PatentDe  


Dokumentenidentifikation EP0880029 22.02.2001
EP-Veröffentlichungsnummer 0880029
Titel Immunoassay für Glucuronid-Metabolite von LSD
Anmelder Microgenics Corp., Pleasanton, Calif., US
Erfinder Coty, William A., Davis, US;
Shindelman, Jeffrey E., Concord, US;
Morrill, Paul R., Springfield Road, Cambridge, CB4 1HY, GB
Vertreter Rechts- und Patentanwälte Lorenz Seidler Gossel, 80538 München
DE-Aktenzeichen 69800486
Vertragsstaaten CH, DE, ES, FR, GB, IT, LI
Sprache des Dokument EN
EP-Anmeldetag 20.05.1998
EP-Aktenzeichen 983040130
EP-Offenlegungsdatum 25.11.1998
EP date of grant 17.01.2001
Veröffentlichungstag im Patentblatt 22.02.2001
IPC-Hauptklasse G01N 33/94
IPC-Nebenklasse C07K 16/16   C07K 16/44   

Beschreibung[en]
RELATED APPLICATIONS

This application claims the priority benefit of U.S. Provisional Application 60/047,256 (Sigler et al.) filed May 21, 1997; and U.S. Application 09/026,780 (Sigler et al) filed February 20, 1998.

BACKGROUND OF THE INVENTION

Although there is widespread public perception that use of LSD is no longer a societal problem, there is considerable evidence that this illicit drug continues to be used, and in some segments of the population, its use is increasing. See Bonner, Drug Detection Report. 1:5 (1992). LSD was one of the 20 controlled substances most commonly encountered in emergency rooms across the nation in 1985, reflecting continuing abuse and trafficking of this illicit drug. In the United States, seizures of LSD by the Drug Enforcement Agency doubled in 1990 over the previous year, and in England, seizures of LSD have steadily increased since mid-1988. See Microgram 23:228 (1990). Further cause for concern are reports that LSD is particularly popular among adolescents, and in some areas, it exceeds cocaine in popularity. See Seligmann, Newsweek, February 3rd, p. 66, (1992). Factors that have contributed to the continued use of LSD are its wide availability, low cost, and the difficulty of detecting LSD use by analysis of body fluids.

Despite the long history of abuse associated with LSD, little is known concerning the disposition of LSD in humans. The lack of pharmacokinetic data on LSD is partly due to the technical difficulty of detecting and measuring the drug in physiological specimens. LSD is not considered highly toxic, although at least two cases where death was apparently a result of LSD toxicity have been reported. However, the major reason many consider LSD to be highly dangerous is that it can have serious psychological and psychotic effects which sometimes cause users to commit irrational acts resulting in injury or death. LSD is an extremely potent psychedelic drug that acts primarily on the central nervous system; only the d-isomer of the drug is pharmacologically active. Oral doses as low as 25 µg can cause central nervous system disturbances such as hallucinations, distortions in sensory perception, mood changes and dream-like thought processes, as well as psychotic reactions in apparently predisposed individuals. Therefore, concentrations of LSD and LSD metabolites in blood and urine are likely to be very low. The detection of LSD in body fluids of users is especially difficult because the quantities typically ingested are very small and because the drug is rapidly and extensively converted to metabolic products. Furthermore, the drug's low volatility, its thermal instability, and its tendency to undergo adsorptive losses during gas chromatographic analysis all contribute to the difficulty of developing a method for confirmation of LSD in body fluids.

LSD is a natural product of the rye fungus Claviceps and was first prepared synthetically in 1938. Its psychological effects were discovered following accidental ingestion. Chemically, LSD is an ergot alkaloid and, like other compounds of this class, contains lysergic acid as the basis of its structure. Structurally similar to serotonin (5-hydroxytryptamine), LSD is thought to exert its psychotomimetic effects through antagonism of serotonin activity in the brain stem. Little is known about the tissue distribution, metabolism and excretion of LSD in humans. LSD is absorbed fairly rapidly by the gastrointestinal tract, and its plasma half-life has been calculated to be about 3 hours in man. Animal studies indicate that LSD is inactivated via hepatic oxidation. It is extensively metabolized with only negligible amounts of unchanged drug appearing in the urine and feces, with most of the metabolites being excreted in the urine. Possible metabolic transformations may be hydrolysis to lysergic acid, N-demethylation to nor-LSD and oxidation to 2-oxo-LSD. Studies with urine samples from human volunteers receiving LSD demonstrate that the drug or its closely related metabolites can be detected in the urine by radioimmunoassay (RIA) for several days following administration.

Although continued illicit use of LSD has stimulated efforts to develop effective analytical methods for the detection of the drug and its metabolites in body fluids from suspected LSD users, the methods currently available are complicated, time-consuming, expensive to perform and plagued by other problems. These methods include high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS) and radioimmunoassay. One problem faced by laboratories involved in the determination of LSD is the strong tendency for LSD and derivatized LSD to undergo adsorptive losses when subjected to gas chromatography. This behavior often prevents detection of the drug at the sub-nanogram/milliliter concentrations normally encountered in body fluids from LSD users.

Commercial RIAs for LSD are available from several sources, including ABUSCREEN LSD assay (® Roche Diagnostics Systems, Nutley, NJ) and COAT-A-COUNT LSD assay (® Diagnostic Products Corp., Los Angeles, CA), and these products serve as a useful and relatively inexpensive method of screening for the presence of the drug. However, RIAs are not totally specific for LSD, so that an RIA-positive specimen still has to be confirmed by a second and more specific assay if the results of the analysis could have punitive consequences. The manufacturers' recommended cut-off concentration for considering a sample positive for LSD is 0.5 ng/ml, although lower cutoffs have been used in investigations where legal consequences were not a concern. The actual concentration of LSD in RIA-positive urine specimens is generally lower than that indicated by the RIA, and often considerably lower. Presumably the higher concentrations indicated by RIA are due to the cross-reactivity of LSD metabolites to the RIA antisera, but this conclusion cannot be substantiated until the major LSD metabolites in urine have been identified and their cross-reactivities determined.

In testing for other drugs of abuse, immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as β-galactosidase and peroxidase, fluorescent molecules such as fluorescent compounds, and radioactive compounds such as 125I are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.

One type of competitive binding immunoassay is based upon the reassociation of enzymatically inactive polypeptide fragments to form active enzyme as a step of generating a detectable signal utilized to determine the amount of analyte present in a sample. This type of assay, known as cloned enzyme donor immunoassay (CEDIA), is described in U.S. Patent No. 4,708,929. In particular, a β-galactosidase enzyme donor polypeptide combines with a β-galactosidase enzyme acceptor polypeptide to form active β-galactosidase enzyme. Conjugating a hapten, or a small analyte or an analyte analog, to the enzyme donor polypeptide at certain sites does not affect the ability to form active β-galactosidase by a complementation reaction and hence does not affect the rate of β-galactosidase activity when in the presence of a substrate for β-galactosidase. However, when the enzyme donor-hapten conjugate is bound by anti-analyte antibody, the complementation rate is impeded, and thereby the enzyme-catalyzed reaction rate during the initial phase of the reaction is reduced. This reduction in enzyme-catalyzed reaction rate can be monitored and has been used successfully to determine a plurality of analytes using the principle of competitive inhibition whereby enzyme donor-analyte conjugate present in a reaction mixture and analyte present in a sample compete for anti-analyte antibody prior to the addition of enzyme acceptor. The complementation rate of β-galactosidase formation, and hence enzyme-catalyzed reaction rate, is increased as the amount of analyte present in the sample is increased.

For the development of non-isotopic immunoassays which detect LSD and LSD metabolites, defined hapten derivatives are needed for preparation of immunogens and labeled conjugates. In particular, hapten derivatives at the 1-position of the lysergamide moiety are desirable because generally accepted strategy for preparing immunogens involves attachment of a hapten to the carrier substance at a position that is distant from the site in the molecule where immuno-specificity is desired, i.e., a site on the other side of the molecule from the ethyl side chains at the 8-position.

The preparation of antibodies to LSD for use in immunoassays to determine the drug has been accomplished by several different approaches. One approach has been to couple the carboxyl group of lysergic acid directly to an immunogenic carrier protein, i.e. poly(L-lysine) or human serum albumin using carbodiimides. See Van Vunakis, Proc. Nat. Acad. Sci., 68:1483-87 (1971); Loeffler, J. Pharm. Sci. 62:1817-20 (1973); and Voss, Psychopharmacologia 26:140-45 (1972). This approach was used in developing early RIA methods for LSD determination, but the antibodies that were produced were characterized by poor specificity for LSD and high cross-reactivities with other ergot alkaloids.

A second approach has been to couple LSD to an immunogenic carrier protein via one of the ethyl side chains at the 8-position. See Ratcliffe, Clin. Chem. 23:169-74 (1977). In another approach, bis-diazo benzidine was used to couple the carrier proteins via an aromatic substitution. See Luderer, Bull. New Jersey Acad. Sci. 19:8-10 (1974).

Finally, LSD has been coupled to an immunogenic carrier protein via a linker arm using a reaction between LSD, formaldehyde and bovine serum albumin. See Castro, Res. Commun. Chem. Pathol. Pharmacol. 6:879-86 (1973); Taunton-Rigby, Science 181:165-6 (1973); and Ratcliffe, Clin. Chem. 23:169-74 (1977). These authors reported the reaction to be a Mannich aminoalkylation reaction, the product of which they postulated to be substituted at the N-1, or indole nitrogen, position. More recent investigation, however, based upon known condensation reactions of indole groups with an aldehyde, suggests that the 2-position was more probably the actual site of reaction. Several references describe the Mannich reaction with indole, and these references teach that no bond is formed at the N- 1 position when there is an opportunity for reaction at another position on the indole molecule. When the 3-position and 2-position are both available, the site of reaction is at the 3-position, with the indole nitrogen remaining unreactive. This is described in Orchin, The Vocabulary of Organic Chemistry, John Wiley & Sons, NY, p. 385; Furniss, Vogel's Textbook of Practical Organic Chemistry, 4th Ed., Longman Scientific & Technical and John Wiley & Sons, NY, p. 813 (1978); and Mundy, Name Reactions and Reagents in Organic Synthesis, John Wiley & Sons, NY, p. 137 (1988). When the 3-position of the indole ring is blocked, however, as is the case in the LSD molecule, the reactive site involves the 2-position, with no bond being formed at the indole nitrogen, N-1. This is described in Orchin, The Vocabulary of Organic Chemistry, John Wiley & Sons, NY, p. 501, Figure 13.790. Orchin describes the indole alkaloid tryptamine undergoing a Mannich condensation reaction with an aldehyde (secologanin), with the resulting substitution occurring at the 2-position on the indole group.

SUMMARY OF THE INVENTION

This disclosure provides a variety of reagents and methods related to the detection or measurement of LSD or a related substance in samples obtained from subjects who may have been exposed to LSD. In various embodiments, the system allows for detection of both the parent substance and natural metabolites as they may be formed within the subject or secreted into a biological fluid, particularly urine. The sensitivity and specificity of the reagents can be used in diagnostic-grade immunoassays for screening of drugs of abuse in a clinical setting.

Featured embodiments described in the disclosure include systems that permit detection of glucuronide metabolites of LSD, either alone or in combination with LSD itself. The testing of a urine sample permits the practitioner to detect possible use of LSD by the subject as far as 36, 48, or even 72 hours after the occurrence.

Particular embodiments of the invention include an antibody for use in an assay for determination of a glucuronide metabolite of LSD excreted into a urine sample. Also included is an antibody for determination of LSD or an LSD metabolite excreted into a urine sample from a subject over a period extending to about 48 hours after exposure of the subject to the LSD. The antibody can be specific for glucuronide metabolites of LSD (as distinct from LSD), or it can be specific for both LSD and the metabolites.

Other embodiments of the invention include a reagent system for use in an assay, comprising an antibody of this invention along with a labeled competitive binding compound (typically an LSD derivative) that competes with the substance being tested for binding to the antibody. The reagent system can also optionally include additional components useful in conducting an assay, such as reagents for developing a detectable signal from the labeled compound following the competition reaction, buffers, standards, or written instructions. Different reagents can be premixed in any workable combination. This invention includes the use of an antibody and other reagents of this invention to provide kits or reagent sets for LSD determination.

Also embodied in the invention are assay methods for determining a glucuronide metabolite of LSD in a sample, or evidence of previous exposure to LSD within a period extending to about 48 hours before collection of the sample from the subject. Such methods involve preparing a reaction mixture comprising the sample, an antibody of this invention, and a labeled competitive binding compound capable of competing with a glucuronide metabolite of LSD. The amount of label bound to the antibody in the reaction mixture is then determined by a suitable detection technique, such as separating the complexes and measuring the label, or measuring an effect on the labeled compound as a result of being bound to antibody, such as a change in fluorescence or enzyme activity. The amount can be correlated with exposure of the subject to LSD, and if appropriate, further steps can be taken to distinguish between LSD and glucuronide metabolites.

Featured reagents include the following: competitive binding compounds in which the label is conjugated to the LSD derivative via the indole ring (such as at the N-1 or the 2 position); homogeneous assay systems; and LSD conjugated enzyme donors of β-galactosidase. Assay methods are featured wherein the median relative level of LSD and LSD metabolites measurable in the immunoassay in sequential urine samples of a pharmacokinetic study taken from a subject 36 hours after administration of LSD is at least about 5% of the highest levels of LSD and LSD metabolites measurable in urine samples from the same subject within the first 24 h after the administration. Antibodies may be specific for glucuronide metabolites of LSD (as opposed to LSD itself), or may be specific for both, in which case the cross-reactivity to the glucuronide metabolite is preferably at least about 10%. Also featured are antibodies with less than 1% cross-reactivity with chlorpromazine or ergotamine, and diagnostic assay systems in which the frequency of false positive reactions with clinical urine samples not containing LSD in the immunoassay is less than 4%.

Further aspects of the invention and desirable characteristics of the reagents and assay methods will be apparent from the description that follows and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood by reference to the following detailed description of the invention when considered in combination with the drawings that form part of the specification, wherein:

Fig. 1 illustrates a particular synthetic scheme for preparing N-1 -carboxymethyl-LSD.

Fig. 2 illustrates a particular synthetic scheme for preparing the N-hydroxysuccinimide ester of N-1-carboxymethyl-LSD.

Fig. 3 illustrates a particular synthetic scheme for preparing N-1-(maleimidoethylamino-carbonylmethyl)-LSD.

Figs. 4 (a) and 4 (b) are graphs showing dose response curves at varying levels of LSD using enzyme donor conjugates and antibodies of the present invention.

Fig. 5 is two graphs showing reverse-phase HPLC analysis of material immunopurified from urine using anti-LSD antibody. Upper panel shows total substance; lower panel shows LSD reactivity measured in the LSD enzyme immunoassay.

Fig. 6 is three graphs showing reverse-phase HPLC/MS/MS separation of a standard mixture of LSD related compounds. Upper panel: Total ion chromatograph; Middle panel: 310→209; Lower panel: 324→223.

Fig. 7 (Upper Panel) is a graph showing reverse-phase HPLC/MS/MS separation of LSD and its metabolites immunoextracted from urine (Total ion chromatograph). Six major peaks are observed. The two early eluting peaks have mass values equal to LSD plus oxygen plus glucuronic acid. Fig. 7 (Lower Panels) is three graphs showing LC/MS/MS analysis of peaks treated with glucuronidase. From top to bottom, the panels show LSD-O-glucuronides 516→340; 2-oxo,3-OH LSD 356→237; and O-LSD 340→239. Major metabolites in urine are 13-hydroxy LSD glucuronide and 14-hydroxy LSD glucuronide. The ability to detect these metabolites extends the period over which a previous exposure to LSD can be detected.

DESCRIPTION OF PREFERRED EMBODIMENTS

Considerable advancement in the field of detecting drugs of abuse was attained by invention of non-isotopic immunoassays specific for LSD. International application PCT/US96/19266 describes such assays and certain preferred embodiments in which immunogens and enzyme conjugates are made using LSD derivatized through the indole ring.

It has now been discovered that certain monoclonal antibodies raised against LSD-carrier conjugates are able to detect a previous exposure to LSD in a urine sample taken several days later. This contrasts with the normal pharmacokinetic profile of LSD, which is excreted mainly in the first 24 hours. The activity was traced primarily to 13-hydroxy LSD glucuronide and 14-hydroxy LSD glucuronide. Siddik et al. once described the fate of [14C]LSD and of [14C]iso-LSD in animals. The present disclosure explains how glucuronide metabolites can be measured in human urine by immunoassay, and illustrates that the pharmacokinetic profile of glucuronide metabolites is delayed compared with the LSD parent compound. The methods and reagents provided in the disclosure considerably extend the window in which a previous LSD exposure can be detected.

Some of the technical terms used in this disclosure can be understood as follows:

A "hapten" is a partial or incomplete antigen. It is a protein-free substance, usually a low molecular weight substance, which is not capable of stimulating antibody formation, but which does react with antibodies. LSD is an exemplary hapten.

A "glucuronide metabolite" of LSD is a modified form of LSD or one of its close pharmacologic relatives, formed by conjugation of the parent compound with one or more glucuronide residues by a mammal to which the parent compound is administered. The metabolite may appear in blood (tested as serum or plasma) or other internal bodily fluid, or excreted into any external bodily fluid, such as urine, bile or saliva. Glucuronide metabolites of LSD include but are not limited to 13-hydroxy LSD glucuronide and 14-hydroxy LSD glucuronide.

A "carrier", as the term is used herein, is an immunogenic substance, commonly a protein, that can join with a hapten, thereby enabling the hapten to stimulate an immune response. Carrier substances include proteins, glycoproteins, complex polysaccharides and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from the host. An exemplary carrier is keyhole limpet hemocyanin (KLH). The term "immunogenic" as used herein refers to substances capable of producing or generating an immune response in an organism.

As used herein, a "label" is an identifying tag which, when attached to a carrier substance or molecule, can be used to detect an analyte. A label may be attached to its carrier substance directly or indirectly by means of a linking of bridging moiety. Examples of labels include enzymes such as β-galactosidase and peroxidase, enzyme fragments (particularly an enzyme donor), fluorescent compounds such as rhodamine and fluorescein isothiocyanate (FITC), luminescent compounds such as dioxetanes and luciferin, and radioactive isotopes such as 125I.

A "peptide" is any compound formed by the linkage of two or more amino acids by amide or peptide) bonds, and is usually a polymer of α-amino acids in which the α-amino group of each amino acid residue (except the NH2-terminal) is linked to the α-carboxyl group of the next residue in a linear chain. The terms peptide, polypeptide and poly(amino acid) are used synonymously herein to refer to this class of compounds without restriction as to size. The largest members of this class are typically referred to as proteins.

A "complex" is an association of chemical compounds or moieties together by non-covalent bonds, possibly in a reversible fashion. Examples are an enzyme-substrate complex (an association of an enzyme and one or more substrates that is the reacting moiety in an enzyme-catalyzed reaction), an antigen-antibody complex, a hapten-antibody complex, or an active enzyme complex of β-galactosidase formed by complementation of an enzyme donor and an enzyme acceptor. A "stable complex" is a complex that persists at least as long as it takes the presence of the complex to be measured by the intended method.

An "enzyme acceptor" (EA) is an enzymatically inactive, polypeptide fragment of an enzyme. Exemplary is an EA of β-galactosidase produced by a deletion mutant of the β-galactosidase gene. When the EA is combined or associated with an enzyme donor, it is capable of forming active enzyme by the process of complementation.

An "enzyme donor" (ED) is an enzymatically inactive polypeptide fragment of an enzyme. Exemplary is an ED of β-galactosidase. The ED comprises a peptide sequence capable of combining or associating with an EA to form active enzyme.

The term "derivative" refers to a chemical compound or molecule obtainable from a parent compound or molecule such as LSD by one or more chemical reactions. When one substance is described as "conjugated" to another, then they are attached together in a stable arrangement, preferably through a covalent linkage, and optionally through a bridging structure.

The term "enzyme immunoassay" includes any immunoassay in which an enzyme is part of the detection system. The enzyme may be simply a tag for an active component in the reaction mixture, or it may be assembled, disassembled, activated, or deactivated in the course of the reaction. The presence of the analyte of interest in the sample may be directly or inversely correlated with enzyme activity.

The term "antibody" as used in this disclosure refers to both polyclonal and monoclonal antibodies. The ambit of the term explicitly encompasses not only intact immunoglobulin molecules, but also such fragments and derivatives of immunoglobulin molecules as may be prepared by techniques known in the art, and retaining the antibody activity of an intact immunoglobulin, or selected from a library of antibody fragments (e.g., a phage display library) having the desired binding specificity.

An antibody is said to be "specific" for a particular substance such as LSD if it is capable of forming a stable complex with the substance in preference to a number of unrelated substances in a typical test sample. Certain antibodies of this invention can be specific for more than one substance, such as LSD and an LSD metabolite.

"Cross reactivity" is determined in a quantitative immunoassay by establishing a standard curve using known dilutions of the target analyte. The standard curve is then used to calculate the apparent concentration of the interfering substance present in various known amounts in samples assayed under similar condition. The cross reactivity is the apparent concentration divided by the actual concentration multiplied by 100. The preferred immunoassay for determining cross-reactivity is a CEDIA® type enzyme complementation assay.

A "competitive binding compound" in the context of an immunoassay for an analyte in a sample refers to a compound which binds the detecting antibody of the immunoassay in a manner that is inhibitable by the analyte.

A "substance of abuse" is a chemical not naturally occurring in the body and administered to the body in contravention of the provisions of a criminal or disciplinary code, terms of employment, terms of participation in a particular activity such as an athletic competition, or which seriously impairs an activity (such as the operation of a vehicle) to the peril of the public or those in the vicinity of the abuser, or which provides the abuser with an unfair physical or intellectual advantage in a competitive arena. LSD is exemplary, especially when taken for reasons other than the bone fide prevention or treatment of disease.

Particular chemical structures represented in this disclosure include all stereoisomers, tautomers, salts, and protonated and deprotonated forms unless otherwise indicated.

The chemical structure of LSD can be represented by the following formula:

Materials useful in the practice of this invention can be prepared as follows:

N-1-carboxyalkyl derivatives of LSD or LSD metabolites are prepared by a method in which N-1-alkylation is favored over N-6 alkylation, i.e. quaternization. This method involves the treatment of LSD with a molar excess of a strong base, e.g. sodium hydride, followed by addition of alkyl-haloalkylcarboxylate. Hydrolysis of the ester yields N-1-carboxyalkyl LSD. The latter derivative may be conjugated to amino groups on immunogenic carrier proteins to yield immunogens directly or may be conjugated to amino groups on linkers, i.e., maleimidoalkylamines, to give adducts suitable for conjugation to thiol groups of enzyme donor polypeptides, immunogenic carrier proteins or labeling groups. LSD can also be conjugated through the 2-position of the indole ring via the Mannich reaction, or through the 6 position according to standard chemistry.

In preparing immunogen, enzyme, or enzyme donor conjugates, a maleimide adduct can first be formed with an aminoalkyl-maleimide derivative. These aminoalkyl-maleimide derivatives are synthesized by the methods of Huber as described in PCT publication WO 90/15798 (Dec. 27, 1990). The maleimide adducts are reacted with thiol groups on the immunogen, enzyme or enzyme donor to give thioether-linked conjugates.

In a preferred approach, a thiol-containing carrier poly(amino acid) or other substance having immunogenic properties is coupled to the maleimide hapten. Although thiolated keyhole limpet hemocyanin (KLH) is an especially preferred antigenic poly(amino acid), or carrier protein, it should be understood that various protein carriers may be employed, including albumins, serum proteins, e.g., globulins, ocular lens proteins, lipoproteins and the like. Illustrative protein carriers include bovine serum albumin, egg ovalbumin, bovine gammaglobulin, thyroxine binding globulin, etc. Alternatively, synthetic poly(amino acids) having a sufficient number of available sulfhydryl groups such as cysteine may be employed, as may other synthetic or natural polymeric materials bearing reactive functional groups. In particular, carbohydrates, yeasts, or polysaccharides may be conjugated to the hapten to produce an immunogen.

Conjugates of the activated hapten and a labeling group such as an enzyme, a substance having fluorescent properties, or a radioactive label may also be prepared and used as reagents in immunoassays. As with the immunogen and enzyme donor conjugates, the label employed must have available thiol-containing groups to be suitable for conjugation via the maleimide linker embodiment of the present invention. The thiol groups may be naturally occurring or they may be artificially introduced using a thiolating agent such as N-succinimidyl-3-(acetylthio) propionate (SATP) or 2-iminothiolane.

In order to generate antibodies, the immunogen is conveniently prepared for injection into a host animal by rehydrating lyophilized immunogen to form a solution or suspension of the immunogen. The immunogen solution is then combined with an adjuvant such as Freund's. The immunogen may be administered in a variety of sites, at several doses, one or more times, over many weeks.

Preparation of polyclonal antibodies using the immunogen may follow any of the conventional techniques known to those skilled in the art. Commonly, a host animal such as a rabbit, goat, mouse, guinea pig, or horse is injected with the immunogen mixture. Further injections are made, with serum being assessed for antibody titer until it is determined that optimal titer has been reached. The host animal is then bled to yield a suitable volume of specific antiserum. Where desirable, purification steps such as affinity adsorption or depletion may be taken to remove undesired material such as nonspecific antibodies before the antiserum is considered suitable for use in performing assays. Monoclonal antibodies may be obtained by hybridizing mouse lymphocytes, immunized as described above, and myeloma cells using a polyethylene glycol method such as the technique described in Methods in Enzymology 73 (Part B), pp. 3-46 (1981).

Assay reagents useful in the practice this invention include a detecting antibody raised by linking the following activated LSD derivative to KLH, and an enzyme donor made using the same derivative:

Antibody producing cell lines with sensitivity and specificity for LSD can be identified by any suitable screening technique. One such method as illustrated below is to conduct test immunoassays using corresponding competitive binding compounds, such as an LSD enzyme or enzyme donor conjugate made using the same activated form of LSD. It has been discovered that a proportion of monoclonal antibodies raised against LSD and screened in this fashion have sufficient binding activity for LSD glucuronide metabolites to provide for their detection in urine. Exemplary is a detecting antibody with laboratory designation 19A7, the properties of which are illustrated in the Example section that follows. This antibody is specific for both LSD and glucuronide metabolites of LSD.

In view of this discovery, it is readily appreciated that antibody activity to LSD glucuronide metabolites can be measured directly in the screening assay using the metabolite as the test analyte. Glucuronide metabolites can be purified using the techniques described in the example section from any suitable source, such as clinical urine samples, or samples of urine or bile from experimental animals administered with LSD. It will also be appreciated that antibodies specific for glucuronide metabolites can be prepared by using an LSD glucuronide derivative in the immunogen preparation. The recommended conjugation position is away from the alkyl amide group, such as the N-1, 2, or 6 position. The N-1 position is particularly recommended. The conjugation position of the label in the competitive binding compound is usually optimal if it matches that of the immunogen.

In certain embodiments of the invention, the detecting antibody is specific for both LSD and glucuronide metabolites of LSD; the cross-reactivity for the glucuronide metabolites (on a wt/wt basis) being typically at least about 5% of LSD itself, and more preferably at least about 10%, 50%, or even 100% of the LSD. In other embodiments of the invention, the detecting antibody binds the glucuronide metabolites of LSD in preference to LSD, in which case the cross-reactivity for the LSD is no more than 50% of the glucuronide metabolites, and more preferably no more than about 10% or even 2%. Such antibodies can be used to distinguish between glucuronide metabolites and LSD, or can be mixed with anti-LSD antibodies to provide an assay system capable of detecting either type of compound.

Immunoassay procedures typically entail combining the sample with the antibody under conditions that permit the formation of a stable complex between the substance to be tested and the antibody. This is followed by detecting any antigen-antibody complex that is formed. In certain immunoassay methods of this invention, the detecting antibody binds a labeled competitive binding compound (typically a labeled form of LSD), in a manner that is inhibitable by glucuronide metabolites of LSD and optionally also the parent LSD compound. In this embodiment, the substance to be tested intercedes between the labeled competitive binding compound, reducing the amount of labeled compound bound to antibody in a competition reaction.

Immunoassays for measuring LSD and its metabolites include both separation-based and homogeneous assays. Generally, in separation based assays, the detecting of the complex involves a process wherein the complex formed is physically separated from either unreacted analyte, unreacted antibody, or both. See, e.g., U.S. Patent No. 3,646,346. Radioimmunoassays are typically of the separation type. Generally, in homogeneous assays, the complex is typically not separated from unreacted reaction components. Instead, the presence of the complex is detected by a property which at least one of the reactants acquires or loses as a result of being incorporated into the complex. Homogeneous assays known in the art include systems involving fluorochrome and fluorochrome quenching pairs on different reagents (U.S. Patent Nos. 3,996,345, 4,161,515, 4,256,834, and 4,261,968); enzyme and enzyme inhibitor pairs on different reagents (U.S. Patent Nos. 4,208,479 and 4,233,401); and chromophore and chromophore modifier pairs on different reagents (U.S. Patent No. 4,208,479).

Homogeneous assay methods of this invention are exemplified by enzyme complementation assays, particularly the cloned enzyme donor immunoassay system described in U.S. Patent No. 4,708,929. Other assay systems of interest are enzyme immunoassays based on glucose-6-phosphate dehydrogenase (G6PD). Examples are provided in International Patent Application WO 94/24559 and EP Patent Application 487,301-A. Other assay systems of interest are fluorescence polarization immunoassays; the general approach being described in U.S. Patent 5,354,693 and E.P. Patent 371,253-B.

For the purposes of screening for prior LSD exposure, an assay capable of detecting both LSD and LSD glucuronide metabolites can be used; the ability to detect the metabolites having the effect of extending the window of detection. For legal purposes, once a positive screening test result is obtained, it may be desirable to distinguish between LSD and LSD glucuronide in the sample. Options for distinguishing include immunoassays or immunoadsorptions using antibodies specific for only one of the two types of compounds, complex physicochemical techniques such as combinations of liquid chromatography and GC/MS, or combinations of physicochemical techniques such as GC/MS in combination with treatment using glucuronidase.

This invention includes various reagent sets, which are effective in performing an assay of this invention, or in adapting an assay for particular determination or purpose. Reagent sets may include any or all of the following in various combinations: a specific antibody for glucuronide metabolites or a cocktail thereof, a labeled competitive binding compound, other components of a signal generation system, buffers, or standards. For sets that include a plurality of reagents, it is not necessary that the reagents all be sold together or by the same distributor, so long as they have the desired functionality. It may be appropriate to distribute the reagents separately, for example, when the shelf life of one reagent is shorter than that of the rest of the system.

This invention also includes kits, in which one or more reagents are provided in suitable packaging. The reagents are optionally aliquoted so that they can be readily used in a clinical system. A kit will often contain written instructions for the performance of the assay method. This can be limited to a simple indication on the packaging that the ingredients are suitable for measuring the analyte of interest. Preferably, the written instructions will also indicate important steps and conditions for performing the tests, and data useful in interpreting the tests.

Further illustration of the development and use of reagents and assays according to this invention are provided in the Example section below. The examples are provided as a guide to a practitioner of ordinary skill in the art, and are not meant to be limiting in any way.

Example 1. Thiolation of KI H

A solution of keyhole limpet hemocyanin (KLH) (12.3 mg, 12.3 nmoles) was reconstituted in 50 mM phosphate buffer (2.5 ml), pH 7.6. To this was added 2-iminothiolane hydrochloride (2-IT) (3.4 mg, 24.6 nmole). The solution was vortexed and allowed to stand at ambient temperature for 60 minutes. The thiolated KLH (KLH-SH) was desalted with 50 mM phosphate buffer (3.5 ml), pH 7.0, over a PD-10 prepacked SEPHADEX G-25 ion exchange column (® Pharmacia, Inc.) pre-equilibrated with 50 mM phosphate buffer, pH 7.6, to remove excess, unreacted 2-IT.

Example 2. Preparation of N-1-carboxymethyl-LSD

N-1 -(ethyl-carboxymethyl)-LSD (formula II) was prepared as a starting material by treating LSD (formula I) with a molar excess of sodium hydride followed by the addition of ethylbromoacetate. The ester formed was hydrolyzed to yield N-1-carboxymethyl-LSD (N-1-CM-LSD, formula III). The synthetic scheme, described in detail below, is illustrated in Fig. 1.

Because LSD was found to be difficult and hazardous to weigh by transfer due to static charge effects, a method for reconstituting a total vial and weighing by subtraction was adopted. Working in a glove box which had been purged with nitrogen gas, 7.4 mg (308 µmol) sodium hydride was weighed directly into a tared 2.5 ml conical reaction vial. Dimethylformamide (DMF), 400 µl, was added to the reaction vial, along with a magnetic stir bar, open cap, and septum with TEFLON (synthetic resin polymer)-coated face. The vial was placed in a beaker containing crushed dry ice for approximately 10 min. DMF, 300 µl, was added to a tared, 50 mg vial of LSD, and the vial was capped and inverted several times until a complete solution was obtained. The LSD solution was transferred to a small culture tube (12 x 75 mm), capped and placed in the dry ice for approximately 10 min. The empty LSD vial was then rinsed with acetone, dried and weighed to obtain the net weight of LSD removed by subtraction, 50 mg (154 µmol). After removing the reaction vial from the dry ice and placing on a magnetic stir plate, the LSD solution was injected while vigorously stirring the sodium hydride suspension in the vial. Evolution of hydrogen gas and bright yellow coloration was noted. The suspension was allowed to warm with stirring for about 5 min, at which point the gas evolution had subsided. Ethylbromoacetate, 17 µl (164 µmol), was then injected and stirred for approximately 2 minutes. After removing the vial from the glove box, a 1-2 µl aliquot was removed for HPLC analysis, and. the vial was then placed in a -70°C freezer while the HPLC was being run. The HPLC aliquot was diluted in a 12 x 75 mm culture tube with 20 µl acetonitrile and 20 µl of 0. 1 M triethylamine acetate (TEA-Ac). The sample was injected on a C4 analytical column (Vydac) and the following program was run: 0-5 min, 100% 0. 1 M TEA-Ac (pH 7); 5-55 min, 0-50% acetonitrile/0.1 M TEA-Ac; 55-60 min, 100% acetonitrile; 60-70 min, 100% 0. 1 M TEA-Ac. The flow rate was 1 ml/min, with UV detection at 320 and 280 nm. The HPLC showed nearly complete conversion of LSD eluting around 30-32% acetonitrile to a major product eluting around 40-42% acetonitrile which showed a slight back shoulder. The N-6 quaternized side-product elutes right after LSD, i.e., 32-33% acetonitrile.

The product was isolated by preparative HPLC on a 2.2 x 25 cm C4 column using the following program: 0-10 min, 10% acetonitrile/0. 1 M TEA-Ac; 10-60 min, 10-60% acetonitrile/0.1 M TEA-Ac; 60-65 min, 65% acetonitrile/0.1 M TEA-Ac; 65-75 min, 10% acetonitrile/0.1 M TEA-Ac. The flow rate was 8 ml/min. The load solution was prepared by diluting the cold reaction mixture with 1 ml 0.1 M TEA-Ac, filtering the resultant, slightly turbid solution through a 1 µm syringe filter, and injecting the clear filtrate, 1.8 ml, on a 2 ml loop. The desired product eluted toward the end of the gradient with a back shoulder. Fractions were collected manually over the major peak, taking care to change fractions at the back shoulder. This back shoulder corresponds to partially resolved N-1-(ethyl-carboxymethyl)-isoLSD, i.e. epimerized at the 8-position. Analytical HPLC was performed on the major fractions and those which were free of the isoLSD shoulder were pooled and lyophilized. The fraction was analyzed by 1H-NMR in acetonitrile-d3 and identity confirmed by mass spectrometry (MS). The NMR spectrum of N-1-(ethyl-carboxymethyl)-LSD showed an absence of the LSD 1-position NH at 9.0 ppm. However, all other LSD resonances were seen at approximately the same position as in the parent drug. This strongly indicated that the 1-position was substituted. In addition, new resonances were noted for the carboxymethyl CH2 (4.9 ppm, 2 proton singlet) and the ethyl ester (CH2 at 4.2 ppm, 2 proton quartet, and CH3 at 1.3 ppm, triplet overlapped with diethylamide resonances). The recovered yield was calculated by comparing UV/visible in acetonitrile/water (50:50) using MW = 409.5 and Emax = 5895 for the peak around 320 nm and found to be 30 mg.

N-1-(ethyl-carboxymethyl)-LSD starting material (28.5 mg, 70 µmol) was dissolved in 3.5 ml of ethanol and transferred to a small vial equipped with a septum/needle attached to an inert gas line and small stir bar. The reaction vial was purged with argon gas. Sodium hydroxide, 75 µl of a 1N solution, was then injected with stirring. The reaction was monitored using the analytical system described above for preparing the starting material. The product eluted at about 24-25% acetonitrile as a sharp peak. A small amount of isoLSD derivative side product was noted which appeared as a back shoulder on the major peak. The reaction was complete in approximately 2 hr. The reaction mixture was then neutralized by adding one equivalent of acetic acid and 1 ml of water, and the resulting solution was clear. The product was isolated and desalted by HPLC on a preparative C4 column using 20 mM TEA-Ac, pH 7, and acetonitrile according to the following program: 0-5 min, 0% acetonitrile/20 mM TEA-Ac; 5-55 min, 0-50% acetonitrile/20 mM TEA-Ac; 55-60 min, 100% acetonitrile. The flow rate was 8 ml/min. The major peak, which eluted around 28-29% acetonitrile, was collected and fractions changed on the back side of the peak to eliminate any shoulder for isoLSD derivative. The pooled fractions were lyophilized and re-lyophilized 2 times from water/acetonitrile 4:1 to get ride of the TEA-Ac and convert the product to a zwitterion. The product was analyzed by 1H NMR in a mixture of acetonitrile-d3 and deuterium oxide. The ethyl ester resonances noted above were confirmed to absent in the NMR whereas the other resonances as noted above were intact. The product was also analyzed by MS and confirmed to have a molecular ion peak corresponding to the theory molecular weight (MW) of 381. The recovered yield was calculated by UV in acetonitrile-H20 as described above for the starting material using MW = 381 and Emax = 5895. The yield of N-1-CM-LSD was found to be between 20 mg.

Example 3. Preparation of N-1-(carboxymethyl)-LSD N-hydroxysuccinimide ester)

As illustrated in Fig. 2, N-1-(carboxymethyl) LSD N-hydroxysuccinimide ester (N-1-CM-LSD-NHS, formula V) was prepared by dissolving a sample of N-1-carboxymethyl-LSD (6.6 mg, 17.3 µmoles, formula IV) in 1.0 ml dimethylsulfoxide (DMSO). To that solution N-hydroxysuccinimide (NHS) (14 mg, 121 µmoles) and 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (23.2 mg, 121 µmoles) was added. The solution was vortexed and incubated at ambient temperature overnight. The activated N-1-CM-LSD-NHS (Formula V) was used in Examples 4 and 5 without any purification.

Example 4. Conjugation of N-1-(carboxymethyl)-LSD N-hydroxysuccinimide ester to KLH

A solution of KLH (15.0 mg, 15 nmoles) in 4.0 ml phosphate buffer was added to a solution of N-1-CM-LSD-NHS (5.7 mg, 15.0 µmoles) in 1.0 ml DMSO, prepared as in Example 3. The resulting solution was vortexed gently for two minutes and allowed to stand at ambient temperature for 8.0 hours. The immunogen [N-1-CM-LSD]n-KLH was dialyzed against water/MeOH (80:20 v/v) for three days. The immunogen was then freeze-dried to form a lyophilized powder material. Loading of the immunogen, calculated as described in Example 7 below, was determined ta be approximately 199, using an extinction coefficient of 17,060 M-1 cm-1 at 250 nm for N-1-CM-LSD.

Example 5. Preparation of N-1-(maleimidoethylamino-carbonylmethyl)-LSD

As illustrated in Fig. 3, a 2-fold excess of maleimidoethylamine hydrochloride (MEA HCl, formula V, 6.1 mg, 34.6 µmoles) in 1.5 ml phosphate buffer was added to the activated N-1-CM-LSD-NHS from Example 3 (6.6 mg, 17.3 µmoles). MEA HCl was synthesized by the method of Huber as described in PCT publication WO 90/15798 (Dec. 27, 1990). The progress of the reaction was monitored by HPLC. The N-1 (maleimidoethylamino-carbonylmethyl)-LSD (N-1-MEA-CM-LSD, formula VI) was then purified by HPLC.

Example 6. Conjugation of N-1-(maleimidoethylamino-carbonylmethyl)-LSD to KLH

KLH-SH solution (7.0 mg, 7.0 µmoles) in 2.1 ml of phosphate buffer from Example 1 was added to a solution of N-1-MEA-CM-LSD (3.5 mg, 7.0 mmoles) in DMSO (0.60 ml). The resulting solution was vortexed gently for two minutes and allowed to stand at ambient temperature for 5 hours. The protein conjugate [N-1-MEA-CM-LSD]n-KLH was dialyzed against H2O:MeOH (80:20 v/v) for three days, then freeze-dried to form a lyophilized powdered material.

The number of N-1-MEA-CM-LSD adducts incorporated into the protein carrier KLH (loading of immunogen) was determined as follows: One milligram of [N-1-MEA-CM-LSD]n-KLH was dissolved in 1.0 ml of 1.0 N NaOH. The spectrum of the solution was recorded between 200 and 400 nm against the same solvent as reference. Using the extinction coefficient of 5,899 M-1 cm-1 at 320 nm for N-1-MEA-CM-LSD in which there is no absorption for KLH, the molar ratio was calculated to be 226:1.

Example 7. Preparation of N-1-(maleimidopentylamino-carbonylmethyl)-LSD:ED Conjugate

N-1-(maleimidopentylamino-carbonylmethyl)-LSD (N-1-MEA-CM-LSD) was first prepared. To a solution of N-1-CM-LSD-NHS (50.11 mg, 131.4 µmoles) prepared as described in Example 3, an approximately equivalent number of moles of maleimidopentylamine hydrochloride (MPA) (28.78 mg, 131.6 µmoles) in dimethylformamide (DMF) was added. To keep the reaction mixture at neutral pH, 300 µl of triethylamine (TEA) was added and the pH checked. The progress of the reaction was monitored by HPLC. The N-1-MPA-CM-LSD was then HPLC purified and used in the preparation of the ED conjugate.

A solution of thiolated ED28 (5.0 mg, 0.5 µmoles) was prepared and desalted in 3.5 ml phosphate buffer. This solution was then added to the solution of N-1-MPA-CM-LSD (1.67 mg/1.5 ml DMF). The resulting mixture was vortexed gently for 2 minutes and allowed to stand at ambient temperature for 55 minutes. The protein conjugate N-1-MPA-CM-LSD:ED was HPLC purified using a Vydac semipreparative C4 column.

Example 8. Preparation of Monoclonal Antibodies

Preparation of the immunogen and immunization of the host animal were accomplished using techniques which will be well known to those skilled in the art. Each of the immunogens prepared in Examples 4 and 6 were administered to mice in a series of injections. Hybridoma cell lines were then developed from fusions using immunized spleens. Supernatant antibody was evaluated as described below, and ascites was produced from useful clones. Ascites was then purified, yielding monoclonal antibody. All of the biological and purification methods were performed in a manner well known to those skilled in the art.

In this example, supernatant antibodies were selected from 96-well culture plates using a CEDIA homogeneous assay. As previously described, the CEDIA assay utilizes two genetically engineered, enzymatically inactive fragments of β-galactosidase. The smaller polypeptide, the enzyme donor, can recombine spontaneously, with the larger fragment, the enzyme acceptor, to form active β-galactosidase, in a process called complementation. When a specific antibody to the ligand or analyte attaches to the enzyme donor conjugate, complementation is inhibited. The addition of free ligand to this system will modulate the inhibition of complementation. This assay principle was used to screen fusion products in a 96-well format.

A primary screening of the fusion products was first performed to evaluate the ability of the antibodies to bind to the enzyme donor conjugate prepared in Example 8 and to inhibit complementation. The number of inhibition-positive clones were then narrowed further by performing a secondary screening assay to determine whether the free drug would modulate or compete with the enzyme donor conjugate for the antibody. The modulation assay also identified specific clones when screened against cross-reacting analytes. The clones which modulated with the specific analytes of choice were then grown for further study. The culture supernatants containing the monoclonal antibodies were collected and evaluated on the HITACHI 717 analyzer (®Boehringer Mannheim Corp., Indianapolis, IN) as described in Example 9 below.

Example 9. Assay for LSD

A CEDIA assay for LSD was performed using the enzyme donor conjugate prepared in Example 7 and the antibody produced according to Example 4.

A preferred assay method comprises the steps of:

  • (a) contacting the sample with:
    • (i) an enzyme donor polypeptide conjugate, preferably having the structure
      wherein Z is an enzyme donor polypeptide of beta-galactosidase;
  • (ii) an enzyme acceptor polypeptide wherein said enzyme acceptor polypeptide is characterized by forming with said enzyme donor polypeptide conjugate an active enzyme complex having beta-galactosidase activity in the absence of an antibody to LSD;
  • (iii) an antibody specific for LSD, preferably raised against the structure
       wherein Z is an immunogenic carrier substance; wherein said enzyme donor conjugate is capable of competitively binding to said antibody, thereby inhibiting the formation of active enzyme complex; and
    • (iv) a substrate capable of reacting with said active enzyme complex, such that the rate of conversion of said substrate by said active enzyme complex can be monitored; and
  • (b) measuring the rate of conversion of said substrate by said active enzyme complex as a measure of the amount of LSD in said sample.

The following reagents are prepared: Antibody reagent: Antibody 57 ng/ml PIPES (piperazine-N,N-bis-[2-ethanesulfonic acid]) 100 mM NaCl 500 mM Fetal bovine serum 0.5 % EGTA 10 mM Magnesium acetate 10 mM Sodium azide 20 mM pH 6.90 Donor reagent: Enzyme donor conjugate 0.487 nM CPRG (chlorphenylred-β-D-galactopyranoside) 3 mg/ml PIPES 100 mM NaCl 400 mM EGTA 10 mM Fragmented BSA 2 mg/ml Sodium azide 20 mM pH 6.90 Acceptor reagent: Enzyme acceptor 880 U/ml Magnesium acetate 10 mM NaCl 400 mM PIPES 100 mM EGTA 10 mM Sodium azide 20 mM pH 6.90

Assays were performed using an HIIACHI 911 analyzer. The instrument dispensed 12 µl of sample containing LSD, and 100 µl of antibody reagent was added. The mixture was allowed to incubate at 37°C for 60 seconds, after which 100µl of donor reagent was added and allowed to incubate for 530 seconds. Finally 140 µl of the acceptor reagent was added. The absorbance rate was measured over the time period of 295 sec to 375 sec following the addition of the acceptor reagent. The primary wavelength used was 570 nm, with 660 nm used as the secondary wavelength. The absorbance rate at 570 nm was plotted against LSD concentration to construct the dose response curve shown in Fig. 4 (a). The results obtained with an LSD specific monoclonal antibody raised against the immunogen of Example 4 are as follows: Dose, ng/ml Rate, mAU/min 0.0 112.4 0.3 132.2 0.5 145.0 0.7 155.3 2.0 207.0 4.0 224.8 100 228.7

Example 10. Assay for LSD

In a similar manner to that described in Example 9, a CEDIA assay for LSD was performed using the enzyme donor conjugate prepared in Example 7 and an LSD specific monoclonal antibody raised against the immunogen of Example 6. The results obtained are as follows: Dose, ng/ml Rate, mAU/min 0.0 63.6 0.3 67.6 0.5 69.9 0.8 72.6 1.6 80.8 4.0 107.4
The response curve obtained by plotting the LSD dose against the rate is shown in Fig. 4 (b).

It is readily appreciated from the data in this Example and in Example 9 that antibodies with very high affinity can be produced using either immunogenic conjugates of the type exemplified in Example 4 or those exemplified in Example 6. Antibodies raised against either conjugate are produced that bind LSD-enzyme donor conjugates in a manner that is detectably inhibitable at an LSD dose of 0.3 ng/mL, and inhibitable at about the 50% maximum level at an LSD dose of about 1 ng/mL. By calculating the effective concentration of each of the assay components at a time when the LSD, conjugate, and antibody are interacting, it is apparent that the affinity of the antibodies for LSD used in these examples is at least about 1010 M-1. More generally, antibodies of an affinity of only 109 M-1 can be used in assays of this type. For an assay of good sensitivity in the range of interest, affinities of at least about 1010 M-1 are preferred, and affinities of at least about 1011 M-1 are even more preferred.

Example 11. Cross-reactivity testing

Using the methods outlined, monoclonal antibodies were identified that had a modulation in the CEDIA (LSD dose of 0.5 ng/mL) of up to about 24%. Antibodies with modulations between 12-24% were subjected to further testing. Different antibody clones had different cross-reactivity profiles for potential interfering substances. Cross-reactivity was tested by spiking various potential cross-reactants into human urine containing a preservative.

Tables 1 and 2 show the cross reactivity data for one monoclonal antibody with the laboratory designation 19A7. This antibody has a modulation with LSD of 16%. Compound Concentration Added (ng/mL) Concentration Observed (ng/mL) % Cross-Reactivity d-LSD 0.5 0.5 100.00 Alpha Ergotryptine 500,000 0.25 0.00000 Dihydroergotamine 125,000 ≤0.01 0.00005 d-LAMPA 0.5 0.29 58.7 Ecogonine 100,000 ≤0.01 0.00000 Ecogonine Methyl Ester 100,000 ≤0.01 0.00000 Ergonovine 10,000 0.58 0.00700 Ergotamine 100,000 ≤0.01 0.00000 Iso LSD 2,500 0.93 0.04000 Lysergic Acid 100,000 ≤0.01 0.00000 Lysergol 5,000 0.01 0.00034 Methysergide Maleate 50,000 0.74 0.00147 Psilocybin 10,000 ≤0.01 0.00000 Psilocyn 10,000 ≤0.01 0.00000 Seretonine 1,000,000 ≤0.01 0.00000 Tryptophan 100,000 ≤0.01 0.00000 2oxo-3hydroxy LSD 30 0.55 1.82
Percent cross-reactivity is expressed as: 100 x Observed Dose / (Concentration of Cross-reactant Added) Compound Concentration Added (ng/mL) Concentration Observed (ng/mL) % Cross-Reactivity Acetaminophen 500000 0.0 <0.00001 Acetylsalicylic Acid 500000 0.0 <0.00001 Amoxicillin 100000 0.0 <0.00001 Amphetamine 500000 0.0 <0.00001 Benzoylecgonine 500000 0.0 <0.00001 Captopril 500000 0.0 <0.00001 Chlordiazepoxide 100000 0.0 <0.00001 Cimetidine 500000 0.0 <0.00001 Codeine 500000 0.0 <0.00001 Diazepam 500000 0.0 <0.00001 Digoxin 100000 0.0 <0.00001 Enalapril 500000 0.0 <0.00001 Fluoxetine 400000 0.48 0.00012 Ibuprofen 500000 0.0 <0.00001 Levothyroxine (T4) 50000 0.0 <0.0001 Methamphetamine 200000 0.45 0.00019 Morphine 100000 0.0 <0.00001 Nifedipine 500000 0.0 <0.00001 Phencyclidine 500000 0.11 0.00002 Phenobarbital 500000 0.0 <0.00001 Ranitidine 500000 0.15 0.00003 Salicyluric acid 500000 0.0 <0.00001 Secobarbital 500000 0.0 <0.00001 Tolmetin 500000 0.0 <0.00001 Verapamil 500000 0.45 0.00009 11-nor-Δ9-THC-COOH 10000 0.0 <0.0001

As for any diagnostic test, it is desirable for an LSD assay to have selectivity for the intended analyte over potential interfering substances. Amongst substances that can interfere are naturally excreted products, excreted forms of pharmaceuticals used for treatment, and excreted forms of other substances of abuse. Structures with a resemblance to LSD are in general more likely to interfere. When designing an immunoassay for LSD, it is preferable to select an anti-LSD antibody that has a cross reactivity of less than 0.1%, preferably less than 0.01%, and still more preferably less than 0.001% for any of the substances listed in Table 1 and Table 2, except for dLAMPA or 2-oxo-3-hydroxy LSD. Also preferred are antibodies having less than 5%, preferably less than 2% cross-reactivity with 2-oxo-3-hydroxy LSD. Cross-reactivity with nor-LSD is optional. In one embodiment of this invention, the cross-reactivity with nor-LSD is at least about 20%, more typically over 30%. In another embodiment, the cross-reactivity with nor-LSD is less than about 10%, preferably less than 5%, more preferably less than 1%. Another class of potential interfering drugs is phenothiozines, exemplified by chlorpromazine. Preferably, the antibody has a cross-reactivity with such drugs of less than 1%, more preferably less than 0.1%, and still more preferably less than 0.01%. The cross-reactivities indicated in this section are also desirable properties for a plurality of assay reagents when used in a combination in the context of a particular assay system. Examples are a monoclonal or polyclonal antibody used in combination with an LSD-protein conjugate, including an LSD-enzyme conjugate or an LSD-enzyme donor conjugate. Cross-reactivities are generally more prominent in assays conducted at higher concentration, in a homogeneous system, in a shorter time period, or in a non-equilibrium situation. Reagents developed for less stringent assays are not necessarily suited for assays conducted under these conditions without further testing or development.

On the other hand, reactivity with the metabolite derivatives of LSD besides the parent compound will clearly be of benefit in the testing for LSD abuse. Putative urinary metabolites of interest include but are not limited to hydroxy-LSD-glucuronide. An additive effect of several derivatives will generally improve the sensitivity when biological samples are tested. In addition, if a particular derivative has a longer metabolic profile than LSD itself, then monitoring can continue for a longer period after the suspected dose.

Example 12. Performance testing on clinical samples

The performance of an assay with clinical samples is affected not only by the known drugs, but also by a wide spectrum of unknown metabolites. In establishing the specificity of a diagnostic assay, it is standard practice to evaluate a large panel of samples from different subjects.

The enzyme assay for LSD described in the previous examples underwent specificity testing using urine samples known to be free of LSD. Results using two different monoclonal antibodies raised against an N-1 LSD conjugate are shown in Table 3. 19A7 Summary RANGE ng/ml # of Samples % of Samples -0.75 to -1.00 0 0.00% -0.50 to -0.75 0 0.00% -0.25 to -0.50 1 0.05% -0.00 to -0.25 1359 67.54% 0.00 to 0.25 647 32.17% 0.25 to 0.50 4 0.20% 0.50 to 0.75 0 0.00% 0.75 to 1.00 1 0.05% >1.0 1 0.05% Total 2012 10E6 Summary RANGE ng/ml # of Samples % of Samples -0.75 to -1.00 1 0.05% -0.50 to -0.75 13 0.67% -0.25 to -0.50 387 19.88% -0.00 to -0.25 1128 57.94% 0.00 to 0.25 416 21.37% 0.25 to 0.50 3 0.15% 0.50 to 0.75 2 0.10% 0.75 to 1.00 1 0.05% >1.0 1 0.05% Total 1952

Using a cut-off level of 0.5 ng/mL, two of 2012 samples (0.1%) gave a false positive reaction for LSD when using antibody 19A7, and four of 1952 samples (0.2%) gave a false positive reaction for LSD when using antibody 10E6. Generally, a false positive rate of less than about 1% is used, with rates of less than 0.5%, 0.2%, and 0.1% being progressively more preferred.

These two antibodies were also tested against a panel of 379 samples positive for methadone, another substance of abuse. The presence of methadone in these urine samples was confirmed using an immunoassay specific for methadone. The number of false positives at a level above 0.5 ng/mL was 16 out of 379 (4%) for the 10E6 antibody and 0 out of 379 for the 19A7 antibody (<0.3%). Generally, a false positive rate with methadone-positive samples of less than 1% is desirable, with a rate below 0.3% being preferred.

Example 13. Fractionation of a urinary metabolite recognized by an LSD antibody

An immunoaffinity extraction method was developed to purify LSD and any LSD derivatives present in urine samples from individuals administered LSD.

Monoclonal antibody 19A7 specific for LSD was attached to aldehyde-derivatized Sepharose® 4B by reductive amination. Urine samples containing LSD were incubated with affinity resin, the resin was washed, and bound drug was eluted in a small volume of methanol. Recovery of LSD added to drug-free urine was >95%, while recovery of the weakly-bound LSD metabolite, 2-oxo, 3-hydroxy LSD was 65%. Lysergic acid methylpropyl amide (LAMPA) or deuterated LSD can be added as internal standard prior to immunoaffinity extraction on order to monitor recovery.

Samples were processed by GC/MS by evaporation of the methanol and derivatization with N, O-bis (trimethylsilyl)trifluoroacetamide (BSTFA). GC/MS analysis of the derivatized immunoassay extracts gave dramatically reduced background signal as compared with conventional solid-phase extraction. An LOD of 50 pg/mL and an LOQ of 61 pg/mL were obtained using conventional equipment (CG/MS model # HP 5971) under routine conditions.

The immunoaffinity extraction method was also used to isolate potential urinary metabolites of LSD. Urine samples testing positive for LSD by immunoassay and GC/MS were extracted, fractionated by reverse-phase HPLC, and the fractions were analyzed by a CEDIA® assay for LSD. A number of samples contained a fluorescent immunoreactive peak (exitation at 320 nm; emission at 400 nm), eluting earlier than the parent LSD (Fig. 5). Preliminary characterization of this peak by mass spectrometry show that the metabolite has a molecular weight of 516, which corresponds to hydroxylation of LSD followed by glucuronidation. A substance of 340 molecular weight was also seen, corresponding to loss of the glucuronide. Treatment of the metabolite in the HPLC peak with E. coli β-glucuronidase resulted in a shift of mobility on HPLC to an elution volume between that of the original peak and parent LSD.

These observations are consistent with the hypothesis that a glucuronic acid conjugate of LSD is a principal metabolite excreted into the urine of subjects administered with LSD. The metabolite is recognized by the antibody used for affinity chromatography and in the assay.

Further confirmation of the identity of the metabolites was obtained by reverse-phase HPLC/MS/MS. Fig. 6 shows the separation of various LSD metabolites (2-oxo,3-hydroxy LSD, nor-LSD) and analogs (iso-LSD, nor-iso-LSD) in a standard mixture. Although the separation of these compounds was not complete (Upper Panel), they could be further differentiated by their parent masses and the masses of the predominant fragment (Middle & Lower panels).

The immunoaffinity extract from pharmacokinetic study samples was analyzed by this HPLC/MS/MS method. Six peaks were observed (Fig. 7, Upper Panel):

  • Two partially resolved, early eluting peaks (0.81 and 1.02 min), with mass values equal to LSD plus oxygen plus glucuronic acid (i.e., hydroxy-LSD glucuronide);
  • A large peak corresponding to 2-oxo, 3-hydroxy LSD (a known metabolite of LSD);
  • A small peak corresponding to 2-oxo LSD; and
  • Small peaks corresponding to LSD and iso-LSD

Although unlikely, the first pair of peaks could have included a glucuronide of 2-oxo LSD. This was ruled out by treatment of the peaks with glucuronidase, followed by analysis by LC/MS/MS. The results (Fig 7, Lower 3 panels) show that the hydroxy-LSD compounds produced by glucuronidase treatment (mass = 340) elute at 1.15 and 1.73 min, which is distinct from 2-oxo LSD.

These results demonstrate that the 19A7 antibody recognizes the LSD metabolites 13-hydroxy LSD glucuronide and 14-hydroxy LSD glucuronide. This is believed to be the first antibody demonstrated to have substantial cross-reactivity with glucuronide derivatives of LSD. It is surprising that the bulky, negatively charged glucuronic acid side chains would be considered likely to interfere with binding of the metabolite to the antibody.

As illustrated in the following examples, it is advantageous to be able to detect glucuronide derivatives of LSD, since this extends the time window in which a previous administration of LSD can be detected. The glucuronide derivatives can be obtained by immunoaffinity purification of human urine samples, as illustrated above, or from the urine or bile of experimental animals administered LSD. Antibodies with substantial cross-reactivity to glucuronide can be detected not only by immunoextraction, but by using the glucuronide derivative in the screening assay of antibody clones raised either against LSD, or against the glucuronide derivatives themselves conjugated to an immunogenic carrier, preferably through the N-1 position, the 2-position (via the Mannich reaction), or the 6-position. Glucuronide reagent conjugates can be used with the selected antibodies in a direct assay for the metabolite. Alternatively, an antibody that cross-reacts with both the LSD and the glucuronide derivatives (such as the 19A7 antibody) can be used in an immunoassay with LSD conjugated to a protein, enzyme polypeptide (such as an intact enzyme or enzyme donor), a solid surface, a radioisotope, or a fluorescent label. Because of the cross-reactivity of the antibody, both LSD and the metabolites will be detected.

Example 14: A competitive immunoassay detecting a distinct urinary metabolite

In this example, experiments were performed to better define the time course of excretion of LSD metabolites in urine, and the ability of various immunoassays to detect them.

Individuals were administered LSD, and urine samples were collected over a 4-day period. The samples were assayed using the LSD enzyme-complementation immunoassay described in Examples 9 and 10 using monoclonal antibody 19A7. The samples were also measured using the DPC radioimmunoassay ("Coat-A-Count"® LSD assay, by Diagnostic Products Company) according to manufacturer's directions, and by GC/MS.

Results are shown in the following table in terms of LSD equivalents (translating the signal generated using a standard curve obtained with known concentrations of LSD). Time hrs CEDIA LSD ng/ml DPC RIA LSD ng/ml GC/MS LSD ng/ml SW U1 0 0.00 0.00 0.00 SW U2 2 3.40 0.93 0.33 SW U3 4 3.60 1.12 0.54 SW U4 6 12.20 3.79 * 0.52 SW U5 8 3.30 0.98 0.71 SW U6 10 10.70 4.12* 0.76 SW U7 12 12.50 8.34* 1.77 SW U8 24 12.70 13.49* 1.42 SW U9 36 1.01 0.38 0.11 SWU10 48 0.67 0.15 0.04 SW U11 60 0.14 0.00 0.02 SW U12 72 0.13 0.00 0.00 SW U13 84 0.09 0.00 *** SW U14 96 0.09 0.04 0.01 JS U1 0 0.00 0.06 0.00 JS U2 2 1.70 0.60 *** JS U3 4 8.30 3.01 0.39 JS U4 6 7.70 2.17 0.37 JS U5 8 5.20 1.40 0.38 JS U6 10 10.60 5.99* 0.64 JS U7 12 6.00 3.09* 0.52 JS U8 24 6.40 3.49* 0.34 JS U9 36 0.34 0.06 0.03 JS U10 48 0.26 0.25 0.01 JS U11 60 0.15 0.08 0.00 JS U12 72 0.73 0.50 0.02 JS U13 84 0.03 0.00 0.00 JS U14 96 0.07 NA 0.00
* The RIA high calibrator is 3.0 ng/ml, values above 3.0 ng/ml are only estimates *** No value available for this sample

The first sample shows a significant improvement in LSD sensitivity with 48 h time point still above the 0.5 ng/mL cut-off, as it reads 0.67 ng/mL in the CEDIA® assay. The DPC assay reads 0.38 ng/mL at 36 h, which is below the cut-off for this assay which is 0.5 ng/mL. The DPC assay detects LSD or LSD metabolites only up to the 24 h time point. The GC/MS results indicate that LSD has essentially disappeared after 24 h. The sensitivity of the CEDIA® assay after about the 24 h time point is attributable principally to an LSD metabolite.

This invention includes amongst its embodiments a set of reagents for use in an immunoassay for quantifying LSD or excreted metabolites of LSD, such as may be present in a urine sample of an individual suspected of having been administered LSD. The reagents include an antibody, preferably a monoclonal antibody, capable of binding the conjugate of the first reagent in a manner that is measurably inhibitable by LSD and one or more LSD metabolites.

Preferred characteristics of the antibody, the reagent combination, and the assay performed using the reagents are that the median relative level of LSD and LSD metabolites measurable in urine samples taken from a subject 36 hours after administration of LSD by the competitive immunoassay is at least 3%, preferably 5%, and more preferably 10% of the highest levels of LSD and LSD metabolites measurable in urine samples from the same subject within the first 24 h after the administration. Other preferred characteristics are that the median relative level of LSD and LSD metabolites measurable in urine samples taken from a subject 48 hours after administration of LSD by the competitive immunoassay is at least 1%, preferably 3%, and more preferably 5% of the highest levels of LSD and LSD metabolites measurable in urine samples from the same subject within the first 24 h after the administration. Other preferred characteristics are that the ratio of LSD measured at 48 hours to the level measured at 24 hours is at least about 2 times, preferably at least about 4 times, more preferably at least about 10 times what is measured by GC/MS.

Other preferred characteristics of the antibody are that it be specific for a glucuronic acid conjugate of LSD, particularly hydroxy-LSD glucuronide, wherein the glucuronic acid conjugate is at least 5% cross-reactive, preferably at least about 10% cross-reactive, more preferably at least about 20% cross reactive, even more preferably at least about 50% cross reactive, still more preferably 100% cross reactive or more, when used to determine the concentration of LSD or LSD metabolite in a competitive immunoassay. Cross reactivity is determined in a quantitative immunoassay by establishing a standard curve using known dilutions of the target analyte, then using the standard curve to calculate the apparent concentration of the interfering substance present in various known amounts in samples assayed under similar condition. It will be appreciated that antibodies with the ability to bind LSD metabolites can also be used in assays to measure LSD metabolites directly; for example, in a competitive radioimmunoassay wherein the metabolite in the sample competes with radioisotopically labeled LSD glucuronide.

The ability of the materials and methods of this invention to detect LSD metabolites is demonstrated in experiments using a sample containing the metabolite but known by GC/MS to be essentially free of the LSD parent compound. In the following experiment, the results of the CEDIA® are compared with those of the DPC assay, and with those obtained using the "Abuscreen"® radioimmunoassay by Roche. While the amount of metabolite in the sample is not known in absolute terms, the behavior is tested by dilution analysis. Results are given in terms of LSD equivalents.

The data indicate that the reagents and methods of the invention recognize the metabolite in the sample about 10-fold better than does the DPC assay, and about 2.5 fold better than the Abuscreen® assay.

The ability to detect a urinary metabolite with a longer half-life is of considerable benefit in evaluating an individual's exposure to LSD. Immunoassay kits of the invention can be used for determining whether a subject has been administered LSD by conducting an immunoassay on a urine sample and then correlating any LSD or LSD metabolite detected with administration of LSD to the subject. A metabolite with a longer biological half-time will provide evidence of a previous exposure even if the sample has been obtained several days after the exposure. Where the sample contains the metabolite, the time between exposure and sample collection can be at least 2 days, and as much as 3 or 4 days later, depending on the amount of LSD administered, urine storage time, and the metabolic rate of the individual tested. To the extent that the metabolite also appears in blood before excretion by the kidney, the materials of this invention can also be used to measure its presence in plasma or serum samples or other biological fluids, and then correlate its presence with LSD exposure. Ultimate interpretation of the results is the responsibility of the managing clinician.

Where permitted by law, all publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be understood that the specification and examples are illustrative but not limiting of the present invention, and that modifications and changes will suggest themselves to those skilled in the art but will not depart from the spirit and scope of the appended claims.


Anspruch[en]
  1. An antibody specific for a glucuronide metabolite of LSD for use in an immunoassay for determination of a glucuronide metabolite of LSD in a biological sample.
  2. An antibody specific for a glucuronide metabolite of LSD for use in an immunoassay for determination of LSD or an LSD metabolite excreted into a urine sample from a subject over a period extending to about 48 hours after exposure of the subject to the LSD.
  3. A reagent system for use in an immunoassay for determination of a glucuronide metabolite of LSD excreted into a biological sample, comprising the antibody according to claim 1 and a competitive binding compound capable of competing with the glucuronide metabolite of LSD for binding to the antibody, said competitive binding compound being conjugated to a detectable label.
  4. A reagent system for use in an immunoassay for determination of LSD or an LSD metabolite excreted into a urine sample from a subject over a period extending to about 48 hours after exposure of the subject to the LSD, comprising the antibody according to claim 2 and a competitive binding compound capable of competing with the glucuronide metabolite of LSD for binding to the antibody, said competitive binding compound being conjugated to a detectable label.
  5. An immunoassay method for determining the possible presence of a glucuronide metabolite of LSD in a biological sample, comprising the steps of:
    • a) preparing a reaction mixture comprising the sample, an antibody according to claim 1, and a competitive binding compound capable of competing with the glucuronide metabolite of LSD for binding to the antibody, said competitive binding compound being conjugated to a detectable label;
    • b) determining the amount of label bound to the antibody in the reaction mixture;
    • c) correlating the amount determined in step b) with the amount of LSD and/or glucuronide metabolite of LSD in the sample; and
    • d) if a detectable amount of LSD and/or glucuronide metabolite of LSD is detected in the sample, then optionally distinguishing between LSD and glucuronide metabolite of LSD in the sample.
  6. An immunoassay method for determination of LSD or an LSD metabolite in a urine sample from a subject, comprising the steps of:
    • a) preparing a reaction mixture comprising the sample, an antibody according to claim 2, and a competitive binding compound capable of competing with the glucuronide metabolite of LSD for binding to the antibody, said competitive binding compound being conjugated to a detectable label;
    • b) determining the amount of label bound to the antibody in the reaction mixture; and
    • c) correlating the amount determined in step b) with exposure of the subject to LSD within a period extending to about 48 hours before collection of the sample from the subject.
  7. Use of an antibody according to claim 1 in the manufacture of a kit or reagent set for conducting an immunoassay for determination of a glucuronide metabolite of LSD in a biological sample.
  8. Use of an antibody according to claim 2 in the manufacture of a kit or reagent set for conducting an immunoassay for determination of LSD or an LSD metabolite excreted into a urine sample from a subject over a period extending to about 48 hours after exposure of the subject to the LSD.
  9. The reagent system of claim 3-4 or the assay method of claim 5-6, wherein the detectable label is selected from the group consisting of:
    • i) a fluorescent label or fluorescent quench label,
    • ii) an enzyme, wherein the activity of the enzyme is affected by binding of the antibody to the labeled competitive binding compound; and
    • iii) an enzyme fragment capable of complementing with a second enzyme fragment to form an active enzyme when the labeled competitive binding compound is not bound to the antibody.
  10. The reagent system of claim 3-4 or the assay method of claim 5-6, wherein the competitive binding compound is an LSD derivative, and the label is conjugated to the LSD derivative via the indole ring of the LSD derivative.
  11. The reagent system of claim 3-4 or the assay method of claim 5-6, wherein the detectable label is an enzyme donor of β-galactosidase.
  12. The product, method or use of any preceding claim, wherein the immunoassay is a homogeneous immunoassay.
  13. The product, method or use of any preceding claim, wherein the antibody is specific for both LSD and a glucuronide metabolite of LSD.
  14. The product, method or use of claim 13, wherein the antibody has at least about 10% cross-reactivity with glucuronide metabolites of LSD, compared with LSD.
  15. The product, method or use of any preceding claim, wherein the median relative level of LSD and LSD metabolites measurable in the immunoassay in sequential urine samples of a pharmacokinetic study taken from a subject 36 hours after administration of LSD is at least about 5% of the highest levels of LSD and LSD metabolites measurable in urine samples from the same subject within the first 24 h after the administration.
  16. The product, method or use of any preceding claim, having one or more of the following features:
    • i) the antibody has less than 1% cross-reactivity with chlorpromazine, compared with LSD or glucuronide metabolites of LSD;
    • ii) the antibody has less than 1% cross-reactivity between LSD or glucuronide metabolites of LSD, and ergotamine; or
    • iii) the frequency of false positive reactions with clinical urine samples not containing LSD in the immunoassay is less than 4%.






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