reagents, calibrators and controls for a preferred fluorescence polarization immunoassay of the present invention can be found in Example 27 infra.
  • the oligonucleodtide When used to label oligonucleotides, the oligonucleodtide is 10-100 bases in length. A preferred length is 15-30 bases. Various levels of complementarity of the oligonucleotide may be used. In general, the oligonucleotide is usually perfectly complementary but occasionally a non-match is tolerated and may be preferred. Generally, an oligonculeotide is specific for only the target of interest but sometimes it may be a consensus oligonucleotide for detecting more than one target sequence.
  • Methods of using the labeled oligonucleotides include the performance of specific hybridizations, such as sandwich hybridizations known in the art. For example, see U.S. 4, 486,539 (Ranki) and GB 2 169403 (Orion).
  • the haptenated oligonucleotides may also be used in amplification techniques, such as PCR and LCR.
  • An illustrative use of a haptenated primer in PCR is described in EP-A-357 011 (Abbott ); the use of a haptenated probe in LCR is described in EP-A-320 308 and in EP-A-0439 182.
  • Other potentially useful known techniques include those described in EP-A- 332435, US 4,883,750 and US 5,185,243.
  • AMForAMF aminomethyl fluorescein a fluorophore
  • BSA Bovine Serum Albumin an immunogenicity conferring carrier.
  • DMEM Dulbecco's Minimum Essential Medium a cell culture medium.
  • the ester was dissolved in methanol (5mL) and 6N aqueous KOH (5mL) and stirred at ambient temperature for several hours.
  • the reaction was acidified to pH2 with 6N hycte jhloric acid and extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure giving 2.0g of the acid.
  • the product was purified by column chromatography (silica gel, ethyl acetate/methylene chloride/hexanes/acetic acid, 4:3:3:0.5). Residual acetic acid was removed by coevaporation with toluene/methanol (1:1) giving the product as a white solid.
  • Mass spectrum FAB, mH + @ m/z 323.
  • 1,2,4-Butanetriol (20.0 g, 188 mmoles) was combined with benzaldehyde dimetiiyl acetal (25.5 mL, 170 mmoles) and stirred vigorously for several hours under nitrogen atmosphere at room temperature. The residue was applied to a flash chromatography column (silica gel, 200-400 mesh) using methylene chloride and eluted with hexanes (IL), ethyl acetate/hexanes, 1:3, (IL), and finally ethyl acetate. The yield of the aldehyde was 20 g (61%). Step b: Conversion to the aldehyde
  • Example 9 6-[N-(8-quinolinesulfonyI)amino]-l,3-hexanedioI, a tethered hapten.
  • a generalized method for reacting an aminodiol with a hapten of the present invention is described in Scheme 1, above.
  • 6-Amino-l,3-hexanediol (0.16 gram, 1.2 mmoles, prepared as described in Example, 8 above) was dissolved in 10% sodium carbonate (3 mL) and THF (5 mL).
  • 8- Quinolinesulfonyl chloride (0.328 g, 1.44 mmoles) was added and die reaction stirred at room temperature, under nitrogen atmosphere, for 18h.
  • Example 10 6-[N-(8-quinolinesulfonyl)aminoH-(0-4,4'-dimethoxytrityl),3- hexanediol, a tethered hapten.
  • Example 11 6-[N-(8-quinolinesulfonyI)amino]-l-[0-(4,4'- dimethoxytrityl)],3- ⁇ [0-(0-(2-cyanoethyl)-N,N-diisopropylphosphoryl)] ⁇ - hexanediol, a tethered hapten.
  • Example 12 4-HydroxyprolinoI-DMT, a tether.
  • Step a Protection of the amino function of hvdroxyproline.
  • trans-4-hydroxy-L-proline 100 g, 763 mmole was dissolved in water (IL) and sodium carbonate was added (323 g, 3 moles).
  • IL water
  • sodium carbonate 323 g, 3 moles
  • tetrahydrofuran 800 mL
  • benzyl chloroformate 220 mL, 1.5 mole
  • the reaction was stirred at 15-30 °C, under an inert atmosphere using a mechanical stirrer. After 24 h, the solution was extracted witii ethyl acetate (2 x IL) then acidified to pH 3.0 witii 6 N HCl and die solid isolated on a fritted glass Buchner funnel.
  • Step b Formation of the diol Into a 3L round-bottom flask, equipped with addition funnel, N-CBZ- hydroxyproline (159 g, 599 mmole) and tetrahydrofuran (IL) was added. To this solution boran-methyl sulfide complex (114 mL, 1.2 mole) was added dropwise using the addition funnel.
  • N-CBZ-hydroxyprolinol (16.9 g, 67 mmole) was dissolved with dry pyridine (250 mL). The solvent was removed in vacuo and the residue dissolved with another 250 mL of dry pyridine. To this solution was added diisopropylethylamine (29 mL, 168 mmole) and 4- dimethylaminopyridine (0.82 g, 6.7 mmole). An addition funnel was added to the reaction flask and charged with 4,4'-dimethoxytrityl chloride (25 g, 74 mmole) dissolved in tetrahydrofuran (200 mL).
  • Step d 4-Hydroxyprolinol-DMT. a tether
  • N-CBZ-hydroxyprolinol-DMT (10 g) is dissolved in methanol and then 10% Palladium on carbon (2 g) is added.
  • the solution is pressurized with hydrogen atmosphere to 50 psi using a Parr hydrogenation apparatus.
  • the solution is shaken for 18 h then flushed with nitrogen gas and filtered through Celite to remove the catalyst.
  • the solvent is removed in vacuo giving the product.
  • Example 13 Quinoline-hydroxyprolinol phosphoramidite, a tethered intermediate.
  • Example 14 Quinoline KLH immunogen (F252): KLH (0.25g) was dissolved in sodium phosphate buffer (lOmL, pH8, 0.05M) then NMP (lmL) was added. To this solution was added 8-quinolinesulfonyl chloride witii vigorous stirring. After 4h at ambient temperature, the solution was diluted with the same buffer (40mL), and then it was dialyzed against 10% ethanol in sodium phosphate buffer (pH8, 0.1M, 2x6.6L). After dialyzing against distilled water (2x6L), the solution was lyopholized, giving an off white powder (0.25g).
  • KLH (0.5g) was dissolved in sodium phosphate buffer (lOmL, pH8.0, 0.05M) then NMP (4mL), and the active ester solution were added with vigorous stirring. After 72h, the solution was dialyzed against 10% ethanol in sodium phosphate buffer (pH8, 0. IM, 3x6.6L), then against distilled water (3x6L). The solution was lyopholized giving a light gray powder (0.5 lg).
  • BSA 0.5g
  • sodium phosphate buffer pH8, 0.05M
  • NMP 5mL
  • the active ester soluion was added to the BSA solution and the reaction was agitated by rotation for 18h at ambient temperature.
  • the solution was dialyzed at 4-8°C against 10% ethanol in sodium phosphate buffer (pH8, 0.1M, 3x6.6L) then against distilled water (3x6L).
  • the solution was lyopholized giving a white powder (0.49g).
  • 8-Quinolinesulfonyl chloride (0.025g, O.llmmol), 5-[6-amino- pentanecarboxamido]-fluorescein trifluoroacetate (0.065g, O.llmmol) andDlEA (0.115mL, 0.66mmol) were dissolved in acetonitrile (0.5mL), and NMP (0.5mL).
  • Quinoline-8-(5-carboxypentyl)-sulfonamide (0.05g, O.l ⁇ mmol), from example 6 (F245), HOSu (0.021g, 0.19mmol), and DCC (0.035g, 0.17mmol) were dissolved in NMP (0.9mL) and stirred at ambient temperature for 18h to form the active ester. The reaction was filtered and the solution used as is.
  • Quinoline-8-(5-carboxypentyl)-sulfonamide (0.05g, 0.16mmol), from example 6 (F245), HOSu (0.021g, 0.19mmol), and DCC (0.035g, 0.17mmol) were dissolved in NMP (0.9mL) and stirred at ambient temperature for 18h to form the active ester. The reaction was filtered and the solution used as is.
  • 6-Amino-l,3-hexanediol hydrochloride (1.39 gram, 8.2 mmoles, prepared as described above) was dissolved in 10% sodium carbonate (40 mL) and THF (20 mL). With stirring, dansyl chloride (2.21 g, 8.2 mmoles) was added with additional THF (20 mL). The sealed reaction was stirred for 18h at room temperature, protected from light. The organic layer was separated and the aqueous layer washed with ethyl acetate (2x25 mL). The combined organic extracts were dried with sodium sulfate and die solvent removed under reduced pressure. The residue was dissolved in minimum methylene chloride and applied to a flash chromatography column. Elution was done with ethyl acetate/hexanes, 2:8 (250 mL), then with etiiyl acetate. The yield was 1.0 gram (33%).
  • Example 24 Example 23 is repeated using the immunogens of example 15-18.
  • Example 25 Four to six week old female BALB/c mice are injected subcutaneously at four weeks intervals with 0.2 mL of the immunogen from example 14 ( 5mg mL; 0.06 mL of immunogen) in.1.88 mL saline; with lOOmg of monophosphoryl lipid A and trehalose dimycloate adjuvant (Rib ⁇ Immunochem Research, Lie).
  • the spleen is removed aseptically and placed in a plastic Petri dish with 5 mL of cold Dulbecco's Minimal Essential Medium (DMEM),with 2.0 mM L-glutamine (Medium A).
  • DMEM Dulbecco's Minimal Essential Medium
  • the spleen is dissociated into a single cell suspension; the cells are centrifuged to a pellet and the red cells lysed by resuspension in 2 mL of 0.83% ammonium chloride in 10 mM Tris buffer. After letting stand for 2 min., 20-30 mL of fresh medium A is added. The cells are washed by centrifugation and resuspended in 10 mL of fresh medium A.
  • HGPRT- EC2.4.2.8
  • the myeloma cell line is maintained in medium A with 20% fetal calf serum added.
  • 0.1 mM 8-azaguanine is added to the myeloma cells in order to kill any HGPRT+ revertants.
  • the myeloma cells are harvested, washed once in medium A, and resuspended in 5 mL medium A.
  • the myeloma and previously harvested spleen cells are counted using a hemacytometer and their viability assessed by Erytiirosin B stain exclusion.
  • the fusion technique used is modified from that of Gefter et. al, Somatic
  • the cells are resuspended gently in PEG solution over a period of 1 minute by slowly aspirating up and down using a 1 mL pipette.
  • the tube is held in the hand for an additional 1 minute and then 1 mL of medium A is added slowly to dilute die PEG.
  • the cells are allowed to stand for an additional 1 minute without agitation or mixing.
  • An additional 20 mL of medium A is added over a period of 3 to 5 minutes, and the cells pelleted at 1400 rpm for 5 minutes.
  • the supernatant is aspirated off and the cells resuspended in 20 mL of medium A witii 20% fetal calf serum, lxlO -4 M hypoxanthine, 4x10 ⁇ 7 M aminopterin and 3x10 ⁇ 6 M thymidine (medium C or HAT selective medium).
  • Aminopterin is toxic for cells that lack the enzyme HGPRT and therefore kills all unfused myeloma cells. Fused cells (hybridomas) survive in HAT because they obtain HGPRT from the B lymphocyte (spleen cell) fusion partner.
  • the cell suspension from example 25 above is transferred into a 75 cm ⁇ T-flask and incubated at 37 °C in a 5% CO2 incubator for 1-3 hours.
  • the cell suspension is then diluted to 1x10 ⁇ spleen cells/mL with medium C, and 1 mL volumes of the cell suspensions are added to each well of a 24 well Costar plates. These plates are incubated for 24 hours at 37 °C and 5% CO2. After the incubation period 1 mL volumes of feeder cell (non-immunized BALB/c mouse spleen cells) suspension in medium C at 2-3x10-5 cells/mL is added to each of the 24 wells of the Costar plates and incubated at 37 °C, 5% C02 for 14 -17 days.
  • Example 27 Cloning of Hybridoma Culture mat Produces Monoclonal Antibodies The cells in antibody secreting wells are diluted in a volume of Medium
  • a and 15% fetal calf serum (Medium B) to a concentration of 10 cells mL and 100 mL of each diluted cell suspension are aliquoted into the wells of three Costar plates of 96 wells each. 100 mL volumes of feeder cells in medium B at 5x10-5 cells/mL are added to each well and the plates incubated at 37 °C, 5% C02 for 14 days. Supematants are again tested for antibody activity using the same protocol as in Example 21. The antibody producing clones are then expanded without feeder cells in 24 well Costar plates and finally in 25 cm2 T- flasks. 32x10 ⁇ cells/mL samples of the clone are then stored in medium B with 10% glycerol added, in liquid nitrogen. 1-2 mL samples were then further evaluated for displacement on the TDx instrument protocol and one clone is selected for ascites production.
  • Example 26 An in vivo method for obtaining large amounts of monoclonal antibodies involved the adaptation of Example 26 to grow as an "ascites" tumor.
  • Female BALB/c mice are "primed by intraperitoneal injection of 0.5 mL of pristane (2,6,10,14-tetra-methylpentadecane).
  • Pristane is a sterile irritant which elicits a serous secretion ("ascites”) in the peritoneal cavity of mice which acts as a growth medium.
  • ascites a sterile irritant which elicits a serous secretion
  • aliquots containing 1.5 x 10 ⁇ actively growing hybridoma cells harvested from in vitro cultures as described in Example 21 are innoculated into the peritoneal cavities of primed mice.
  • Seven days following hybridoma cell injection 5 - 10 mL of ascites fluid is harvested from each mouse. Upon purification by ammonium sulfate precipitation
  • the reagents for the FPIA of the present invention comprise tracers and antibodies raised against immunogens of the present invention, specific for tethered intermediates.
  • conventionally used assay solutions including a dilution buffer, and quinoline derivative calibrators and controls are prepared.
  • test sample can be mixed with a pretreatment solution and antibody in dilution buffer before a background reading is taken.
  • the tracer is then added to the test solution. After incubation, a fluorescence polarization reading is taken.
  • the fluorescence polarization value of each calibrator, control or test sample is determined and printed on the output tape of the TDx, ADx or IMx instrument.
  • the instrument also generates a standard curve by plotting the polarization of each calibrator versus it's concentration, using a nonlinear regression analysis. The concentration of each control or sample is read off the stored curve and printed on the output tape.
  • the following reagents are used in the preferred automated quinoline derivative assays.
  • the tracer diluted in 50% methanol in potassium phosphate buffer (0.15 M phosphate buffer, pH 7.5).
  • the antibody comprising rabbit antisera or mouse monoclonal antibody raised against a quinoline derivative immunogen, diluted in TDx buffer (0.1 M phosphate buffer, pH 7.5, containing 0.01% bovine gamma globulin and 0.1% sodium azide) with 30% glycerol;
  • a diluent buffer comprising TDx buffer, 5) a sets of calibrators
  • the fluorescence polarization due to tracer binding to the antibody is obtained by subtracting die polarized fluorescence intensities of the background from the final polarized fluorescence intensities of the mixture; and 5) die polarization value for the unknown test sample is compared to a standard curve prepared using calibrators of known quinoline derivative content.

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Abstract

Cette invention concerne de nouveaux haptènes et des conjugués apparentés à base de quinoline ainsi que des procédés de préparation et d'utilisation de ces conjugués. Des haptères basés sur la structure de noyau de quinoline peuvent être substitués aux positions 5 et 8. Des intermédiaires comprenant une amarre en position 8, des immunogènes, des traceurs, des supports solides et des oligonucléotides étiquetés sont décrits ainsi que des procédés d'utilisation desdits intermédiaires pour préparer les conjugués, des procédés d'utilisation des conjugués pour fabriquer et purifier des anticorps, sous forme de traceurs d'analyse, et dans des techniques d'hybridation d'acide nucléique. Cette invention présente également des kits contenant des oligonucléotides hapténés et des conjugués anti-haptènes.
PCT/US1993/002899 1992-03-27 1993-03-26 Haptenes, traceurs, immunogenes et anticorps destines a la quinoline WO1993020094A1 (fr)

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FR2728687A1 (fr) * 1994-12-21 1996-06-28 Bio Merieux Procede de dosage d'un haptene selon une technique de competition
US5989831A (en) * 1994-12-21 1999-11-23 Bio Merieux Hapten assay by a competition-based method
WO1996019729A1 (fr) * 1994-12-21 1996-06-27 Bio Merieux Procede de dosage d'un haptene selon une technique de competition
JP4669704B2 (ja) * 2005-02-02 2011-04-13 学校法人東京理科大学 紫外線分解化合物
JP2006213615A (ja) * 2005-02-02 2006-08-17 Tokyo Univ Of Science 紫外線分解化合物
EP1782826A1 (fr) * 2005-11-08 2007-05-09 GBF Gesellschaft für Biotechnologische Forschung mbH PQS, c-diGMP et leurs conjugués utilisés comme adjuvants et leur emploi dans des compositions pharmaceutiques
WO2007054283A2 (fr) * 2005-11-08 2007-05-18 Helmholtz-Zentrum für Infektionsforschung GmbH Pqs et leurs conjugues utiles en tant qu'adjuvants et leurs utilisations dans des compositions pharmaceutiques
WO2007054283A3 (fr) * 2005-11-08 2007-08-09 Helmholtz Infektionsforschung Pqs et leurs conjugues utiles en tant qu'adjuvants et leurs utilisations dans des compositions pharmaceutiques
EP2444807A3 (fr) * 2006-11-01 2013-02-27 Ventana Medical Systems, Inc. Haptènes, conjugués de haptène, compositions de haptène, procédé de fabrication et utilisation
US11092594B2 (en) * 2006-11-01 2021-08-17 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US20100297725A1 (en) * 2006-11-01 2010-11-25 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US8618265B2 (en) * 2006-11-01 2013-12-31 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US8846320B2 (en) 2006-11-01 2014-09-30 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US9719986B2 (en) 2006-11-01 2017-08-01 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof preparation and method for their preparation and use
US20180120306A1 (en) * 2006-11-01 2018-05-03 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof and method for their preparation and use
EP2444806A3 (fr) * 2006-11-01 2013-02-27 Ventana Medical Systems, Inc. Haptènes, conjugués de haptène, compositions de haptène, procédé de fabrication et utilisation
US11447451B2 (en) 2016-07-15 2022-09-20 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
WO2019238817A1 (fr) * 2018-06-13 2019-12-19 University Of Dundee Molécules bifonctionnelles pour cibler rpn11
CN112533918A (zh) * 2018-06-13 2021-03-19 安菲斯塔治疗有限责任公司 用于靶向Rpn11的双功能分子
US11925690B2 (en) 2018-06-13 2024-03-12 Amphista Therapeutics Limited Bifunctional molecules for targeting Rpn11
WO2020063824A1 (fr) * 2018-09-29 2020-04-02 江苏亚虹医药科技有限公司 Promédicament de nitroxoline et utilisation associée

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