WO1993020074A1 - Haptenes, traceurs, immunogenes et anticorps d'acides acridine-9-carboxyliques - Google Patents

Haptenes, traceurs, immunogenes et anticorps d'acides acridine-9-carboxyliques Download PDF

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Publication number
WO1993020074A1
WO1993020074A1 PCT/US1993/002832 US9302832W WO9320074A1 WO 1993020074 A1 WO1993020074 A1 WO 1993020074A1 US 9302832 W US9302832 W US 9302832W WO 9320074 A1 WO9320074 A1 WO 9320074A1
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hapten
conjugate
group
antibody
oligonucleotide
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PCT/US1993/002832
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English (en)
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Phillip G. Mattingly
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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
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/02—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with only hydrogen, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems 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 carbon atoms of the ring system
    • 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

Definitions

  • the present invention relates to novel acridine-9-carboxylic acid 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.
  • a label- phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis. For example, see Thuong, N. T. et al., 7et.
  • 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
  • Methodology for preparing tracer molecules also is known.
  • 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 diffeientiable from the signal from the free form, so that the amount of analyte-hapten can be estimated.
  • One method for differentiating the signals is by fluorescence polarization immunoassay (FPIA), in which the "millipolarization", the “span” or the “relative intensity” can be measured as described in the literature and below.
  • FPIA fluorescence polarization immunoassay
  • EP-A-273 115 (Abbott Laboratories) describes chemiluminescent acridinium and phenanthridinium compounds. These are conjugated to binding pair memebers such as haptens, antibodies or oligonucleotides. The complex of the binding pair member with its partner can be detected by the luminescent label.
  • EP-A-368273 (Ishikawa) and EP-A- 257 541 (Hoechst) are other examples.
  • NTIS Order No.7-246,688 (“Cohen") describes oligonucleotides incorporating a 2-methoxy-6-chloroacridine at the 5' end.
  • the acridine moiety is added during oligonucleotide synthesis by preparing an acridine-phosphoramidite reagent.
  • Acridine serves as an intercalator in the antisense oligonucleotides described. Creech and O'Connell, Cancer Res.
  • the present invention is derived from the class of compounds which are based on acridine-9-carboxylic acid derivatives of the following structure:
  • a and a' are independently selected from the group consisting of: hydrogen (H), hydroxy (-OH), protected hydroxy (-OZ), mercapto (-SH), protected mercapto (-SZ), nitro (-NO2), sulfo (-SO3”), and 3-nitrobenzyloxy (-O-CH2C6H4-
  • B is selected from the group consisting of -hydroxy (-OH), protected hydroxy (-OZ), amino (-NR"R" ⁇ where R" and R'" may independently be H, alkyl or aryl), thiol (-SH), protected thiol (-SZ), and a leaving group, wherein Z is a protecting group.
  • protecting group Z may be selected from among the many known protecting groups for O and/or S.
  • the compound of formula I has a and a' at the 3 and 6 positions.
  • the tethered intermediate acridine-9-carboxylic acid compounds have the following structure:
  • 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 about 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.
  • Possible reactive functionalities y are described below.
  • 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 (III).
  • antibodies may be prepared by the process of injecting an immunogen (III) 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 antibody 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.
  • hapten are often used interchangeably- For example, 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 ac ⁇ idine-9-carboxylic acid core structure shown below:
  • 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.
  • BTG bovine thyroglobulin
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • BGG bovine gamma globulin
  • TSG thyroxine binding globulin
  • hapten-specif ⁇ c 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 Cj)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).
  • each group Z may be independently selected from the various protecting groups. Indeed, one of ordinary skill in the art will know which protecting groups are routine for which functional groups.
  • B may be a "leaving group”.
  • halides are the most useful leaving groups in nucleophilic reactions
  • 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- ⁇ -icyclohexylcarbodiimide 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 about 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.
  • 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 -B.
  • x is also a functional group (selected from the same group as y) which can react with functional groups on the hapten or -B.
  • 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 intermediate
  • 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.
  • exemplary Michael acceptors include:
  • a particularly preferred Michael acceptor is maleimide:
  • Solid supports refer to a wide variety of support materials.
  • Polymeric plastics such as polystyrene, polypropylene, andpolytetrafluoroethylene, 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.
  • Both 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 by eliciting an immune response as before. Following appropriate incubation and booster injections, B-lymphocyte cells are removed from the spleens of the animals by standard procedures, and the B-lymphocyte cells are then fiised 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: fluorescein amine, carboxyfluorescein, a- iodoacetamidofluorescein, 4'-aminomethylfluorescein, 4'-N- alkylaminomethylfluorescein, 5-aminomethylfluorescein, 2,4-dichloro- 1 ,3,5-triazin- 2-yl-aminofluorescein (DTAF), 4-chloro-6-methoxy-l,3,5-triazin-2-yl- aminofluorescein, fluorescein isothiocyanate.
  • 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. A. Reg-rents
  • Haptens which are structurally similar to acridine-9-carboxylic acid have the general structure (I) given above.
  • Substituents having a lone pair of electrons or heteroatoms e.g. alkoxy, nitro, sulfo, etc); and/or substituents having relatively rigid chemical structures may be preferred for increased antigenic avidity.
  • Haptens according to the invention may be synthesized according to the procedures described in the example section, below.
  • substituted acridine-9-carboxylic acids may be prepared according to the general procedures outlined in R. M. Acheson, Acridines, John-Wiley, New York, 1973 and A. Albert, The Acridines, St. Martin Press, New York, 1966. Two general routes to acridine-9- carboxylic acids are shown.
  • the first route relies on the base induced ring opening and rearrangement of N- phenylisatins.
  • a preparation of substituted N-phenylisatins is the subject of co- pending application serial no. 07/443869 [Mattingly and Dombrowski], herein incorporated by reference.
  • the second route proceeds by the addition of cyanide to a 9-unsubstituted acridine to produce a 9-cyano-dihydroacridine species which then oxidizes on standing to the 9- cyanoacridine, Hydrolysis of the cyano group produces the desired carboxylic acid.
  • 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 molecme 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
  • Immunogens can be produced from a wide variety of tethered intermediates.
  • the immunogens of the present invention have the general structure (--II) shown above, wherein Q represents an immunogenicity conferring carrier. Typical carriers were previously described.
  • the tether functionality, y, of tethered intermediates (II) 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.
  • phosgene or a phosgene equivalent such as di or triphosgene or carbonyld ⁇ midazole
  • 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.
  • 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 or 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 acridine-9-carboxylic acid 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).
  • phosgene or a phosgene equivalent such as di or triphosgene or carbonyldiimidazole
  • oligonucleotide conjugates can be produced from a wide variety of tethered intermediates.
  • the oligonucleotides of the present invention have the general structure (DI) wherein Q represents an oligonuclotide
  • 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).
  • haptens e.g. biotin, fluorescein, dansyl, acetylaminofluorene and iodo- acetylaminofluorene, etc.
  • probes labeled with the hapten of this invention are particularly useful in multiplex versions of PCR and LCR.
  • 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.
  • reagents such as Aminomodifier® (Clontech, Palo Alto). Hydroxyl functions are typically formed during automated synthesis.
  • Several means are available for derivatizing oligonucleotides with one or more functionalities that may later be reacted with appropriately derivatized acridine dyes. References disclosing methods for derivatizing oligonucleotides with amino or thiol functionalities include Connolly, Nucleic Acids Research. 15: 3131-39 (1987) Ruth, DNA. 3: 123-29 (1984), and Smith et al., Nucleic Acids Research.
  • 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; Rl and RlO are independently hydrogen, alkyl of from 1-10 carbon atoms, an amino protecting group, or aryl, alternatively Rl or RlO when taken together with W and the nitrogen atom to which they are attached may form a cyclic amine.
  • 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
  • Compound 1 is carboxyl-activated (step 1) via a nucleophilic acyl substitution reaction, followed by reaction with Meldrum' s acid (2,2-dimethyl- 1,3- dioxane-4,6-dione, 2) and R 2 OH (R 2 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 to form the tethered hapten 7.
  • the tethered hapten is then phosphoramidated at the secondary alcohol (step 8) 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.
  • Detailed descriptions of procedures for solid phase synthesis of oligonucleotides are widely available, e.g., U.S. Patent Numbers 4,401,796 and 4,458, 066 which are incorporated by reference.
  • synthesis of acridine-labeled oligonucleotides is accomplished by reacting an acridine 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).
  • a tethered hapten (II) is reacted in the presence of carbon tetrachloride with a phosphonate group already incorporated into 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:
  • 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 millipolarization 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.
  • 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.
  • 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 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.
  • Example 1 Preparation of ⁇ -(5-carboxypentyl)-acridine-9-carboxamide; a hapten.
  • Acridine-9-carboxylic acid (5g) (Aldrich Chemical Co. Milwaukee WI) was refluxed in thionyl chloride for 4 hours. After cooling the excess reagent was removed in vacuo and the residue dissolved in hot methylene chloride (lOOmL). The mixture was filtered and added to heptane (lOOmL). On cooling a precipitate formed which was collected by filtration. After drying in vacuo over P2O5 there remained 4.78g of acridine- 9-carboxylic acid chloride hydrochloride.
  • N-(5-carbomethoxypentyl)-acridine-9-carboxamide was saponified in methanolic sodium hydroxide to give 0.65g of N-(5-carboxypentyl)-acridine-9-carboxamide. Mass spectrum: (M+H)+ 337.
  • 3-Nitrobenzyl 3-(3-nitrobenzyloxy)-acridine-9-carboxylate 400mg was saponified with aqueous methanolic sodium hydroxide at 65°C for 48 hours to give 3-(3- nitrobenzyloxy)-acridine-9-carboxylic acid (150mg).
  • 3-(3-Nitrobenzyloxy)-ac ⁇ idine-9-carboxylic acid (150mg) was dissolved in thionyl chloride and heated to reflux for 4 hours. The excess reagent was removed in vacuo The crude acid chloride was taken up in tetrahydrofuran.
  • Methyl 6-aminocaproate 80mg
  • triethylamine 167 ⁇ L
  • the solution was evaporated in vacuo and chromatographed on silica gel eluting with ethyl acetate/cyclohexane (1 to 1) yielding 3-(3-nitrobenzyloxy)-N-(5-carbomethoxypentyl)-acridine-9-carboxamide (147mg).
  • Example 3 Preparation of 8-amino-3-hydroxy- 1 -(4,4'-dimethoxytrityloxy)octane; a tether.
  • the title compound is prepared according to Scheme 1, steps 1-6, above.
  • the bold-faced compound numbers refer to the compounds in that scheme.
  • Step a Preparation of Methyl 8-(N-Cbz-amino)-3-oxo-octanoate.
  • 6-N-Cbz-aminohexanoic acid (1, R 1 is Cbz, R 0 is H, W is -(CH2)5-, 50g, 0.188 moles), anhydrous methylene chloride (1 L), Meldrum' s Acid (2, 27.15 g, 0.188 moles), and triethyl amine (68.6 mL, 0.5 moles) were added in a reaction flask equipped with a magnetic stirbar and latex septum. While stirring under nitrogen, diethylcyanophosphonate (30.8 mL, 0.188 moles) was added.
  • Step b Preparation of 8-(N-Cbz-amino)-l,3-octanediol Methyl 8-(N-Cbz-amino)-3-oxo-octanoate (3, 51.5 g, 0.16 moles) was dissolved in anhydrous tetrahydrofuran (350 mL) in a reaction flask equipped with a magnetic stirbar, reflux condenser, pressure equalizing addition funnel and nitrogen gas inlet While stirring the mixture under nitrogen sodium borohydride (15.1 g, 0.4 moles) was cautiously added. The mixture was then heated to reflux and methanol was added dropwise over 90 minutes. Stirring was continued at reflux temperature for 60 minutes after the addition was complete.
  • Step c Preparation of 8-(N-Cbz-amino)-3-hydroxy-l-(4,4'-dimethoxytrityloxy)octane 8-(N-Cbz-amino)-l,3-octanediol (4, 25 g, 85 mmoles ) was dissolved in anhydrous pyridine (200 mL),evaporated in vacuo and redissolved in anhydrous pyridine (200 mL). Diisopropylethylamine (38.1 mL, 88 mmoles) and 4- dimethylaminopyridine (170 mg) were added.
  • Example 5 Preparation of 8-N-[5-O-(O-2-cyanoethyl-N,N-diisopropylphosphatidyl)-8- (4,4 l -dimethoxytrityloxy)octyl]-9-acrididinecarboxamide; a tethered hapten.
  • the title compound is prepared according to Scheme 1, steps 7-8, above.
  • the bold-faced compound numbers refer to the compounds in that scheme.
  • reaction mixture was stirred for 12h, evaporated and chromatographed [Whatman PLKC18F, 1mm, 20x20cm reverse phase plates, methanol/1% aq. acetic acid, 60:40 or MERCK Silica Gel 60 F-254, 2mm, 20x20cm , chloroform/methanol, 85: 15].
  • Example 10 N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 4'-aminomethylfluorescein to give a tracer of structure:.
  • Example 11 N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 4'-N-(glycylaminomethyl)fluorescein to give a tracer of structure:.
  • Example 12 N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 5-aminomethylfluorescein to give a tracer of structure:.
  • Example 13 N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 5-[N-(2-aminoethyl)carboxamido]fluorescein to give a tracer of structure:.
  • Example 14 N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 6-[N-(2-aminoethyl)carboxamido]fluorescein to give a tracer of structure:.
  • Example 15 N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 5-[N-(6-aminohexyl)carboxamido]fluorescein to give a tracer of structure:.
  • Example 16 3-(3-Nitrobenzyloxy)-N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 4'-aminomethylfluorescein to give a tracer of structure:.
  • Example 17 3-(3-Nitrobenzyloxy)-N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 4'-N-(glycylaminomethyl)fluorescein to give a tracer of structure:.
  • Example 18 3-(3-Nitrobenzyloxy)-N-(5-carrx>xypentyl)-aoidine-9-carboxamide was reacted according to example 9A with 5-aminomethylfluorescein to give a tracer of structure:.
  • Example 20 3-(3-Nitiobenzyloxy)-N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 5-[N-(2-aminoethyl)carboxamido]fluorescein to give a tracer of structure:.
  • Example 21 3-(3-Nitrobenzyloxy)-N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 6-[N-(2-aminoethyl)carboxamido]fluorescein to give a tracer of structure:.
  • Example 22 3-(3-Nitroben2yloxy)-N-(5-carboxypentyl)-acridine-9-carboxamide was reacted according to example 9A with 5-[N-(6-aminohexyl)carboxamido]fluorescein to give a tracer
  • mice Four to six week old female BALB/c mice were injected subcutaneously at four week intervals with 0.2 mL of the immunogen from Example 7 ( 5mg mL; 0.06 mL of immunogen) in.1.88 mL saline; with lOOmg of monophosphoryl lipid A and trehalose dimycloate adjuvant (Ribi Immunochem Research, Inc).
  • the donor mice are killed by cervical dislocation three days following the last immunization; 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 centrif uged 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.
  • An immunoglobulin non-secreting mouse myeloma cell line (SP 2/0) deficient in the ctyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT-, EC2.4.2.8), as disclosed by Kearney, Journal of Immunology, 1979,223,1548, which is incorporated herein by reference, is used as the fusion partner.
  • 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 Erythrosin B stain exclusion.
  • the fusion technique used is modified from that of Gefter et. al., Somatic Cell Genetics, 1977, 3, 231, which is hereby incorporated by reference.
  • To a sterile 50 mL conical centrifuge tube was added 1-I.5xl0 8 spleen cells with an equal number of SP 2/0 myeloma cells.
  • the myeloma-spleen cell suspension was centrifuged at 1400 rpm for 5 minutes to pellet the cells together. The supernatant was aspirated off and the tube tapped gently to loosen the cell pellet and I mL of 50% polyethylene glycol (PEG, MW 1000, Sigma) in DMEM, without serum, M, added to the cell pellet.
  • PEG polyethylene glycol
  • the cells were resuspended gently in PEG solution over a period of 1 minute by slowly aspirating up and down using a 1 mL pipette. The tube was held in the hand for an additional 1 minute and then 1 mL of medium A was added slowly to dilute the PEG. The cells are allowed to stand for an additional 1 minute without agitation or mixing. An additional 20 mL of medium A was added over a period of 3 to 5 minutes, and the cells pelleted at 1400 rpm for 5 minutes.
  • the supernatant was aspirated off and the cells resuspended in 20 mL of medium A with 20% fetal calf serum, lxlO -4 M hypoxanthine, 4xl0- 7 M aminopterin and 3xl0" 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.
  • Example 25 Selection of Hybridomas Producing Monoclonal Antibodies to Ac ⁇ idine-9- carboxylic acid Immunogen of Example 7.
  • the cell suspension from example 24 above is transferred into a 75 cm2 T-flask and incubated at 37°C in a 5% CO2 incubator for 1-3 hours.
  • the cell suspension is then diluted to lxlO 6 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% CO 2 .
  • hybridoma suspensions are chosen for further cloning by picking those supernatants with tracer binding in mP units greater than 20% over background.
  • the cells from wells chosen for containing antibody activity are cloned by limiting dilution within 24 hours of sampling.
  • Example 26 Cloning of Hybridoma Culture that Produces Monoclonal Antibodies to acridine-9-carboxylic acid derivatives.
  • 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 s cells/mL are added to each well and the plates incubated at 37 °C, 5% CO 2 for 14 days. Supernatants are again tested for antibody activity using the same protocol as in Example 24.
  • the antibody producing clones are then expanded without feeder cells in 24 well Costar plates and finally in 25 cm2 T-flasks. 32xl0 6 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 6 actively growing hybridoma cells harvested from in vitro cultures as described in Example 24 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 precipit
  • Example 28 Monoclonal Antibodies from KLH 'immunogens.
  • the KLH immunogens from Example 8 are processed according to the procedure of Example 26 to produce hybridomas.
  • the hybridomas are cloned according to the procedure of Example 26 or grown in vivo according to the procedure of Example 27.
  • 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 acridine-9-carboxylic acid 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 acridine-9-carboxylic acid derivative assays.
  • the pretreatment solution 2) 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 acridine-9-carboxylic acid 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;
  • the fluorescence polarization due to tracer binding to the antibody is obtained by subtracting the polarized fluorescence intensities of the background from the final polarized fluorescence intensities of the mixture; and 5) the polarization value for the unknown test sample is compared to a standard curve prepared using calibrators of known acridine-9-carboxylic acid derivative content.

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Abstract

Nouveaux haptènes et conjugués apparentés à base d'acides acridine-9-carboxyliques, ainsi que procédé servant à préparer et à utiliser lesdits conjugués. Les haptènes à base de la structure centrale décrite ci-dessus peuvent être substitués au niveau des positions 3 ou 6. L'invention décrit en totalité l'utilisation d'intermédiaires restreints, d'immunogènes, de traceurs, de supports solides et d'oligonucléotides marqués; elle décrit également des procédés d'utilisation des intermédiaires, afin de préparer les conjugués, ainsi que des procédés d'utilisation desdits conjugués afin de préparer et de purifier des anticorps, en tant que traceurs d'essai, ainsi que dans des essais d'hybridation d'acide nucléique. Elle décrit également des kits contenant des oligonucléotides possédant des caractéristiques d'haptène et des conjugués anti-haptène.
PCT/US1993/002832 1992-03-27 1993-03-26 Haptenes, traceurs, immunogenes et anticorps d'acides acridine-9-carboxyliques WO1993020074A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000284A1 (fr) * 1994-06-24 1996-01-04 Behringwerke Aktiengesellschaft Procede de stabilisation de molecules ou de parties de molecules sensibles a l'hydrolyse
WO1996019729A1 (fr) * 1994-12-21 1996-06-27 Bio Merieux Procede de dosage d'un haptene selon une technique de competition
US5834206A (en) * 1994-12-10 1998-11-10 Behring Diagnostics Gmbh Immunoassays for haptens and hapten tracer-antibody complex which can be used therefor, and process for the preparation thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0273115A2 (fr) * 1986-10-22 1988-07-06 Abbott Laboratories Sels d'acridines et de phénantridines chimiluminescents
EP0330433A2 (fr) * 1988-02-26 1989-08-30 Gen-Probe Incorporated Marqueurs chimiluminescent protégés
EP0407816A2 (fr) * 1989-07-14 1991-01-16 Abbott Laboratories Nucléosides à base modifiée

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0273115A2 (fr) * 1986-10-22 1988-07-06 Abbott Laboratories Sels d'acridines et de phénantridines chimiluminescents
EP0330433A2 (fr) * 1988-02-26 1989-08-30 Gen-Probe Incorporated Marqueurs chimiluminescent protégés
EP0407816A2 (fr) * 1989-07-14 1991-01-16 Abbott Laboratories Nucléosides à base modifiée

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Title
JOURNAL OF BIOLUMINESCENCE, Vol. 4, issued 1989, SEPTAK, "Acridinium Ester-Labelled DNA Oligonucleotide Probes", pages 351-356. *
US PATENT APPLICATION, COHEN et al., 15 February 1989 (See the "Characterization of Acridine Containing Oligomers" section). *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000284A1 (fr) * 1994-06-24 1996-01-04 Behringwerke Aktiengesellschaft Procede de stabilisation de molecules ou de parties de molecules sensibles a l'hydrolyse
AU689920B2 (en) * 1994-06-24 1998-04-09 Dade Behring Marburg Gmbh Method of stabilizing molecules, or parts of molecules, which are sensitive to hydrolysis
US6010868A (en) * 1994-06-24 2000-01-04 Behring Diagnostics Gmbh Method for stabilizing hydrolysis-sensitive molecules or molecular moieties
US5834206A (en) * 1994-12-10 1998-11-10 Behring Diagnostics Gmbh Immunoassays for haptens and hapten tracer-antibody complex which can be used therefor, and process for the preparation thereof
WO1996019729A1 (fr) * 1994-12-21 1996-06-27 Bio Merieux Procede de dosage d'un haptene selon une technique de competition
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

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