WO1993020094A1 - Haptens, tracers, immunogens and antibodies for quinoline - Google Patents

Abstract

Novel haptens and related conjugates based on quinoline, as well as methods for making and using such conjugates. Haptens based on the above core structure may be substituted at positions 5 and 8. Using intermediates with a tether in the 8 position, immunogens, tracers, solid supports and labeled oligonucleotides are all described; as are methods for using the intermediates to prepare the conjugates, methods of using the conjugates to make and purify antibodies, as assay tracers, and in nucleic acid hybridization assays. Kits containing haptenated oligonucleotides and anti-hapten conjugates are also described.

Description

HAPTENS, TRACERS, IMMUNOGENS AND ANTIBODIES FOR

QUINOLINE

This application is a continuation-in-part of U.S. Serial No. 07/858,820 filed March 27, 1992, the whole of which is incorporated by reference.

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.

I. BACKGROUND OF THE INVENTION:

It is commonly known that many small molecules will not elicit an antibody response by themselves but, when coupled to an appropriate immunogenicity conferring carrier molecule (to become an immunogen), antibodies can be prepared against the hapten. This technology is discussed in many textbooks. See Erlanger, B. F. in Methods of Enzymology, 70:85-105 (Academic Press 1980); and Hum, B. A. L., et al., in Methods of Enzymology, 70:105- (Academic Press 1980).

Many methods of adding haptens to oligonucleotide probes are known in the literature. A review of such conjugate literature is found in Goodchild, Bioconjugate Chemistry, 1(3):165-187 (1990). Enzo Biochemical (New York) and Clontech (Palo Alto) both have described and commercialized probe labeling techniques, including techniques for labeling probes with biotin or similar haptens. In addition, co-pending applications U.S. Serial Nos. 625,566, filed December 11, 1990 and 630,908, filed December 20, 1990 teach methods for labeling probes at their 5' and 3' ends respectively. The entire disclosures of the aforementioned co-pending applications are incorporated by reference. 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).

According to one known method for labeling an oligonucleotide, 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., Tet. Letters, 29(46):5905-5908 (1988); or Cohen, J.S. et al., U.S. Patent Application 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688) (1989). However, DNA synthesis reaction conditions are quite severe (e.g. iodine oxidation and ammonium hydroxide cleavage) and many haptens (e.g. biotin and fluorescein) do not readily withstand these conditions without modification. In another approach useful for labile haptens, 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.

Automated synthesis of oligonucleotides (See e.g. Beaucage and Caruthers, Tet. Letters, 22(20):1859-1862 (1981) and U.S. Patents 4,973,679 and 4,458,066) is often the most efficient method of preparing probes. However, the hostile conditions required during automated synthesis limit the choice of labels available for labeling by this method. The present invention overcomes these drawbacks by describing novel haptens which will withstand the rather rigorous conditions of DNA synthesis. Thus, using the haptens of the invention, an oligonucleotide can be directly labeled during automated synthesis, without involving an intervening isolation or a secondary labeling reaction.

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.

Methodology for preparing tracer molecules also is known. For example, fluorescence polarization assays require tracers comprising an analyte-hapten coupled to a fluorescent molecule. Typically, 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. 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. The technique of FPIA has been described, for example, in Jolley, M.E., J. Analyt. Toxicol, 5:236-240 (1981) and in Blecka, L. J. Amer. Assoc. Clin. Chem. pp. 1-6 (March 1983) ,the entire disclosures of which are incorporated herein by reference.

Japanese patent publication 1169357 (July 1989) 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.

In addition, several researchers have made immunogens from — and antibodies against - quinoline derivatives in an attempt to produce antibodies that could be used in an ELISA type assay for the antimalarial quinoline drugs. These drugs are known to cause side effects and it is desirable to monitor the blood levels of the drugs to ensure therapeutic effect at the lowest possible doses.

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

8-hydroxyquinoline (8-HQ). The same authors report their findings in English in Mol. Immunol., 23(8):793-797 (1986). The antibodies they raised reacted with 7-chloro-4-aminoquinoline compounds but were not inhibited by 7-MQ or 8-HQ, presumably because of the lack of the 7-chloride atom. G. Christie, et al., in Biochem. Pharmacol, 38(9):1451-1458 (1989), describe antibodies prepared against amodiaquine (a 7-chloro-4-(3' diethylamino-4'-hydroxyanilino)-quinoline derivative) in an effort to determine the mechanism of side effects caused by this drug.

B. Ravindran, et al., in Med. Sci. Res., 16:161-162 (1988), describe antibodies prepared against primaquine. Primaquine is another antimalarial drug, but is an 8-(4-amino-l-methyl-butylamino)-6-methoxy derivative. Based on inhibition studies, the antibodies showed some cross reactivity with chloroquine, a 7-chloro-4-(4 diethylamino-1-methylbutylamino) derivative. However, applicants are unaware of any art demonstrating the antigenicity or immunogenicity of the claimed quinoline derivatives. π. SUMMARY OF THE PRESENT INVENTION:

In one aspect, the present invention is derived from the class of compounds which are based on the quinoline core structure:

wherein 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

( ₂)- • In a specific embodiment, the compound of formula I has a at the 5 position.

In another aspect, the invention relates to a tethered intermediate quinoline compound having the following structure:

wherein a is selected from the group consisting of hydrogen (H), hydroxy (OH), protected hydroxy, mercapto (-SH), protected mercapto, nitro (- N0₂), 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. Typically, 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 in detail below, but include hydroxyl (-OH), thiol (-SH), carboxy (-C(=O)OH), amino (-NH2), aldehyde (-CH(=O)), leaving group, Michael acceptor, phosphoramidite, phosphonate and protected forms of these functional groups. Where more than one protecting group is used, each 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.

When Z is O, A may ideally comprise a lower alkyl substituent having a reactive y functional group. Such a ZA substituent is logicaly referred to generally as an alkoxy quinoline.

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.

In another aspect, the present invention relates to conjugates of the following structure:

wherein a, Z and A are defined as above; except that A becomes -L-, the y having been reacted with Q; and wherein Q is a conjugation partner. 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). In another aspect, 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. In addition, the invention relates to the following methods of using the above compounds:

1. Use of compounds (II) to prepare immunogens, tracers, labeled oligonucleotides and affinity solid supports;

2. Use of compounds (IE: Q= immunogenicity conferring carrier) to raise antibodies;

3. Use of compounds (HI: Q=solid support) to isolate or purify antibodies;

4. Use of compounds (HI: Q=oligonucleotide) for detection of nucleic acids complementary to the oligonucleotide; and

5. Use of compounds (HI: Q=detectable signal moiety) for detection of hapten-analog analytes. Finally, the invention also relates to kits containing compounds of the invention (e.g. H:A=phosphoramidite or HI: Q=oligonucleotide), in combination with an antibody reactive with the compounds, said antibody being attached to or adapted for attachment to either solid supports or detectable labels. In the second example, the oligonucleotide probe may be hybridized with a target and the antibody may be used to separate or detect it. In the first example, the phosphoramidite may be used to label one's own oligonucleotide during its synthesis, while the anitbody is used as before.

m. DETAILED DESCRIPTION:

The following definitions are applicable to the present invention: "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. 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. For purposes of this application, "hapten" is defined as any compound having the quinoline core structure shown below:

wherein 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.

As suggested above, the term "immunogen" refers to a conjugate of a hapten or antigen and a carrier molecule. The carrier is often a protein or peptide. Known 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. Alternatively, synthetic poly(amino-acids) can be utilized such as polylysine, etc. However, any molecule which is capable of conferring antigenicity to a hapten is an "immunogenicity conferring carrier."

The term "hapten-specific binding member", as used herein, 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.

In general, terms like "alkyl", "alkenyl" and "aryl" have the meanings usually attributed to them by persons skilled in the art of organic chemistry. For example, 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_nH2n-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 π electron clouds of each carbon remaining on opposite sides of the plane. Aryl substituents satisfy the Huckel (4n+2) π 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). Hydroxyl functions are routinely protected as alkyl or aryl ethers (Z= alkyl, aryl, alkenyl), silyl ethers (Z=silyl), esters (Z=acyl), carbonates (Z= - C(=O)-0-alkyl, -C(=O)-O-aryl, -C(=O)-O-alkenyl) and carbamates (Z=-C(=O)- NH-alkyl, -C(=0)-NH-aryl, -C(=0)-NH-alkenyl). Amino functions are routinely protected as carbamates (Z= -C(=O)-O-alkyl, -C(=O)-O-aryl, -C(=0)- O-alkenyl), amides (Z=-C(=O)-alkyl, -C(=O)-aryl, -C(=O)-alkenyl), cyclic imides (Z=phthaloyl), N-benzyl derivatives (Z=-CH(_n)aryl(3__n), n=l-3), imine derivatives (__= =CH(_n)alkyl(2-_n). =CH(_n)aryl(2-_n) n=0-2), silyl derivatives (Z=silyl), N-sulfenyl derivatives (Z= -S-aryl, -S-CH(_n)aryl(3__n), n=0-3), and N- sulfonyl derivatives (Z= -SO2-aryl, -SC_>-alkyl). Thiol functions are routinely protected as thioethers (Z= -CH(_n)aryl(3__n), n=l-3, aklyl), thioesters (Z=acyl), thiocarbonates (Z= -C(=0)-0-alkyl, -C(=0)-0-aryl, -C(=0)-0-alkenyl), thiocarbamates (Z=-C(=O)-NH-alkyl, -C(=O)-NH-aryl, -C(=O)-NH-alkenyl), and disulfides (Z= -S-alkyl, aryl). Where more than one protecting group is called for, it will be understood that 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.

A "leaving group" is defined in the art as follows: "In a reaction in which a substrate molecule becomes cleaved, part of it (the part not containing the carbon) is usually called the leaving group." (J. March, Advanced Organic Chemistry, 2ndEd.,McGraw-Hill, NY, 1977, p 187). Exemplary leaving groups are given in March, supra, and include halo (-C1, -Br, -I), alkyl sulfonate esters (-OS(=0)2-alkyl) and aryl sulfonate esters ( -OS(=0)2-aryl) among others. The choice of an appropriate leaving group may be left to one of ordinary skill in the art. Commonly, halides are the most useful leaving groups in nucleophilic reactions, The hydroxyl group in carboxylic acids (-C(=OVOH) may be converted to a halide. For example, an acid

is formed by the reaction of a carboxylic acid with thionyl chloride or oxalyl chloride/DMF. Alternatively,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.

As used herein, the "linking moiety" A is also referred to as a "tether". These terms refer to the spacer molecule having the formula:

-L-y where 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. Typically 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. In formulas where A is already linked to Q, the group y is dropped from the formula, leaving A = -L-. Typically, y is chosen from the group consisting of hydroxy (-OH), thiol

(-SH), carboxy (-C(=O)OH), amino (-NH2 aldehyde (-CH(=O)), leaving group, Michael acceptor, phosphoramidite, phosphonate and protected forms of these functional groups. In synthesis, 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. 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 α,β-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. Thus, exemplary Michael acceptors include:

wherein a, b and c can independently be hydrogen, alkyl, or aryl, and wherein U is chosen from -CH(=O), -C(=O)Rt, -C(=O)NH₂, -CN, -NO2, -

S(=O)Rt, and -S(=O)2Rt.and Rt is again alkyl or aryl. A particularly preferred Michael acceptor is maleimide:

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. First, 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 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 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" as used herein 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. The term "detectable label" or "signalling" label or moiety is used when the intent is to differentiate from hapten-type labels or "hooks". The term "tracer" 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. Preferably, 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. In an FPIA, 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. In the present invention, the preferred fluorescent labels are fluorescein and fluorescein derivatives. These compounds provide fluorescent response when excited by polarized light of an appropriate wavelength and thereby enable the fluorescence polarization measurement. For example, 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.

The term "oligonucleotide" (sometimes abbreviated to "oligo") 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. Reagents

1 Haptens a. Structure of Haptens

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 (O₂)- . For subsequent use, the available bond to Z is occupied by a linker or a second molecule as described below.

b. Synthesis of Haptens

Hapten starting materials may be purchased (in some cases) or synthesized as described in the example section below, c. Tethered intermediates

The haptens are converted according to methods known to those skilled in the art using linker molecules x-L-y, (containing reactive functional groups x and y capable of coupling to complementary reactive groups on another molecule or macromolecule) to produce hapten intermediate compounds with a tether or sidechain, A= -L-y. Of course, 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. As is known in the art, it is sometimes desirable to activate a functional group on Q, and sometimes a second linker is used. Depending on the desired y group, it may also be preferred (for ease of synthesis) to interchange one y for another after the tether has been attached to the hapten.

Specific examples follow for various means for preparing desired intermediates and products. Alternative general activation/coupling schemes are given below.

The hapten (I) with an activated carboxyl group (B= -OH) is reacted with a linker or tether molecule x-L-y, wherein x is selected from -OH, -SH, and -NHR'-, R' being selected from H, alkyl, aryl, substituted alkyl and substituted aryl; wherein L is spacer group as defined above; and wherein y is chosen from the group consisting of hydroxy (-OH), thiol (-SH), carboxy (-C(=O)OH), amino (-NH2), aldehyde (-CH(=O)), leaving group, Michael acceptor, phosphoramidite, phosphonate and protected forms of these functional groups to form the tethered intermediate (H).

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. For example hapten (T)[ B=carboxy (-C(=0)OH)] may be activated with oxalyl chloride and DMF to produce the acid chloride [I, B= -C(=O)Cl]. Further reaction with the linker, methyl 6-aminocaproate

-CO2CH3] gives the tethered intermediate [II, A= - NH(CΗ2)5Cθ2CH3]. Saponification of the methyl ester with sodium hydroxide gives the tethered intermediate [II, A= -NH(CH2)5Cθ2H] which is ready to be activated and coupled to Q. Other specific examples may be found in the EXAMPLES section.

2 Immunogens a. Structure of Immunogens

Immunogens can be produced from a wide variety of tethered intermediates. The immunogens of the present invention have the following general structure:

wherein a and Z are as defined above; A is a tether as defined above except that the reactive function y has already been reacted with, Q, an immunogenicity conferring carrier. Typical carriers were previously described. Immunogens find principal use in the raising of antibodies. b. Synthesis of Immunogens

In the immunogens of the present invention, the tether functionality, y, of tethered intermediates (π) 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. Alternatively, 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. One of ordinary skill in the art will recognize that there are many reagents that can be used to form amide bonds other than those listed.

A tethered intermediate with a terminal amine functionality [II, y= -NH2] can be transformed into a highly reactive N-hydroxysuccinimide urethane by reaction with N,N'-disuccinimidyl carbonate in a suitable solvent, such as acetonitrile or dimethylformamide. The resultant urethane is then reacted with the immunogenicity conferring carrier in a buffered, aqueous solution to provide an immunogen. A tethered intermediate with a terminal aldehyde functionality [II, y=-

CH(=O)] can be coupled to the immunogenicity conferring carrier in a buffered, aqueous solution and in the presence of sodium cyanoborohydride, by reductive amination according the methods known to those skilled in the art.

Alternatively, tethered intermediates containing an alcohol group [II, y=- OH] can be coupled to the immunogenicity conferring carrier by first reaction it 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 the immunogenicity conferring carrier in a buffered, aqueous solution to provide an immunogen.

In a manner analogous to immunogens, 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.

2 Antibodies

Methods for antibody preparation are generally known and have been summarized above. Specific examples using the haptens and immunogens of the present invention are given in the EXAMPLE section below. 3 Tracers a. Structure of Tracers

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:

wherein a and Z are as defined above; A is a spacer/linker as defined above except that the reactive function y has already been reacted with, Q, a detectable label. As mentioned above, 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. For tracers of the present invention it is preferred that 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. b. Synthesis of Tracers

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. Or 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. Alternatively, 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.

Alternatively, 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:

wherein a and Z are as defined above; A is a tether as defined above except that the reactive function y has already been reacted with, Q, an oligonucleotide in this case. Oligonucleotides were previously defined. As noted below, 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. 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. b. Synthesis of Tethered Oligonucleotides

In the oligonucleotides of the present invention, 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.

For example a tethered hapten intermediate (II, y= phosphoramidite) where the phosphoramidite, y, is

is prepared from the tethered hapten by reaction with N,N-diisopropyl-0-(2- cyanoethyl)chlorophosphoramidite as described in Scheme I below.

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.

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. In one embodiment of the present invention synthesis of quinoline-labeled oligonucleotides is accomplished 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

, R²OH

4. Reduction

-W.

HR¹⁰N' O-DMT ₅ Dimethoxytritylation

OH 6. N-Deprotection

Phosphoramidite-linked Hapten 8

Alternatively, a tethered hapten intermediate (II, y= phosphonate) where the phosphonate is

is prepared from the tethered hapten (H, y=-OH] by reaction with phosphorous trichloride followed by hydrolysis. Both the phosphoramidite and phosphonate derivatives are readily incorporated into oligonucleotides during synthesis using standard protocols.

Alternatively, 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.

Of course, it is now fairly routine practice to make oligonucleotides by synthetic methods in automated synthesizers that are commercially available, for example Applied Biosystem's DNA Synthesizer 380B. B. FPIA Assay Methods

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;

2) a known concentration of tracer is then added to the tube;

3) a known, limiting concentration of analyte-specific antibody, produced using the immunogen as described above, is added to the tube;

4) the reaction mixture is incubated, wherein the tracer and analyte compete for limited antibody binding sites, whereby tracer-antibody and analyte- antibody complexes form; and

5) 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 ADx™ Abused Drug System, IMx® Fluorescence Polarization and Microparticle Enzyme Immunoassay (MEIA) Analyzer all of which are available from Abbott Laboratories, Abbott Park, Illinois. When the TDx, ADx, or IMx systems are used, the assays are fully automated from pretreatment to final reading once the test sample has been prepared. Manual assays, however, can also be performed. Although the principles of the invention are applicable to manual assays, the automated nature of the TDx , ADx and IMx systems assures minimal technician time to perform assays and interpret data. 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. For fluorescein tracers, 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 TDxFLx™ 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.

If a manual assay is being performed, 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, TDxFLx™ 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.

With respect to the foregoing preferred procedure, it should be noted that 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.

C. Methods of Use

Methods of using the novel haptens, tethered intermediates, immunogens, solid supports and tracers have each been described above. 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. Each of the above-mentioned disclosures is incorporated herein by reference.

The invention will now be described by way of examples which are intended to illustrate but not limit the invention.

TV. EXAMPLES

All materials are from Aldrich Chemical, Milwaukee, WL, and all percentages expressed herein are weight/volume unless otherwise indicated. Unless context demands otherwise, the following abbreviations have the meanings given:

ACA aminocaproic acid

AMForAMF aminomethyl fluorescein, a fluorophore

BAE 3 carboxypropyloxy radical

BSA Bovine Serum Albumin, an immunogenicity conferring carrier.

Cbz Carboxybenzyl, an amino protecting group

Celite® A trademark of Manville Products Corporation, for diatomaceous earth

CDI 1,1' caibonyldiimidazole, a coupling reagent

DCC dicyclohexylcarbodimide

DEAD or DE ADC diethylazodicarboxylate

DIEA diisopropylethylamine

DMAP 4-(N,N-dimethylamino)pyridine

DMEM Dulbecco's Minimum Essential Medium, a cell culture medium.

DMF N,N-dimethylformamide

DMT Dimethoxytrityl, a hydroxy protecting group

EBB ethyl 4-bromobutyrate

EEDQ 2-ethoxy-l-ethoxycarbonyl-l^-dihydroquinoline glyme l_2-dimethoxyethane

HNQ 8-hydroxy-5-nitroquinoline

HOSu hydroxysuccinimide

KLH keyhole limpet hemocyanin, an immunogenicity conferring carrier

NHS N-hydroxysuccinimide MP N-methylpyrrolidiπone

PEG polyethylene glycol

TBAF tetrabutylammonϊum fluoride

TEA triethylamine

THF tetrahydrofuran A. Synthesis of Haptens, Tethers, and Tethered Haptens

Example 1: 8-[3-(f-butyldimethyIsiloxy)]-propyloxy-5-nitroquinoline (F174), a hapten:

3-(t-Butyldimethylsiloxy)-l-propanol (F168): 1,3-Propanediol (20.0 g,

169 mmol) was dissolved in dry dichloromethane (200 mL). To this solution was added diisopropylethylamine (29mL, 166 mmol) and the solution cooled in an ice bath. A solution of t-butyldimethylsilyl chloride (17 g, 113 mmol) in dry dichloromethane (lOOmL) was added dropwise. After 18h, the solvent was removed in vacuo an the residue purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:9) giving 17 grams. Mass spectrum: CI, NH3/GC; (m+H)+ @ m/z 191, (m+NH4)+ @ m z 208, and (m+H-H₂O)+ @ m z 173.

8-Hydroxy-5-nitroquinoline (HNQ, 5.0g, 26mmol), triphenylphosphine (8.3g, 32mmol), and tert-butyldimethylsiloxy-3-hydroxypropane (5.0g, 26mmol; from part a above) were dissolved in dry tetrahydrofuran (THF, 50mL). The solution was cooled in an ice bath and then diethylazodicarboxylate (DEADC,6.2mL, 32mmol) was added over 30 minutes. The reaction was removed from the ice bath and heated to 60°C. After 18h the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate/hexanes, 8:2 then t-butylmethyl ether/methylene chloride, 5:95) giving the pure product (7.6g). Mass spectrum: DCI/NH3, (m+H)+ @ m/z 363. Example 2: 8-(3-hydroxypropyloxy)-5-nitroquinoline (F178), a hapten:

The product from example 1 (F174, 7.5g, 20.7mmol) was dissolved in methylene chloride and then cooled in an ice bath. To this solution was added tetrabutylammonium fluoride (TBAF, l.OM in THF, 41mL, 41mmol). The reaction was removed from the ice bath and stirred at ambient temperature for several hours.

The solvent was removed under reduced pressure and the product purified by column chromatography. Mass spectrum: DCI/NH3, (m+H)+ @ m/z 249.

Example 3: 8-[0-(4-oxa-5-oxo-7-carboxyheptyl)]-5-nitroquinoIine (F179), a hapten:

8-[3-Hydroxypropyloxy]-5-nitroquinoline from example 2 (F178, l.Og, 4mmol), and succinic anhydride (0.4g, 4mmol) were dissolved in pyridine (lOmL) and stirred for 18h at ambient temperature. Another equivalent (0.4g) of succinic anhydride was added and the reaction stirred for another 18h before isolating the product. Mass spectrum: DCI/NH3, (m+H)+ @ m/z 349. Exampie 4: 8-oxymethanesulfonyI-5-nitroquinoIine (F163), a hapten:

8-Hydroxy-5-nitroquinoline (l.Og, 5.3mmol) and N,N- dimetiiylaminopyridine (DMAP, 0.064g, 0.53mmol) were dissolved in pyridine (2mL). To this solution was added methanesulfonyl chloride (0.448mL). After several hours, acetonitrile was added to dissolve the solid material. After an additional hour, the reaction was poured into ice water (50mL). The solution was extracted with ethyl acetate, and the solvent removed under reduced pressure. Purification by column chromatography (silica gel, ethyl acetate/hexanes, 1:1) gave yellow crystals. Mass spectrum: DCI/NH3, (m+H)+

@ m z 269.

Example 5: 8-[ N-(3-hydroxypropyl)-amino]-5-nitroquinoIine (F173), a hapten:

A small amount of 8-oxymethanesulfonyl-5-nitro-quinoline from example 4 (F163) was dissolved in dry tetrahydrofuran and to this solution was added a drop of 3-aminopropanol. After 18h at ambient temperature, the product was isolated by preparative TLC. Mass spectrum: DCI/NH3, (m+H)+ @ m/z

248, (m+NH4)⁺ @ m/z 265. Example 6: 8-[ N-(5-carboxypentyl)-suIfonamide] quinoline (F245), a hapten:

8-Quinolinesulfonyl chloride (QSC1, 2.0g, 8.8mmol), methyl-6- aminocaproate hydrochloride (1.6g, 8.8mmol), and diisopropylethylamine (DIEA, 4 mL, 26mmol) were mixed in methylene chloride (30mL). After several hours of stirring at ambient temperature, the solvent was removed under reduced pressure, water was added, and the residue extracted with ethyl acetate. The solvent from the extracts was removed and the residue purified by column chromatography (silica gel, ethyl acetate/hexanes, 20-40%) giving pure material (2.1g) as the methyl ester.

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.

Example 7: 8-[ N-(2-3 dihydroxypropyl)-sulfonamide] quinoline (F245), a hapten:

OH 8-Quinolinesulfonyl chloride (2.0 g, 8.8 mmol) was added to a solution of 3- amino-l,2-dihydroxypropane (0.8 g, 8.8 mmol) in a mixture of 10% aqueous sodium carbonate (15 ml) and tetrahydrofuran (15 ml). After 8 h, the organic layer was separated and the aqueous layer washed with ethyl acetate (20 ml). The combined organic layers were dried over anhydrous sodium sulfate and the solvent removed in vacuo. The yield was 1.8 g of product as a clear glass. Mass spectrum: DCI/NH3, (m+H)+ @ m/z 283.

Example 8. Preparation of 6-amino-l,3-hexanediol, a tether. Step a: Protection of the 1.2 diol function

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

A 250 mL round-bottom flask, charged with oxalyl chloride (2.5 mL, 29 mmoles) in methylene chloride (65 mL) was fitted with two addition funnels; one charged with DMSO (4.0 mL, 57 mmoles) in methylene chloride (15 mL), and the other charged with alcohol 37649-100 (5.0 g, 26 mmoles) in methylene chloride (25 mL). While under nitrogen atmosphere, the flask was cooled to - 78°C and the DMSO was added dropwise over 9 min. After an additional 5 min. the alcohol was added over 30 min. The flask was removed from the cold bath until the precipitate dissolved (about 10 min.), and then recooled. After 15 min., triethylamine (18 mL, 130 mmoles) was added dropwise over 10 min. The flask was removed from the cold bath and after 20 min. 10% aqueous citric acid (125mL) was added. The solution was extracted with methylene chloride (3xl00mL), and the organic layers combined, dried with magnesium sulfate, and the solvent removed under reduced pressure. Drying over phosphorous pentoxide for 18h gave 4.9 g (99%) of the aldehyde which was used without further purification. Step c: Introduction of an amine function into the aldehyde

Diethyl cyanomethylphosphonate (4.33 mL, 27 mmoles) in THF (25 mL) was added dropwise, under nitrogen atmosphere, to a suspension of sodium hydride (0.803 g, 27 mmoles, 80% suspension in mineral oil) in THF (25 mL). After 3h, the aldehyde (4.9 g, 25 mmoles) in THF (20 mL) was added dropwise to the anion. After stirring at room temperature for 18h, the solvent was removed under reduced pressure and the residue dissolved in methylene chloride and washed with water. Some of the methylene chloride was removed and the solution added to a flash chromatography column (silica gel, 200-400 mesh), and eluted with ethyl acetate/hexanes, 2:8. The resulting compound (3.4 g) was hydrogenated with Pt(IV) oxide (0.7 g), and di-tert-butyl dicarbonate (4.0 mL, 1.6 mmoles) in THF (40 mL). After recovering some of the catalyst, the residue was purified by flash chromatography (silica gel, 200-400 mesh), eluting with ethyl acetate/hexanes, 2:8. giving 2.0 g (40%) of the protected aminoalkanediol. The resulting compound (2.0 g, 6.2 mmoles) was dissolved in methylene chloride (20 mL, distilled from P2O5) and then trifluoroacetic acid (5 mL) was added with stirring, under a nitrogen atmosphere. After several hours, the solvent was removed under reduced pressure and residual TFA was removed by azeotroping with toluene giving 6-amino-l,3-hexanediol hydrochloride as an oil (2.0 g) which was used in the next step without further purification. A generalized method for obtaining such a diol is found in Scheme I, above.

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. The organic layer was separated and die aqueous layer extracted with ethyl acetate (2x10 mL). The combined organic layers were dried over sodium sulfate and the solvent removed under reduced pressure. The residue was purified by flash chromatography, eluting with ethyl acetate. The yield of the tethered hapten was 0.40 g for the following structure:

Example 10. 6-[N-(8-quinolinesulfonyl)aminoH-(0-4,4'-dimethoxytrityl),3- hexanediol, a tethered hapten.

A generalized phosphoramidation and deprotection of a tethered hapten is described in steps 7 and 8 of Scheme I, described above. 6-[N-(8- quinolinesulfonyl)amino]-l,3-hexanediol (0.32 gram, 1.0 mmoles) is dissolved in dry pyridine (3.5 mL) and then 4,4'-dimethoxytrityl chloride (0.40 gram, 1.2 mmoles), diisopropylethyl amine (0.24 mL, 1.4 mmoles), and DMAP (6 gram) were added. The solution was stirred at room temperature, under nitrogen atmosphere, protected from light, for 18h. The solvent was removed under reduced pressure and the residue purified by flash chromatography, eluting with ethyl acetate/hexanes, 1:1. The yield was 0.50 gram (85%) for:

Example 11. 6-[N-(8-quinolinesulfonyI)amino]-l-[0-(4,4'- dimethoxytrityl)],3-{[0-(0-(2-cyanoethyl)-N,N-diisopropylphosphoryl)]}- hexanediol, a tethered hapten. 6-[N-(8-quinolinesulfonyl)amino]- l-(0-4,4'-dimethoxytrityl),3- hexanediol (0.45 gram, 0.76 mmoles) was dissolved in methylene chloride (7mL) and then diisopropylethyl amine (1.0 mL, 5.7 mmoles), and chloro-N,N- diisopropylamino-b-cyanoethoxyphosphine (0.50 mL, 2.2 mmoles) were added. The nitrogen sealed reaction was kept at room temperature and protected from lightfor 2h.. Dry methanol (0.10 mL) was added to die reaction and after 30 min. ethyl acetate (25 mL) and TEA (5 mL) were added, the solution washed witii 10% sodium carbonate (2x25 mL), saturated brine (2x25 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The residue was dissolved with TEA (1 mL) and minimum methylene chloride, then applied to a flash chromatography column. Elution was with ethyl acetate/hexanes/TEA, 1:1:0.02. The yield was 460 mg, (78%) of

Example 12. 4-HydroxyprolinoI-DMT, a tether.

Step a: Protection of the amino function of hvdroxyproline.

Into a 3 L round-bottom flask, trans-4-hydroxy-L-proline (100 g, 763 mmole) was dissolved in water (IL) and sodium carbonate was added (323 g, 3 moles). To this solution was added tetrahydrofuran (800 mL) and then 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. The filtrate was extracted witii ethyl acetate (4 x IL), and die combined organic layers dried over anhydrous sodium sulfate. The solution was filtered and the solvent removed in vacuo giving N-CbZ-hydroxyproline as a clear, glassy material (160 g). 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. After the addition, the funnel was replaced with a water-cooled condenser and the reaction heated to gentle reflux. After 19 h, the reaction was allowed to come to ambient temperature and methanol (250 mL) was added very slowly. Solvent was removed in vacuo and any borane complex removed by four coevaporations with methanol (IL each). The residue was purified by flash chromatograhpy (900 g of silica gel, 230-400 mesh, eluted with ethyl acetate) giving 125 g of N-CBZ-hydroxyprolinol as product. Step c: Protection of the primary alcohol moiety

In a IL round-bottom flask, 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). Uder an inert atmosphere the reaction flask was cooled in an ice bath and then the addition was started. The addition was completed in 30 minutes and the ice bath was removed from the reaction. After 18 h, the solvent was removed in vacuo and therasidue purified by flash chromatography (980 grams of silica gel, 230-400 mesh, elution with ethyl acetate/ hexanes/ triethylamine, 6:4:0.5). The yield of N-CbZ-hydroxyprolinol- DMT was 26 grams (67%).

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.

Quinoline-8-sulfonyl chloride (1.0 eq) is dissolved in dichloromediane and added to a solution of 4-hydroxyprolinol-DMT (1.0 eq) in dichloromethane plus triethylamine (2.0 eq) at -15°C. When the adddition is complete, the reaction is allowed to come to room temperature. Stirring is continued under inert atmosphere until TLC indicates the completion of the reaction. The solvent is removed in vacuo and the resulting residue (quinoline-hydroxyprolinol-DMT) purified by flash chromatograhy using silica gel. The residue (1.0 eq) is dissolved in freshly distilled dichlorometiiane and then diisopropylethylamine (4.0 eq) is added. To this solution is added 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (1.4 eq) under a dry inert atmosphere. The reaction is stirred at 15-30°C for 1 hour then dry methanol (1.0 eq) is added. The reaction is stirred for an additional 5 minutes then die solution is transferred to a separatory funnel using ethyl acetate/triethylamine (9:1.5). The solution is washed with 10% sodium carbonate tiien with concentrated brine. The solution is dried over anhydrous sodium sulfate and the solvent removed in vacuo. The residue is purified by silica gel flash chromatography.

B. Synthesis of Immunogens

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).

Example 15: Quinoline BSA immunogen (G53):

BSA (0.5g) was dissolved in sodium phosphate buffer (lOmL, pH8, 0.05M) men NMP (5mL) was added. 8-Quinolinesulfonyl chloride (0.057g) was dissolved in NMP (lmL) then added to the protein solution. The reaction was mixed by rotation for 18h at ambient temperature then dialyzed against sodium phosphate buffer (pH8, 0.1M, 3x6L), and against distilled water (3x6L). After lyopholization, the immunogen was a powder (0.44g).

Example 16: Alkoxy quinoline KLH immunogen (F144):

8-[3-Hydroxypropyloxy]-5-nitroquinoUne from example 2 (F178, 0.9g, 3.6mmol) was stirred in a sealed flask with a phosgene solution (IN in toluene, 20mL) for 18h at ambient temperature. The solvent was removed under reduced pressure giving the chloroformate. KLH (O.lg) was dissolved in sodium phosphate buffer (pH8, 0.05M, lOmL) then NMP (3mL) was added. To this solution was added the chloroformate (0.05g) which had been dissolved in NMP (lmL). The reaction was stirred for 18h at ambient temperature then dialyzed against 25% ethanol in sodium phosphate buffer (pH8, 0.1M, 2x4L), then against distilled water (4L). Lyopholization gave a brownish solid.

Example 17: Sulfonamide quinoline KLH immunogen (F224):

Preparation of active ester: 8-[N-(5-carboxypentyl)-sulfonamide] quinoline (0.161g, 0.5mmol), from example 6 (F245), and N-hydroxysuccinimide (HOSu, 0.069g, 0.6mmol), were dissolved in N-methylpyrrolidinone (NMP, lmL), then N,N'-dicyclohexylcarbodiimide (DCC, 0.113g, 0.55mmol) was added.After several hours, the reaction was filtered and more HOSu (0.069g, O.όmmol) and DCC (0.113g, 0.55mmol) were added. The reaction was stirred at ambient temperature for 18h then the solvent was removed under reduced pressure. The residue was dissolved in methylene chloride and purified by column chromatography (silica gel, ethyl acetate/methylene chloride, 1:1). The pure active ester was isolated and dissolved in NMP (lmL)

Conjugation with carrier: 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).

Example 18: Alkoxy quinoline BSA immunogen (G50):

Preparation of active ester: 8-[N-(5-carboxypentyl)-sulfonamide] quinoline (0.101 g), from example 6 (F245), and N-hydroxysuccinimide (HOSu, 0.054 g), were dissolved in N-methylpyrrolidinone (NMP, 1.5 mL), then N,N'- dicyclohexylcarbodiimide (DCC, 0.084 g) was added. The reaction was stirred at ambient temperature under an inert atmosphere for 18h The solution was then filtered to remove N,N'-dicyclohexylurea. Conjugation with carrier: BSA (0.5g) was dissolved in sodium phosphate buffer (lOmL, pH8, 0.05M) then NMP (5mL) was added. 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). C. Synthesis of Tracers

Example 19: Quinoline Fluorescent tracer (F247):

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). after 18h at ambient temperature, the solvent was removed under reduced pressure and the residue purified by preparative TLC (reverse phase, C18, methanol/water/acetic acid, 6:4:0.1) giving die tracer shown below: The tracer had an FAB mass spectrum with (m+H)+ @ m/z 666, (m+Na)+ @ m z 688.

Example 20: Quinoline Fluorescent Tracer (F248a):

Quinoline-8-(5-carboxypentyl)-sulfonamide (0.05g, O.lόmmol), 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.

5-Aminomethylfluorescein hydrobromide (0.01 g) was dissolved in NMP (O.lrnL) plus DIEA (0.012mL). To this solution was added the active ester solution (0.15mL) from part a. The reaction was stirred at ambient temperature for 48h, the solvent was removed under reduced pressure, and the residue was purified by preparative TLC (reverse phase, CI8, methanol/water, 6:4) giving the tracer shown below: The tracer had an FAB mass spectrum with (m+H)⁺ ions at m/z 666 and (m+Na)+ ions at m/z 688.

Example 21: Quinoline Fluorescent Tracer (F248b):

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.

4'-Aminomethylfluoroscein hydrochloride (0.009g) was dissolved in NMP

(O.lmL) plus DIEA (0.012mL). To this solution was added the active ester solution

(0.15mL) from part a. The reaction was stirred at ambient temperature for 48h, the solvent was removed under reduced pressure, and the residue was purified by preparative TLC (reverse phase, C18, methanol/water, 6:4) giving the tracer shown below: The tracer had an FAB mass spectrum with (m+H)+ ions at m/z 666 and

(m+Na)+ ions at m/z 688.

Example 22: 6-(N-Dansylamino)-l-3-hexanedioI.

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%).

D. Production ofAntisera Example 23: Antisera

Approximately four to five month old female New Zealand White rabbits were injected subcutaneously and intramuscularly with an initial inoculation of 0.2 mg of the immunogen from example 14 (F252) in Freund's Complete Adjuvant followed by a day 14 boost of 0.1 mg of the immunogen and tiiereafter mondily booster injections of 0.05 mg in Freund's Incomplete Adjuvant. Bleeds were taken two weeks following each booster injection and the serum tested for binding to tracers in the TD_X instrument. Antibodies with adequate net millipolarization and span were shown in some bleeds 6 weeks from initial inoculation.

Example 24: Example 23 is repeated using the immunogens of example 15-18.

E. Production ofHybridomas 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 (Ribϊ Immunochem Research, Lie). Three months from initial inoculation, upon testing positive for antibody activity 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). 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.

An immunoglobulin non-secreting mouse myeloma cell line (SP 2/0) deficient in the enzyme hypoxanthine-guanine phosphoribosyl transf erase

(HGPRT-, EC2.4.2.8), as disclosed by Kearney, Journal of Immunology, 1979, 725,1548, which is incorporated herein by reference, is used as die fusion partner. The myeloma cell line is maintained in medium A with 20% fetal calf serum added. For three days prior to fusion, 0.1 mM 8-azaguanine is added to the myeloma cells in order to kill any HGPRT+ revertants. On the day of fusion, 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

Cell Genetics, 1911, 3, 231, which is hereby incorporated by reference. To a sterile 50 mL conical centrifuge tube is added 1-1.5x10^ spleen cells witii an equal number of SP 2/0 myeloma cells. The myeloma-spleen cell suspension is centrifuged at 1400 rpm for 5 minutes to pellet the cells together. The supernatant is aspirated off and the tube tapped gently to loosen the cell pellet and 1 mL of 50% polyethylene glycol (PEG, MW 1000, Sigma) in DMEM, without serum, is added to the cell pellet. 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.

Example 26: Selection of Hybridomas Producing Monoclonal Antibodies

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. During ti is period, on alternate days, 1 mL volumes of medium is removed from each well by aspiration and replaced witii 1 mL of fresh medium C. On day 10 die supematants from the hybridoma containing wells are tested for antibody activity in the TDx instrument using selected tracers (Examples 13-15), 25 mL of hybridoma supernatant. Five hybridoma suspensions are chosen for further cloning by picking those supematants 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 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 28: In Vivo Production of Monoclonal Antibodies

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. Approximately 4-5 weeks following the pristane injection, 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 approximately 24.6 mg of antibody is obtained per mL of ascites fluid.

Example 29: Quinoline Fluorescence Polarization Immunoassays

As described previously, the reagents for the FPIA of the present invention comprise tracers and antibodies raised against immunogens of the present invention, specific for tethered intermediates. In addition, conventionally used assay solutions including a dilution buffer, and quinoline derivative calibrators and controls are prepared.

The preferred procedure is designed to be used in conjunction with the automated TDx, ADx, or IMx systems; however, manual assays can also be performed. In both procedures, the 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.

In the automated assays, 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.

1) the pretreatment solution

2) the tracer diluted in 50% methanol in potassium phosphate buffer (0.15 M phosphate buffer, pH 7.5). 3) 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;

4) a diluent buffer comprising TDx buffer, 5) a sets of calibrators

6) controls comprising 5 mg mL quinoline derivatives All polarized fluorescent measurements are made using the TDx instrument which performed the assay in accordance with the following protocol:

1) 22.5 mL of standard or unknown test sample and 12.5 mL each of the antibody reagent and die pretreatment reagent are delivered into the cuvette and a sufficient volume of diluent buffer is added to raise the volume to 1 mL, and a background intensity reading is taken;

2) 12.5 mL each of pretreatment reagent and antibody, 25 mL of die tracer, and the second 22.5 mL of sample and are added to the cuvette, and a sufficient volume of diluent buffer is added to raise the volume to 2.0 mL;

3) the reaction mixture is incubated;

4) 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.

Claims

WHAT IS CLAIMED IS:

1. A compound having the following structure:

wherein 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); Z is -0-, or -S (0₂)- ; 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.

2. A compound according to claim 1 wherein a is other than hydrogen and is at position 5.

3. A compound according to claim 1 wherein Z is oxygen.

4. A compound according to claim 3 wherein -L- is alkyl of from 1- 6 carbon atoms with a functional y group selected from me group consisting of hydroxyl (-OH), thiol (-SH), carboxy (-C(=O)OH), amino (-NH2), aldehydo, leaving group, Michael acceptor, phosphoramidite, phosphonate and protected forms of these functional groups.

5. A compound according to claim 4 wherein -L-y is -(CH2)nOR wherein n is from 1-10 and R is selected from the group consisting of hydrogen, trimethylsilyl, and -C(0)(CH2)nCO2H wherein n is from 1-10.

O

6. A compound according to claim 1 wherein Z is 11 O

7. A compound according to claim 6 wherein L- is alkyl of from 1-6 carbon atoms or alkylamino wherein the alkyl portion is from 1-6 carbon atoms and y is selected from the group consisting of hydroxyl (-OH), thiol (-SH), carboxy amino (-NH2), aldehydo, leaving group, Michael acceptor, phosphoramidite, phosphonate and protected forms of these functional groups.

8. A compound according to claim 6 wherein the atom of A bonded to the S is a nitrogen atom, thereby forming a sulfonamide.

9. A compound according to claim 8 wherein -L-y is selected from the group consisting of -NH(CH2)n(CHOH)2 and -NH(CH2)_nCθ2H wherein n is from 1-10.

10. A compound according to claim 2 wherein -L-y is -(CH2)nOR wherein n is from 1-10 and R is selected from the group consisting of hydrogen, trimethylsilyl, and -C(0)(CH2)nCO2H wherein n is from 1-10..

11. A compound according to claim 2 wherein -L-y is selected from the group consisting of -NH(CH2)n(CHOH)2 and -NH(CH2)_nC02H wherein n is from 1-10.

12. A compound according to claim 2 wherein y is a phosphoramidite or a phosphonate.

13. A compound according to claim 4 wherein y is a phosphoramidite or a phosphonate.

14. A compound according to claim 8 wherein y is a phosphoramidite or a phosphonate.

15. A conjugate compound having the following structure:

wherein 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-CH2C5H4-NO2);

Zis -0-, or -S (0₂)- ;

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; and wherein Q is an immunogenicity conferring carrier molecule, a detectable label, an oligonucleotide or a solid support.

16. The conjugate of claim 15, wherein Q is an immunogenicity conferring carrier.

17. The conjugate of claim 16, wherein Q is selected from the group consisting of BSA, KLH, thyroglobulin, and ovalbumin.

18. The conjugate of claim 15, wherein Q is a detectable label.

19. The conjugate of claim 18, wherein said detectable label is a fluorescent molecule.

20. The conjugate of claim 19, wherein said fluorescent molecule is a fluorescein derivative selected from the group consisting of 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- 1 ,3,5-triazin-2-yl- aminofluorescein, fluorescein isothiocyanate, and a dansyl chloride.

21. The conjugate of claim 20, wherein said detectable label is an aminomethylfluorescein or a dansyl group.

22. The conjugate of claim 15, wherein Q is a solid support.

23. The conjugate of claim 22, wherein said solid support is selected from the group consisting of beads, tubes, rods, microtitre plates and columns.

24. The conjugate of claim 15, wherein Q is an oligonucleotide.

25. The conjugate of claim 24, wherein said oligonucleotide is a deoxyribonucleotide from about 10 to about 100 bases in length.

26. A method of preparing an oligonucleotide conjugate, comprising the steps of: a) obtaining or preparing a compound according to claim 1 having a reactive y moiety; and b) reacting the compound of step a with an oligonucleotide under conditions such that a conjugate is formed between the moiety y and a functional amino or hydroxyl group of the oligonucleotide.

27. The method according to claim 26 wherein the moiety y of the compound of step a is a phosphoramidite or phosphonate moiety; and in step b, forming a conjugate between the phosphoramidite or phosphonate moiety and a hydroxyl group of the oligonucleotide.

28. A method of preparing an oligonucleotide conjugate, comprising die steps of: a) obtaining or preparing a compound according to claim 1; and b) reacting a compound of step a with an oligonucleotide under conditions such that a conjugate is formed between the moiety y and a phosphorous on the oligonucleotide.

29. A method of detecting a target nucleic acid, comprising: a. mixing a hapte oligonucleotide conjugate according to claim 24 with die target nucleic acid under conditions promoting hybridization, said conjugate having an oligonucleotide component, Q, which is complementary to the target nucleic acid; b. separating unhybridized conjugate from hybridized conjugate; and c. detecting die amount of hybridized hapte oligonucleotide conjugate; wherein at least one of said separating and detecting steps is performed using a specific binding member for said hapten.

30. The method according to claim 29 wherein separation is performed using a solid phase to which is immobilized said specific binding member for said hapten.

31. The method according to claim 29 wherein detection is performed using a conjugate of a detectable label and said specific binding member for said hapten.

32. The metiiod according to claim 29, wherein die target nucleic acid is amplified prior to said separating step.

33. A kit comprising: a. at least one oligonucleotide probe comprising a haptenroligonucleotide conjugate according to claim 24; and b. an antibody reactive with said hapten, said antibody being attached to a solid support or a detectable label, or being adapted for attachment to a solid support or a detectable label.

34. The kit according to claim 33, further comprising at least one second oligonucleotide probe comprising an oligonucleotide conjugated to a second, different hapten; and a second antibody reagent comprising an antibody reactive with the second hapten attached to or adapted for attachment to the otiier of said solid support and detectable label.

35. A kit comprising: a. at least one tediered intermediate compound according to claim 1, said intermediate being useful for labeling a biological macromolecule; and b. an antibody reactive with said hapten, said antibody being attached to a solid support or a detectable label, or being adapted for attachment to a solid support or a detectable label.

36. The kit according to claim 35 wherein said tethered intermediate compound comprises a y moiety selected from the group consisting of a phosphoramidite and a phosphonate.

37. An antibody reactive with a compound of the formula:

wherein 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);

Z is -0-, or -S (0₂)- ; and Z is -0-, or -S (0₂)- ; 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; said antibody being raised by injecting an animal with the immunogen of claim 16.

38. An antibody according to claim 37, wherein the antibody is a monoclonal antibody.