EP1066523A2 - Dosages confirmatifs de drogues constituees de molecules de taille reduite - Google Patents

Dosages confirmatifs de drogues constituees de molecules de taille reduite

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Publication number
EP1066523A2
EP1066523A2 EP98915179A EP98915179A EP1066523A2 EP 1066523 A2 EP1066523 A2 EP 1066523A2 EP 98915179 A EP98915179 A EP 98915179A EP 98915179 A EP98915179 A EP 98915179A EP 1066523 A2 EP1066523 A2 EP 1066523A2
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EP
European Patent Office
Prior art keywords
analyte
antibody
assay
sample
anfibody
Prior art date
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EP98915179A
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German (de)
English (en)
Inventor
Jeffrey E. Shindelman
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Microgenics Corp
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Microgenics Corp
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Publication of EP1066523A2 publication Critical patent/EP1066523A2/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • This invention relates generally to the field of the detection of drugs and drug metabolites in biological samples. More specifically, it provides a system for confirming the presence of a particular analyte in a sample potentially containing interfering substances.
  • Antibodies can be raised that have extraordinarily specificity and sensitivity for small molecules of diagnostic interest. In combination with other reagents that have a separating or labeling function, specific antibodies can be used as part of a rapid screening test for the presence of the small molecule in a clinical sample.
  • Small molecules that can be measured in this way include hormones, natural metabolites, and prescription drugs.
  • substances of abuse include canabinoids, tranquilizers such as barbiturates, stimulants such as amphetamines, hallucinogenic alkaloids such as cocaine and lysergic acid diethylamide (LSD), and anabolic steroids.
  • canabinoids tranquilizers such as barbiturates
  • stimulants such as amphetamines
  • hallucinogenic alkaloids such as cocaine and lysergic acid diethylamide (LSD)
  • anabolic steroids anabolic steroids.
  • U.S. Patent Nos. 4,952,336 and 5,354,693 and E.P. Patent 371,253-B relate to the detecting of analytes in the amphetamine class in an assay using fluorescent tracers and antibodies raised against amphetamine derivatives.
  • an assay method is recited in which the sample is combined with reagent antibodies and protein conjugates for both amphetamine and methamphetamine.
  • Diagnostic kits for measuring amphetamine class compounds are available commercially using the CEDIA® DAU technology of Boehringer Mannheim Corp., the fluorescence polarization immunoassay (FPIA) technology of Abbott Laboratories, the EMIT® II technology of Behring Diagnostics, the COBAS® INTEGRA technology of Roche Diagnostics, and the COAT-A- COUNT® radioassay technology of Diagnostic Products Corporation.
  • Radioimmunoassay kits for LSD are sold in the ABUSCREEN® product line by Roche and the COAT-A-COUNT® product line .
  • International patent application PCT/US 96/19266 provides reagents and procedures for the detection and measurement of LSD in an enzyme immunoassay.
  • a diagnostic kit for LSD based on this technology is commercially available in the CEDIA® DAU product line. More recently, other enzyme-type immunoassays for LSD and related compounds have been developed (Hu et al., Clin. Chem. (1996) 42:S219; Webb et al., J. Forensic Sciences (1996) 41 :938).
  • the touchstone verification test for small molecule drugs is mass spectroscopy — particularly when combined with gas or liquid chromatography (GC/MS or LC/MS), or tandem mass spectrometry (GC/MS/MS or LC/MS/MS). These secondary tests are complex, require expensive instrumentation and highly trained personnel, and therefore are very expensive as compared to the initial screening test. Any reduction in the proportion of false positives selected for verification reduces the overall costs of drug screening.
  • U.S. Patent No. 3,856,469 relates to the removal of ⁇ -hydroxyamine compounds from samples to be tested for amphetamine, by treating with aqueous periodate under mildly basic conditions. Modifications of the periodate treatment method have been described for reducing interference by ephedrine, pseudoephedrine, and phenylpropanolamine. See Elsohly et al., J. Anal. Toxicol. (1992) 16:109; Spiehler et al., J. Anal. Toxicol. (1993) 17:125; and Paul et al., J. Anal. Toxicol. (1994) 18:331.
  • U.S. Patent No. 5,573,955 describes reducing tyramine interference in amphetamine immunoassays by treating with tyramine oxidase.
  • U.S. Patent No. 4,843,020 relates to a method for detecting tetrahydrocannabinol (THC) in human urine by precipitating out any melanin that might be present by treating with nitroferricyanide.
  • U.S. Patent No. 4,477,346 relates to a method for removing interfering substances in theophylline assays, particularly caffeine. The sample to be tested is pre-treated by liquid-liquid extraction using hydrophobic, macroreticular resin slurried in a protic solvent.
  • U.S. Patent No. 5,518,887 relates to competition assays using an insolubilized analog of the substance to be measured. Two different detecting antibodies are used, one of which is selected to have less cross-reactivity for the interfering substance.
  • the disclosure proposes that the method be used to distinguish between phencyclidine (PCP) and structurally related substance, particularly 2- ethylidene-1 ,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), a methadone metabolite.
  • PCP phencyclidine
  • EDDP 2- ethylidene-1 ,5-dimethyl-3,3-diphenylpyrrolidine
  • the interfering substance is proposed to have less effect on the antibody that is more specific.
  • positive inhibition of both antibodies would confirm the presence of the true analyte.
  • U.S. Patent No. 4,722,889 relates to a solid-phase sandwich-type immunoassay for huma ⁇ ⁇ chorionic gonadotropin (hCG).
  • hCG huma ⁇ ⁇ chorionic gonadotropin
  • a scavenger antibody is included in the reaction mixture that prevents cross-reacting protein hormones from reacting with the capture or detecting antibody.
  • U.S. Patent No. 5,296,354 relates to a competition-type immunoassay that is intended to distinguish Angiotensin II from any Angiotensin III that may be present in the sample. Detection of the analyte occurs via an inhibition of the reaction between an anti-Angiotensin II antibody and a labeled Angiotensin II reagent.
  • the reaction mixture includes an antibody more specific for Angiotensin III, which is intended to prevent any Angiotensin III from inhibiting the reaction between the anti-Angiotensin II and the labeled reagent.
  • U.S. Patent No. 5,064,755 is directed to a two-site confirmatory assay.
  • the disclosure relates a method for confirming the presence in a sample of a Chlamydia antigen, which is a complex multi-epitope antigen with variant subspecies.
  • the confirmatory assay involves comparing the results of two different tests.
  • the first test involves developing the sample with a detecting antibody specific for Chlamydia antigen.
  • the second test involves treating a duplicate sample with a confirmatory antibody before the detecting antibody.
  • a predetermined decrease in signal resulting from pretreatment with the confirmatory antibody confirms the presence of the antigen in the sample.
  • the second antibody is chosen so as to be capable of binding a second epitope on the Chlamydia and thereby prevent the binding of the first antibody.
  • U.S. Patent No. 5,308,755 is directed to an assay device for concurrently detecting an analyte and confirming the test result.
  • the exemplary device has two fluid-flow pathways, and is illustrated for use in detecting hepatitis B surface antigen (HBsAg).
  • the first pathway contains a binding member (such as anti-HBsAg) which participates in forming an immobilized labeled complex if analyte is present in the test sample.
  • the second pathway contains in addition a mobile confirmatory reagent (such as anti-HBsAg) which inhibits the binding of the immobilized labeled complex.
  • the amount of labeled analyte complex immobilized in the first pathway is related to the presence of analyte in the sample, which is confirmed if the confirmatory reagent prevents the formation of the immobilized labeled complex.
  • a neutralization assay for confirming HBsAg in a sample is manufactured by Sorin Biomedica (Italy) and distributed in the U.S. by Incstar Corporation. The assay is described in the Instruction Manual for the REAC-801 Confirmatory Test.
  • HBsAg is captured using a first antibody on a solid phase, and developed using a labeled second antibody that reacts against a second epitope on the HBsAg.
  • the confirmatory test involves treating the captured HBsAg with an unlabeled, neutralizing antibody before adding the labeled antibody.
  • the neutralizing antibody is specific for HBsAg, and blocks subsequent binding of the labeled antibody to its specific epitope on HBsAg. If the signal of the neutralized sample is significantly lower than the signal of the non-neutralized sample, the presence of HBsAg in the sample is confirmed.
  • the present invention provides a system for the improved detection of analytes, and the ability to distinguish them from cross-reacting substances.
  • Samples giving a positive reaction in a direct immunoassay test are treated with a neutralizing antibody that inhibits reactivity of the true analyte, but not the interfering substance.
  • samples giving a positive reaction in the direct test but decreased reaction in the confirmation test are marked as containing the true analyte.
  • Samples giving a positive reaction of roughly equivalent magnitude in both the direct and confirmation test are marked as containing an interfering substance.
  • Certain types of confirmatory assays described in this disclosure are classified as bidirectional antibody type confirmatory assays.
  • Certain types of confirmatory assays described in this disclosure are adsorption type confirmatory assays. Exemplary confirmatory assays of this invention meet the requirements of one or both of these classifications.
  • the detecting antibody and the neutralizing antibody recognize the analyte from different orientations.
  • the neutralizing antibody does not impede the analyte detection system, and can optionally be added directly into the final reaction mixture.
  • Embodiments relating to bidirectional antibody type confirmation assays include reagent sets and kits for determining an analyte in a sample in a confirmatory assay.
  • One such embodiment is a set of reagents comprising a detecting antibody for the analyte; and a neutralizing antibody for the analyte.
  • the detecting antibody is raised against a first conjugate in which a hapten is linked to a carrier;
  • the neutralizing antibody is raised against a second conjugate in which a hapten is linked to a carrier through a position of the hapten which is different from that of the hapten in the first conjugate. Binding of the neutralizing antibody to the analyte inhibits binding of the analyte to the detecting antibody.
  • Another such embodiment is a set of reagents comprising a detecting antibody, a neutralizing antibody, and a competitive binding compound.
  • An exemplary competitive binding compound is a hapten derivative, such as a hapten-protein conjugate or a hapten labeled with a radioisotope or fluorochrome.
  • the detecting antibody binds the competitive binding compound in a manner that is specifically inhibitable by the analyte; the hapten derivative preferentially binds the detecting antibody in comparison with the neutralizing antibody; and binding of the neutralizing antibody to the analyte inhibits binding of the analyte to the detecting antibody.
  • the competitive binding compound is a hapten conjugate
  • the detecting antibody preferably binds the hapten of the conjugate in a manner that is specifically inhibitable by the analyte.
  • kits for adapting an assay to permit distinguishing the analyte from an interfering substance, comprising a neutralizing antibody comprising a neutralizing antibody.
  • the assay is conducted using a detecting antibody and a competitive binding compound, and the neutralizing antibody provided in this embodiment performs in the assay according to the properties already outlined.
  • written instructions for conducting the confirmatory assay are included with the kit.
  • the assay method for confirming the analyte involves preparing a reaction mixture comprising the sample, the detecting antibody, a neutralizing antibody, and a competitive binding compound. Binding of the analyte to the neutralizing antibody inhibits binding of the analyte to the detecting antibody, and the competitive binding compound preferentially binds
  • a reaction mixture is prepared, comprising the sample, a detecting antibody, and a competitive binding compound, wherein the detecting antibody binds the compound in a manner that is specifically inhibitable by the analyte.
  • a second reaction mixture is prepared, comprising the sample, the detecting antibody, a neutralizing antibody, and a competitive binding compound, wherein binding of the analyte to the neutralizing antibody inhibits binding of the analyte to the detecting antibody, and wherein the competitive binding compound preferentially binds the detecting antibody in comparison with the neutralizing antibody.
  • the amount of detecting antibody bound to the competitive binding compound is compared between the reaction mixtures.
  • the neutralizing antibody preferentially inhibits binding to the detecting antibody of the analyte compared with the interfering substance.
  • the presence of true analyte is confirmed if there is a significant effect on the result due to use of the neutralizing antibody.
  • the sample is treated with an amount of neutralizing antibody that is sufficient to remove the analyte but not all the interfering substance from the sample, or otherwise prevent its binding to the detecting antibody.
  • the neutralizing antibody is provided in an insolubilized form, and the detection step is performed after removal or neutralization of the analyte.
  • Embodiments relating to adsorption type confirmation assay include reagent sets and kits for distinguishing between an analyte and an interfering substance.
  • One such embodiment comprises a detecting antibody for the analyte, and a neutralizing antibody for the analyte.
  • the neutralizing antibody preferentially binds the analyte in comparison with the interfering substance.
  • the neutralizing antibody is preferably either aliquoted in an amount sufficient to remove the analyte but not all the interfering substance from the sample, or a written indication is provided as to the amount required.
  • the set of reagents also typically comprises a competitive binding compound, with the property that the detecting antibody binds the hapten derivative in a manner that is specifically inhibitable by the analyte and the interfering substance.
  • a competitive binding compound is a hapten derivative, such as a hapten-protein conjugate or a hapten labeled with a radioisotope or fluorochrome.
  • kits for adapting an assay to permit distinguishing the analyte from an interfering substance, comprising a neutralizing antibody comprising a neutralizing antibody.
  • the assay is conducted using a detecting antibody and a competitive binding compound, and the neutralizing antibody provided in this embodiment per orms in the assay according to the properties already outlined. Typically, written instructions for conducting the confirmatory assay are included with the kit.
  • Further embodiments of the invention are assay methods.
  • One such embodiment is an assay method for distinguishing between a small molecule analyte and an interfering substance in a sample.
  • a direct assay is conducted to determine the amount of analyte and/or interfering substance in the sample.
  • the same sample or a duplicate is treated with a neutralizing antibody in an amount sufficient to remove the analyte but not the potential interfering substance, and an assay is conducted on the treated sample. The amount detected is then compared between the treated and untreated sample.
  • a reaction mixture is prepared, comprising the sample, a detecting antibody, and a competitive binding compound, wherein the detecting antibody binds the hapten derivative in a manner that is specifically inhibitable by the analyte.
  • the amount of the detecting antibody bound to the competitive binding compound is measured.
  • the same sample is treated with a neutralizing antibody, or else a duplicate sample is treated with a neutralizing antibody before, during or after the direct test.
  • the neutralizing antibody prevents the analyte but not all the interfering substance in the sample from being available to bind the detecting antibody when an assay is conducted on the treated sample.
  • the results from the direct and the confirmatory test are then compared. The presence of true analyte is confirmed if there is a significant effect on the result due to use of the neutralizing antibody.
  • Such technologies are especially appropriate for use on small molecule drugs, such as substances of abuse and their metabolites.
  • Particular small molecules of interest include amphetamines, LSD, and their respective metabolites.
  • Exemplary detecting and neutralizing antibodies for determining substances of the amphetamine class are raised against amphetamine derivative linked to a carrier through a position in the phenol ring, and through the alkyl amine group in either order.
  • Exemplary detecting and neutralizing antibodies for determining substances of the LSD class are raised against LSD derivative linked to a carrier through the indole ring, and through the alkyl amide group.
  • the technology can be applied to immunoassay detection methods, including but not limited to homogeneous assay systems, such as those involving an enzyme or fluorescent marker in the detection system.
  • Figure 1 is a series of three graphs illustrating a bidirectional antibody type confirmation assay for amphetamines.
  • the upper graph shows a typical standard curve for amphetamine ( ⁇ ) and methamphetamine in a homogeneous assay.
  • the exemplar is an enzyme complementation assay commercially available in the CEDIA® DAU product line.
  • the lower graphs compare the results of the direct assay (left) with the assay adapted to confirm the presence of an amphetamine (right), using a neutralizing antibody specific for both amphetamine and methamphetamine.
  • the neutralizing antibody is specific for the analyte but not the hapten conjugate with which the analyte competes. It neutralizes the true analyte, thereby suppressing the subsequent enzyme signal.
  • Figure 2 is a series of four graphs illustrating the results of the amphetamine confirmation assay for samples spiked with several potential interfering substances. The samples were tested in the absence ( ⁇ ) and presence ( ⁇ ) of the neutralizing antibody.
  • Panel A pseudoephedrine
  • Panel B phenylpropanolamine
  • Panel C phentermine
  • Panel D tyramine.
  • the neutralizing antibody does not eliminate enzyme signal that is due to interfering substance.
  • Figure 3 is a series of four graphs illustrating an adsorption-type confirmation assay for LSD.
  • the upper graph shows the results of a CEDIA ® DAU LSD assay in the direct test ( ⁇ ); or after pretreatment of the sample with the neutralizing antibody ( ⁇ ).
  • the neutralizing antibody is insolubilized, and removes the analyte from the portion of the sample, which is then tested using the detecting antibody. Elimination of the true analyte again suppresses the subsequent enzyme signal.
  • the lower three graphs compares the results of the direct ( ⁇ ) and confirmation ( ⁇ ) test for LSD (Left Panel), 2-oxo-3-hydroxy LSD (Center Panel) or a partially purified preparation containing a mixture of the putative LSD metabolites 13-hydroxy LSD giucuronide and 14-hydroxy LSD giucuronide (Right Panel).
  • Figure 4 is a series of three graphs comparing the results of the direct ( ⁇ ) and confirmation ( ⁇ ) test for LSD for samples spiked with the following potential interfering substances: Left Panel: methamphetamine; Center Panel: fenfluramine; Right Panel: ambroxol.
  • Figure 5 is a series of six graphs in which the results from Figures 3 and 4 are displayed as relative rates.
  • ( ⁇ ) ⁇ Rate 1 difference between direct test and baseline);
  • ( ⁇ ) ⁇ Rate 2 difference between confirmation test and direct test).
  • the present invention is directed to reagents and methods for detecting or confirming the presence of an analyte in a test sample.
  • the analyte is distinguished from one or more cross- reacting interfering substances that may be present in the sample, either as an alternative to or in combination with the analyte.
  • Immunoassays for small molecule drugs are especially susceptible to the problem of cross- reactivity.
  • some samples test positive by immunoassay but fail to confirm by GC/MS.
  • GC/MS GC/MS-based spectroscopy
  • the test range for LSD in urine is about 0.1 to 5 ng/ml
  • potentially interfering drugs such as acetaminophen, fluoxetine, ibuprofen, cimetidine, and chlorpromazine
  • the cross-reactivity of the detecting antibody may be as low as 0.0003% to 0.01% (Example 7)
  • these substances can cause a substantial number of false positives in a standard LSD immunoassay.
  • the materials and methods of this invention provide an improved approach for distinguishing a test analyte from interfering substances in an immunoassay. Rather than trying to remove or neutralize the interfering substance, the invention is aimed at removing or neutralizing the true analyte, and retesting the sample for the interfering substance.
  • the advantages of this invention include the following features:
  • the neutralizing antibody can be added directly to the reaction mixture, without a separation step
  • the test is able to distinguish the analyte even in a multi-fold excess of the interfering substance
  • the neutralizing antibody in the confirmation part of the test need not be more specific for the analyte than the detecting antibody. This last feature is particularly counterintuitive: since the purpose of the neutralizing antibody is to distinguish multiple compounds that all react with the detecting antibody, it would seem that the neutralizing antibody would have to be even more specific. But this is not the case.
  • This disclosure teaches several ways in which a neutralizing antibody can be set up to differentiate between the analyte and the interfering substance, even when its cross-reactivity profile is no better than that of the detecting antibody.
  • Non-limiting illustrations are provided in this disclosure of diagnostic-grade confirmatory assays for amphetamines and for LSD.
  • a direct and a neutralization test are performed.
  • the direct test is an assay (typically an immunoassay) for the analyte or the interfering substance.
  • the neutralization test is an assay (typically an immunoassay) in which the analyte has been removed or inactivated, but the interfering substance has not.
  • Samples giving a positive reaction in the direct test but negative reaction in the confirmation test are marked as containing the true analyte.
  • Samples giving a positive reaction in both the direct and confirmation test are marked as containing an interfering substance.
  • confirmation assay can refer to the neutralization test alone, or both tests together.
  • a “bidirectional antibody type confirmatory assay” is an assay in which the neutralizing antibody and the detecting antibody recognize the analyte from different orientations.
  • An “adsorption type confirmatory assay” is an assay in which the sample is treated with a particular amount of neutralizing antibody that is sufficient to remove the analyte but not all the interfering substance from the sample, or otherwise prevent its binding to the detecting antibody.
  • 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.
  • antibody 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. Examples of antibodies other than intact immunoglobulins are provided below.
  • antibody activity refers to the ability of an antibody to bind a specific antigen in preference to other potential antigens via the antigen combining site located within a variable region of an immunoglobulin.
  • a "detecting antibody” is an antibody that is used in an immunoassay for detecting the presence of an analyte in a sample.
  • the detecting antibody will be able to distinguish between the analyte and other substances that may be present in the sample, although there may be a subset of substances that cross-react.
  • the immunoassay is performed by contacting the antibody with the sample under conditions that permit the antibody to form a complex with any analyte present, and measuring any complex formed.
  • a “neutralizing antibody” is an antibody that is used in an assay for confirming the presence of an analyte in a sample.
  • the neutralizing antibody will be able to bind the analyte and thereby prevent it from giving a positive reaction in an assay, particularly an immunoassay conducted with a detecting antibody.
  • 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 assay.
  • an “analyte” is a substance of interest to be measured in a sample using a particular assay system. It may have any size, structure, or valence irrespective of components used in the assay system, unless otherwise specified or required.
  • a "small molecule analyte” has a size of ⁇ 5,000 mol wt and typically ⁇ 1 ,000 mol wt.
  • 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.
  • Non limiting examples include canabinoids, barbiturates, amphetamines, hallucinogenic alkaloids and anabolic steroids taken for reasons other than the bone fide prevention or treatment of disease.
  • hapten denotes a homogeneous or heterogeneous chemical compound, generally ⁇ 5,000 mol wt and typically ⁇ 1 ,000 mol wt, with the property that a complex between the hapten and one hapten-specific anfibody will inhibit the binding of a second hapten-specific antibody, regardless of whether the second antibody is different from the first or recognizes the hapten from a different orientation.
  • a hapten has a functional valence of one with respect to anfibody binding.
  • a "hapten derivative” denotes a compound that contains a feature of a hapten that is specifically recognizable by an anti-hapten antibody, and has been derivatized to provide it with an additional property of interest.
  • hapten derivatives include a hapten covalently linked to a protein, and a hapten covalently linked to a solid surface (where the linkage is a covalent bond or a linking group of one or more atoms, such as may be formed by chemical synthesis or conjugation using a cross-linking agent).
  • Other examples of hapten derivatives are haptens that have been chemically derivatized with a labeling feature, such as a fluorescent structure, a fluorescent quenching structure, or an enzyme inhibitor.
  • a hapten derivative need not be functionally monovalent with respect to antibody binding — for example, a hapten-protein conjugate can optionally contain a plurality of haptens, depending on its intended role, and the valence will approach that of the conjugation ratio.
  • 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 is said to be “purified” if it is (except from solvent) at least 50%, preferably at least about 90%, and even more preferably at least about 99% pure when analyzed by a suitable technique such as GC/MS.
  • a “synthetic” compound is a compound assembled from component parts by a process that does not involve live organisms or cells.
  • amhetamine when used in the plural or with the indefinite article refers not only to 1-phenyl-2-aminopropane, but also 1-phenyl-2-methylaminopropane (methamphetamine), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), p- hydroxyamphetamine, and other structurally related sympathomimetic amines, especially phenethylamines, with psychostimulant activity.
  • methamphetamine methylenedioxyamphetamine
  • MDMA methylenedioxymethamphetamine
  • p- hydroxyamphetamine p- hydroxyamphetamine
  • other structurally related sympathomimetic amines especially phenethylamines, with psychostimulant activity.
  • a bidirectional anfibody type confirmatory assay is an assay in which the neutralizing antibody and the detecting antibody recognize the analyte from different orientations. Depending on the nature of the assay, this can provide either of the following advantages:
  • neutralizing antibodies can be obtained that can be added directly to the reaction mixture without affecting the detection system. In this embodiment, no preadsorption with the neutralizing antibody is required.
  • the usual reagents for conducting the direct test for analyte are a detecting anfibody for the analyte, and a competitive binding compound which is typically a hapten derivative.
  • the detecting antibody binds the hapten derivative in a manner that is specifically inhibitable by the analyte.
  • the hapten derivative is typically a modified form of the analyte or a closely related chemical structure, adapted so as to provide a signal detection system.
  • the hapten derivative is a fluorescent hapten or a fluorescent quench hapten, typically a chemically modified form of the analyte in which the fluorescent or fluorescent quench group is adapted onto one end.
  • the hapten derivative is a conjugate in which the hapten is linked to an enzyme, either through a covended bond or through a bridging structure, such as may be formed using a cross-linking agent, or using a hapten chemically modified with the bridging structure with a protein-linking group on the far end.
  • the hapten derivative is a conjugate in which the hapten is linked to an enzyme fragment which complements with a second enzyme fragment to form an active enzyme complex.
  • the hapten derivative is a conjugate in which the hapten is linked to an inert substance, such as a large protein, a polymer (such as polystyrene, polyacrylamide, latex, or a high molecular weight carbohydrate), a particulate, or the surface of the reaction vessel.
  • an inert substance such as a large protein, a polymer (such as polystyrene, polyacrylamide, latex, or a high molecular weight carbohydrate), a particulate, or the surface of the reaction vessel.
  • the direct test is conducted by preparing a reaction mixture comprising the sample, the detecting antibody, and the competitive binding compound.
  • the test is completed by measuring the amount of the complex formed between the detecting anfibody and the competitive binding compound, in competition with the analyte from the sample.
  • the amount of complex may correlate positively or inversely with the amount of analyte (or cross-reacting substance) in the sample.
  • the neutralization test is conducted in a similar fashion, using a detecting antibody for the analyte, and a competitive binding compound which is typically a hapten derivative.
  • a detecting antibody for the analyte and a competitive binding compound which is typically a hapten derivative.
  • the neutralization test additionally involves a neutralizing antibody, which has the function of preventing a proportion of the true analyte from reacting with the detection antibody, thereby reducing the assay signal.
  • the neutralization test is typically conducted by preparing a reaction mixture comprising the sample, the detecfing antibody, the neutralizing antibody, and the competitive binding compound or hapten derivative, and then measuring the formation of reaction complexes as in the direct test.
  • the neutralizing anfibody is preincubated with the sample in which the analyte is to be measured. Usually, however, this is not required, and it is sufficient to add the sample to a reaction mixture containing the detecting and neutralizing antibody together.
  • the hapten conjugate reagent is added after the antibodies are in equilibrium with any analyte in the sample. More generally, the reagents may be combined in any order, depending on the kinetic parameters of the reaction system.
  • the neutralization test is completed by measuring the amount of the complex formed between the detecting antibody and the competitive binding compound, and correlating the result with the degree of neutralization of the substance in the original sample. Successful neutralization of a sample testing positive in the direct test indicates the presence of true analyte.
  • the hapten derivative "preferentially" binds the detecfing antibody in comparison with the neutralizing antibody. This means that under assay conditions and in the absence of analyte, the proportion of hapten derivative bound to detecting anfibody is at least about 10 times higher, preferably at least about 100 or 1000 times higher than that the proportion of hapten derivative bound to neutralizing anfibody. Typically, the affinity of the detecting antibody for the hapten derivative is at least 10 times and preferably 100 or 1000 times higher than that of the neutralizing anfibody.
  • the affinity of the detecfing antibody for the hapten derivative will generally have an affinity of at least about 10 8 M '1 , with affinities of at least about 10 9 M " ⁇ 10 10 M 1 , and 10 11 M " being increasingly more preferred.
  • the binding of the neutralizing antibody to the hapten derivative will generally have an affinity of no more than about 10 7 M ⁇ ⁇ with affinities of no more than about 10 6 M “1 or 10 5 M "1 being increasingly more preferred. Binding of the neutralizing antibody to the analyte inhibits binding of the analyte to the detecfing antibody.
  • the binding of the analyte to the detecting antibody and subsequent specific signal generation is reduced by at least about 2-fold, and preferably 5-foid, 25-fold, or 100- fold in order of increasing preference.
  • Higher degrees of inhibition can be obtained by using a neutralizing antibody that has a higher affinity for the analyte than does the detecting antibody.
  • Another option is to preincubate the neutralizing antibody with the sample before adding the detecting anfibody in a non-equilibrium situation. More typically, the amount of neutralizing antibody in the reaction mixture is in excess (preferably 10 fold or even 100 fold higher), thereby increasing " analyte binding to the detecting anfibody by mass action.
  • the neutralizing anfibody also "preferentially" inhibits binding of the analyte to the detecting anfibody, in comparison with a potential interfering substance.
  • the proportion of analyte bound to the neutralizing antibody is at least about 5 times higher, preferably at least about 25, 100, or 1000 times higher than that the proportion of interfering substance bound to neutralizing antibody.
  • Preferential inhibition may occur because the affinity of the neutralizing anfibody for the analyte is at least 10 times and preferably 100 or 1000 times higher than it is for the potential interfering substance.
  • detecting and neutralizing anfibodies are obtained by using immunogen or antibody screening or purifying reagents using different hapten-carrier conjugates, in which a chemical analog of the analyte is linked to the carrier in different orientations.
  • the conjugate used to raise or select the detecfing anfibody is usually one in which the hapten is linked to the carrier in a position that is identical or nearby the position in which the hapten is linked or modified in the hapten derivative used in the assay.
  • the conjugate used to raise or select the neutralizing antibody is selected to enhance the likelihood that the anfibody will not react with the hapten derivative used in the assay, in accordance with the properties described above.
  • the conjugate presents the hapten so as to elicit anfibodies that will be sterically inhibited from reacting with the hapten derivative of the assay (in the case of an enzyme conjugate), or will be faced with the signaling moiety of the hapten derivative (in the case of a fluorescently modified analog) and be unlikely to cross-react.
  • the presentation of the hapten used to raise the detecting and neutralizing antibodies will be from opposite ends of the molecule.
  • Exemplary confirmatory assays are based on the CEDIA® DAU immunoassay technology.
  • the assay method is a homogeneous enzyme complementafion assay, in which enzyme donor and enzyme acceptor fragments of ⁇ -galactosidase combine to form an active enzyme complex.
  • the enzyme donor is conjugated with an analog of the test analyte, and a reagent antibody against the analyte is also present in the reaction mixture.
  • the anfibody binds to the enzyme donor conjugate, which is thereby inhibited from complementing the enzyme acceptor to form an active enzyme.
  • test analyte When test analyte is present, it binds the anfibody, the antibody cannot bind the enzyme donor, and enzyme complementafion occurs.
  • the rate of enzyme catalysis (measured by conversion of a chromogenic substrate) is directly related to the amount of analyte in a test sample.
  • One particular example is based on the CEDIA® DAU immunoassay for LSD.
  • the detecting " antibody in the kit was raised to LSD attached at the indole nitrogen to the immunogenic carrier protein KLH via a linker arm.
  • Structure "A” shows an activated LSD derivative used to prepare the immunogen, in which LSD is linked to KLH through the indole ring; specifically at the N-1 position.
  • anfibodies can also be raised using KLH conjugates formed by the Mannich reaction, in which LSD is linked through the 2 position.
  • the enzyme donor in the kit is a conjugate in which the LSD is linked to the ⁇ -galactosidase through the same N-1 position.
  • Neutralizing anfibodies suitable for use in a confirmation assay can be obtained using an immunogen generated using structure "B", in which LSD is linked to the carrier protein via the alkyl amide group.
  • Another example is based on the CEDIA® DAU immunoassay for amphetamines.
  • This kit measures both 1-phenyl-2-aminopropane (amphetamine) and 1-phenyl-2-methylaminopropane (methamphetamine), using separate detecting antibody-enzyme donor reagent pairs in the same reaction mixture.
  • the two detecfing anfibodies were raised to amphetamine and methamphetamine attached to carrier proteins via the phenyl ring.
  • Neutralizing anfibodies suitable for use in a confirmation assay can be obtained using an immunogen generated using structure "D", in which an amphetamine is linked to the carrier protein via the alkyl amide group.
  • a neutralizing anfibody can be selected that commonly removes both amphetamine and methamphetamine, or separate anfibodies can be obtained each of which removes one of the two compounds.
  • neutralizing antibodies can be obtained that are suitable with other established detection assays for small molecule drugs.
  • a neutralizing antibody is obtained using conjugates of Structure C or Structure D, above, selected to present the amphetamine in an opposite direction from that of the G6PD conjugate.
  • the neutralizing anfibody will be raised or selected using conjugates in which an amphetamine is linked to the carrier protein via the alkyl amine group, and vice versa.
  • anfibodies raised using conjugates in which an amphetamine is linked to the carrier protein via the alkyl amine group are likely to be the most effective neutralizers.
  • anfibodies raised using conjugates in which an amphetamine is linked to the carrier protein via the phenyl ring are likely to be the most effective neutralizers.
  • Antibody pairs for use in bidirectional type confirmatory assays for PCP can be prepared using PCP immunogens according to Structure E and Structure F, shown below.
  • PCP chemistry and assays are described in Ceneste et al. (Bull. Soc. Chim. France 11 :610, 1978) and Katz et al. (Theriogenology 6:2-3, 1976).
  • the hapten conjugate is prepared according to Structure E.
  • the hapten conjugate is prepared according to Structure F.
  • the sample is treated with a particular amount of neutralizing anfibody that is sufficient to remove the analyte but not all of a particular interfering substance from the sample, or otherwise prevent its binding to the detecfing antibody.
  • the neutralizing antibody preferentially binds the analyte in comparison with the interfering substance, meaning that it binds the analyte about 5 times better, preferably at least about 25, 100, or even about 1000 times better, compared with potential interfering substance. Preferenfial inhibifion may occur because the affinity of the neutralizing antibody for the analyte is at least 10 times and preferably 100 or 1000 times higher than it is for the potential interfering substance.
  • the neutralizing antibody can optionally be different from the detecting anfibody. However, it has been discovered that this is not critical to the practice of adsorption type confirmatory assay in a number of its embodiments. The assay can be performed by using the same anfibody for both neutralizing and detecting.
  • the key is to provide enough anfibody at the neutralizing step to remove or inactivate the analyte, but not enough to remove or inactivate the interfering material.
  • the technique takes advantage of the following two features: first, the specificity of the anfibody, as indicated in the preceding paragraph; and second, the fact that the interfering substance must be present in large molar excess in order to have given a positive reaction in the direct test.
  • a simple hypothetical example will illustrate the point.
  • the antibody used to detect the analyte has a high affinity for analyte, and cross-reactivity for the interfering substance of 1%.
  • the interfering substance When the interfering substance is present at 1000 nM, it will give about the same signal in the direct test as analyte at 10 nM.
  • the antibody should adsorb nearly all the analyte in a 10 nM sample, but no more than about 5% of the interfering substance in a 1000 nM sample (probably less, since the affinity for interfering substance is lower).
  • the true analyte will give a negative result in the neutralization test, but the interfering substance will give a positive result.
  • thresholds can be set in the confirmatory part of the test that can be recorded as negative (confirming the presence of analyte) or positive (indicating the presence of interfering substance).
  • the neutralizing antibody can act to prevent the analyte from reacting with the detecting antibody in the detection phase of the confirmatory test by several different mechanisms.
  • the neutralizing antibody simply binds the analyte at the same epitope (in a similar or dissimilar orientation) as the detecfing antibody. Since the neutralizing anfibody should not then go on to substitute for the detecfing antibody, the detection means must involve some difference between the two.
  • the detecting anfibody is conjugated to the labeling system, such as in the enzyme complementafion assay described in US Patent 5,212,064.
  • the neutralizing anfibody could be the same Fab fragment (or the whole anfibody equivalent) in an unconjugated form, and would prevent binding to the detection conjugate.
  • the neutralizing anfibody recognizes the analyte from a different orientation from the detecfing anfibody and is unable to react with the detection system in the same way.
  • the neutralizing antibody is used to pretreat the sample and remove the true analyte in preference to the interfering substance. Then the sample is tested using a detection anfibody and competitive binding compound in a similar fashion to the direct test. Any type of immunoadsorption can be used.
  • the neutralizing anfibody is "insolubilized", which means that it is attached to an insoluble polymer or bead made of a suitable inert material, such as polystyrene, polyacrylamide, cellulose, and the like, or the side of a vessel wall through which the sample is passed or preincubated.
  • An affinity separation step can be performed by column chromatography or filtration. However, elaborate separation procedures are generally not necessary.
  • a convenient procedure is to simply add an affinity matrix into the sample, keep in suspension for sufficient time to adsorb the analyte, and then allow it to settle to the bottom of the reaction vessel. The supernatant can then be assayed for interfering substance.
  • the neutralizing anfibody is not attached directly to a solid surface, but insolubilized after it is added to the sample.
  • a secondary capture anfibody or binding compound can be used for this purpose.
  • the neutralizing antibody is a mouse anti- analyte antibody
  • the capture antibody can be a polyclonal rabbit anti-mouse immunoglobulin reagent bound to a solid phase.
  • the neutralizing anfibody can be provided with a suitable capture ligand, such as fluorescein or biotin.
  • the anfibody (along with bound analyte) is removed using a receptor with the matching specificity: respectively anti-fluorescein antibody, or avidin.
  • the primary anfibody or the capture anfibody is linked to a ferromagnetic particle, which is subsequently removed from the solution (along with the analyte) using a magnetic field.
  • the amount of neutralizing anfibody is adjusted so as to be sufficient to remove the analyte but not all the interfering substance.
  • the neutralizing anfibody is linked to a particulate, it can be diluted as needed by adding additional particulate which has not been activated or which is linked to an alternative, inactive molecule such as bovine albumin. This permits an appropriate amount of antibody to be provided in an easily handled amount of particulate. This is illustrated in Example 6.
  • An assay for distinguishing between the analyte and an interfering substance involves conducting an immunoassay to determine the amount of analyte and/or interfering substance in the sample (the direct test); treafing the same sample or a duplicate of the sample with a neutralizing antibody in an amount sufficient to remove or inactivate the analyte but not the potential interfering substance; and conducting an immunoassay to determine the amount of interfering substance in the treated sample (the confirmatory test).
  • the direct and confirmatory immunoassays are performed by preparing a reaction mixture comprising the sample, a detecfing antibody, and a competitive binding compound (usually a hapten derivative), wherein the detecting antibody binds the hapten derivative in a manner that is specifically inhibitable by the analyte, and then measuring the amount of the detecfing anfibody bound to the hapten derivative.
  • adsorption type confirmatory assays for amphetamine and LSD using a neutralizing antibody insolubilized by attaching directly to a resin such as Sepharose® 4B.
  • Example 7 provides an illustration of an adsorption-type confirmatory test for LSD, in which the detecfing anfibody and the neutralizing anfibody are the same.
  • the commercially available CEDIA DAU® kit contains a detecfing anfibody raised by linking the following activated LSD derivative to KLH, and an enzyme donor made with the same derivative:
  • the detecting antibody has the laboratory designation 19A7.
  • Example 6 illustrates the preparation of an insolubilized form of 19A7 and adjustment of the activity to provide a suitable neutralizing reagent for a confirmatory assay.
  • the assay is illustrated in Example 7.
  • the assay correctly identified the 27 samples that contained LSD, and five samples that were false positive, as verified by GC/MS.
  • Confirmatory assays preferably have a success rate of at least 75% and more preferably at least about 95% in identifying false positives due to interfering substances, within the standard working range of the assay.
  • the preparation of detecting and neutralizing antibodies for use in this invention is performed according to established techniques, using an immunogen that presents the hapten in an appropriate orientation.
  • the hapten is typically a structure that is identical or closely related to the analyte to be measured, linked to an immunogenic carrier such as KLH either directly or through a linking group.
  • Polyclonal anfibodies of this invention are raised by administration of the immunogenic conjugate to a mammalian host, usually mixed with an adjuvant.
  • the immunogen is conveniently prepared for injection by rehydrating lyophilized immunogen to form a solution or suspension.
  • Preferred adjuvants are water-in-oil immersions, particulariy Freund's complete adjuvant for the first administration, and Freund's incomplete adjuvant for booster doses.
  • the preparation is typically administered in a variety of sites, and typically in two or more doses over a course of at least weeks. Serum is harvested and tested for the presence of specific anfibody using a hapten-protein conjugate or other competitive binding compound for the analyte in a standard immunoassay or precipitation reaction.
  • Polyclonal antisera will typically contain anfibodies not reactive with the analyte or having undesired cross-reactivities.
  • Methods for purifying specific anfibodies from a polyclonal antiserum are known, particularly affinity purification using a column of analyte conjugated to a solid phase. The antiserum is passed over the column, the column is washed, and the antibody is eluted with a mild denaturing buffer such as 0.1 M glycine, 0.2 M NaCI, pH 2.5. If the antiserum is passed over the column in a buffer containing potential interfering substances, then the bound and eluted fraction will be enriched for antibodies that don't cross-react.
  • a mild denaturing buffer such as 0.1 M glycine, 0.2 M NaCI, pH 2.5.
  • Monoclonal antibodies of this invention can be prepared by a number of different techniques known in the art. For hybridoma technology, the reader is referred generally to Harrow & Lane (1988), U.S. Patent Nos. 4,491 ,632, 4,472,500, and 4,444,887, and Methods in Enzymoiogy, 73B:3 (1981).
  • the most common way to produce monoclonal anfibodies is to immortalize and clone a splenocyte or other antibody-producing cell recovered from an immunized animal. The clone is immortalized by a procedure such as fusion with a non-producing myeloma, by transfecting with Epstein Barr Virus, or transforming with oncogenic DNA.
  • the treated cells are cloned and cultured, and clones are selected that produce anfibody of the desired specificity.
  • Specificity testing is performed on culture supernatants by a number of techniques, such as using the immunizing antigen as the detecting reagent in an immunoassay.
  • a supply of monoclonal anfibody from the selected clone can then be purified from a large volume of culture supernatant, or from the ascites fluid of suitably prepared host animals injected with the clone.
  • the antibody can be tested for activity as raw supernatant or ascites, and is optionally purified using standard biochemical preparation techniques such as ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography.
  • Immunocompetent means that the cell or particle is capable of expressing an anfibody specific for the anfigen without further genetic rearrangement, and can be selected from a cell mixture by presentation of the anfigen.
  • Immunocompetent eukaryotic cells can be harvested from an immunized mammalian donor, or they can be harvested from an unimmunized donor and prestimulated in vitro by culturing in the presence of immunogen and immunostimulatory growth factors.
  • Cells of the desired specificity can be selected by contacting with the immunogen under culture conditions that result in proliferation of specific clones but not non-specific clones.
  • Immunocompetent phage can be constructed to express immunoglobulin variable region segments on their surface. See Marks et al., New Engl. J. Med. 335:730, 1996; WO patent applications 94/13804, 92/01047, 90/02809; and McGuinness et al., Nature Biotechnol. 14:1149, 1996.
  • Phage of the desired specificity can be selected, for example, by adherence to a hapten-protein complex " attached to a solid phase, and then amplified in E. coli.
  • antibody encompasses not only intact immunoglobulin molecules, but also such fragments and derivatives (including recombinant derivatives) of immunoglobulin molecules with the desired specificity. Fragments and other derivatives of immunoglobulins can be prepared by methods of standard protein chemistry, such as subjecting the antibody to cleavage with a proteolytic enzyme like pepsin, papain, or trypsin; and reducing disulfide bonds with such reagents as dithiothreitol.
  • proteolytic enzyme like pepsin, papain, or trypsin
  • genetically engineered variants of intact immunoglobulin can be produced by obtaining a polynucleotide encoding the anfibody, and applying the general methods of molecular biology to splice encoding sequences or introduce mutations and translate the variant.
  • Antibodies that are engineered variants of particular interest include chimeric and humanized antibodies, Fab-like fragments, single-chain variable region fragments (scFv), and diabodies.
  • Detecfing antibodies for use in enzyme complementation assays in the CEDIA® series are selected on the basis of specificity for the analyte, and also on the basis of three other criteria.
  • One referred to as “inhibition”, relates to how well the antibody binds the enzyme-donor conjugate. Sufficient inhibition (preferably at least about 70%) is needed in order to provide an adequate signal.
  • a second criterion is the titer of the antibody required to obtain the desired level of inhibition. Inhibifion at lower anfibody levels is preferred.
  • a third criterion, referred to as “modulation”, relates to how well the sample analyte is able to compete with the conjugate for enzyme binding.
  • Modulation is calculated as the difference in enzyme rate between a sample having the analyte at a target concentration (moderately chosen within the intended working range) and a sample having no analyte, divided by the rate at the target concentration. Better modulation correlates with better assay sensitivity. Detecfing antibodies for other types of assays are selected by criteria appropriate to provide the desired sensitivity and specificity through the working range.
  • Neutralizing anfibodies are selected on the basis of criteria necessary for their use in the intended assay system. Neutralizing anfibodies that are maintained in the reaction mixture during the detection of any uncomplexed interfering substance, such as in a bidirectional type confirmatory assay, must not react with the other reagents in the mixture and give a false signal. In particular, they must not be able to take the place of detecfing antibody in binding to any hapten derivative involved in signal generation. Thus, neutralizing anfibodies are selected that don't have this activity, either by direct negative selection for the unwanted binding characteristic, or by negative selection in an assay mixture according to the immunoassay method in the absence of detecting anfibody.
  • Anfibodies can be "insolubilized” by attaching them to a vessel wall, to a particulate, or to a large molecular weight carrier that can be kept in suspension but is removable by physicochemical means, such as centrifugation or microfiltration.
  • the attachment need not be covalent, but is at least of sufficient permanence to withstand any separation techniques (including washes) that are part of the assay procedure.
  • Suitable particulate materials include agarose, polystyrene, cellulose, polyacrylamide, latex particles, magnetic particles, and fixed red cells.
  • Suitable commercially available matrices include Sepharose® (Pharmacia), Poros® resins (Boehringer Mannheim Biochemicals, Indianapolis), Actigel SuperflowTM resins (Sterogene Bioseparations Inc., Carlsbad CA), and DynabeadsTM (Dynal Inc., Lake Success, NY).
  • Sepharose® Puracia
  • Poros® resins Boehringer Mannheim Biochemicals, Indianapolis
  • Actigel SuperflowTM resins Steogene Bioseparations Inc., Carlsbad CA
  • DynabeadsTM DynabeadsTM
  • This invention includes various sets of reagents, which are effective in performing a confirmatory assay of this invention, or in adapting a direct assay for distinguishing between an analyte and an interfering substance.
  • 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.
  • 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 a confirmatory assay. This can be limited to a simple indication on the packaging that the ingredients are suitable for confirmatory tests, eliminating false positives, or distinguishing the analyte from named or unnamed interfering substances.
  • the written instructions will also indicate important steps arid " conditions for performing the tests, and data useful in interpreting the tests.
  • the kit preferably contains an indication of the amount of neutralizing antibody to be added to each sample, or the neutralizing anfibody will be pre-aliquoted in the appropriate amount.
  • the assay procedure entails combining the sample with the antibody under conditions that permit the formafion 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.
  • 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.
  • Assays suitable for use or modification in this invention include both qualitative and quantitative assays.
  • Typical quantitative methods involve mixing the analyte with a pre-determined non-limiting amount of the reagent anfibody, and correlating the amount of complex formed with the amount of analyte in the original sample using a relationship determined using standard samples containing known amounts of analyte in the range expected for the sample to be tested.
  • a qualitative assay sufficient complex above or below a threshold level established by samples known to contain or be free of analyte establish the assay result. Unless otherwise required, “measuring” refers both to qualitative and quantitative determination.
  • Assays of this invention include both separation-based and homogeneous 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.
  • the complex can be first formed in the fluid phase, and then subsequently captured by a solid phase reagent or separated on the basis of an altered physical or chemical property, such as by gel filtration or precipitation.
  • one of the reagents can be attached to a solid phase before contacting with other reagents, and then the complex can be recovered by washing the solid phase free of unreacted reagents.
  • Separation-based assays typically involve use of a labeled analog or anfibody to facilitate detection or quantitafion of the complex.
  • Suitable labels are radioisotopes such as 125 l, enzymes such as peroxidase and ⁇ -galactosidase, and fluorescent labels such as fluorescein isothiocyanate.
  • the separation step involves removing labeled reagent present in complex form from unreacted labeled reagent. The amount of label in the complex can be measured directly or inferred from the amount left unreacted.
  • a sample can be tested for amphetamine by mixing with labeled anti- amphetamine antibody and solid-phase amphetamine.
  • the amount of label bound to the solid phase inversely correlates with the amount of amphetamine in the sample.
  • the complex is typically not separated from unreacted reactioTT components, but 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).
  • Assays of this invention include both sandwich and competition assays.
  • Sandwich assays typically involve forming a complex in which the analyte to be measured is sandwiched between one reagent, such as a first antibody used ultimately for separation of the complex, and another reagent, such as a second anfibody used as a marker for the separated complex.
  • one reagent such as a first antibody used ultimately for separation of the complex
  • another reagent such as a second anfibody used as a marker for the separated complex.
  • competition assays are more common.
  • Competition assays involve a system in which the analyte to be measured competes with an analog of the analyte for binding to another reagent, such as an antibody.
  • a “competitive binding compound” refers to a compound that is able to compete with the analyte to be measured in the sample for binding to the detecfing anfibody.
  • Competitive binding compounds are identified functionally, and include radioisotope conjugates, enzyme conjugates, and other protein complexes, and structurally similar chemical analogs.
  • CEDIA® is an example of a competition assay.
  • Homogeneous assay methods of this invention are exemplified by enzyme complementafion assays, exemplified by the cloned enzyme donor immunoassay system described in U.S. Patent No. 4,708,929. Related reagents and methods are taught in U.S. Patent Nos. 5,254,577; 5,444,161; 5,464,747; and 5,514,560. Cloned enzyme donor immunoassays are available commercially under the registered trademark CEDIA®.
  • a cloned enzyme donor immunoassay involves combining the sample with a specific detecfing anfibody; an enzyme donor polypeptide conjugate; an enzyme acceptor polypeptide (wherein the enzyme acceptor polypeptide is capable of forming with said enzyme donor polypeptide conjugate an active enzyme complex in the absence of an anfibody to the analyte), and a substrate capable of being transformed by the active enzyme complex into a product.
  • the amount of product is then measured, usually as a function of time.
  • Preferred enzyme-donor and enzyme-acceptor polypeptides are based on the enzyme ⁇ -galactosidase polypeptide.
  • a " ⁇ -galactosidase polypeptide” is a polypeptide identifiable on the basis of its amino acid sequence or enzymatic activity as being developed from an enzyme with ⁇ -galactosidase activity, and includes naturally occurring ⁇ -galactosidase, fragments, deletion mutants, fusion proteins, mutants, and other variants.
  • Particular ⁇ -galactosidase polypeptides are described in the aforelisted U.S. Patent applications pertaining to cloned enzyme donor immunoassays.
  • ⁇ -galactosidase enzyme acceptors are preferably produced by a deletion mutant of the ⁇ -galactosidase gene.
  • EA22 one of the preferred acceptors, has a deletion of amino acid residues 13-40.
  • Other enzyme acceptor fragments of ⁇ -galactosidase include EA5, EA11 , EA14, EA17, EA18, EA20, EA23 and EA24.
  • the distal end of the deleted segment normally falls between amino " acid positions 26 and 54 of the ⁇ -galactosidase sequence. In EA22, the distal end of the deletion segment is amino acid 40.
  • a particularly preferred ⁇ -galactosidase enzyme donor is ED28.
  • This is a fragment of ⁇ -galactosidase consisting of amino acids 1-46, with cysteine residues at positions 1 and 46.
  • ED28 is described in European Patent Application No. 90308937.3. The two cysteine residues can be used for exact and reproducible placement of maleimide adducts of a chemical hapten, as illustrated in the examples.
  • Preferred substrates for use in immunoassays based on ⁇ -galactosidase include those described in U.S. Patents 5,032,503; 5,254,677; 5,444,161 and 5,514,560.
  • the preferred substrates is chlorophenol ⁇ -D-red galactopyranoside (CPRG).
  • CEDIA® type homogeneous assays for small molecule analytes are often formulated such that an analog of the analyte is attached to the enzyme donor near a site involved in recombination of the donor and acceptor.
  • assays for procainamide and N-acetylprocainamide (NAPA) are described in U.S. Patent Nos. 5,439,798 and 5,525,474. Binding of an antibody in the solution to the analog in the conjugate inhibits recombination into an active enzyme complex. Thus, the presence of analyte in the sample is positively correlated with enzyme activity.
  • NAPA N-acetylprocainamide
  • 5,212,064 describes a different approach, in which the anfibody fragment is conjugated to the enzyme donor, and the analog is conjugated to a macromolecule or insoluble particle. Binding of the enzyme donor to the analog conjugate via the anfibody sterically inhibits recombinafion with the enzyme acceptor. Analyte in the sample competes with the analog conjugate, freeing up the enzyme donor for recombinafion with the acceptor. Again, the presence of analyte in the sample is positively correlated with enzyme activity.
  • G6PD glucose-6- phosphate dehydrogenase
  • Examples are provided in International Patent Application WO 94/24559 and EP Patent Application 487,301-A.
  • a homogeneous assay can be performed, in which G6PD is conjugated with an analog of the analyte in a posifion where binding of an anfibody to the analog inhibits G6PD activity. If the test sample contains the analyte of interest, it competitively binds the antibody, which is then prevented from binding the G6PD. Thus, enzymatic activity correlates positively with the presence of analyte in the sample.
  • the assays in the Behring Emit® II series are homogeneous assays using G6PD conjugates, in which absorbance change of NAD to NADH is measured spectrophotometrically. Assays in this series can be converted for confirmation testing by supplying a neutralizing antibody according to this invention.
  • Other assay systems of particular interest are fluorescence polarization immunoassays.
  • Amphetamine/Methamphetamine II assay in the TDx®/TDxFLx® series of Abbott Laboratories is an example of a fluorescence polarization assay.
  • the assays can be converted for confirmation testing by supplying a neutralizing anfibody according to the invention.
  • Amphetamine analogs shown in U.S. Patent 5,354,693 and E.P. Patent 371,253-B differ in the posifion at which the fluorescent group is attached, and may require different neutralizing anfibodies for use in a bidirectional type assay, as described earlier.
  • the assay methods of this invention can be carried out manually, or on automated equipment.
  • Devices suitable for confirmatory assays generafing an enzymafic signal include analyzers in the Beckman SynchronTM series, Olympus analyzers (AU800, AU5000, AU 5200), Roche COBAS® analyzers, and devices in the Boehringer Mannheim/Hitachi series.
  • Analyzers suitable for performing bidirectional antibody type confirmation assays for amphetamine, similar to those illustrated in Example 4, include BM/Hitachi models 704, 717, 747, 902, 904, 911, 912, 914, and 917.
  • Analyzers suitable for performing adsorption type confirmafion assays for LSD similar to those illustrated in Example 7, include BM/Hitachi models 902, 911, 912, and 717.
  • the experiments described in Example 4 and Example 7 were performed primarily using a BM/Hitachi model 911 automated analyzer.
  • the full detection and confirmation of the analyte involves two tests: a) a direct test for any analyte or interfering substance in the sample, and b) a neutralization or confirmation test, in which the analyte (but not interfering substances) is removed or prevented from generating a signal by treafing with a neutralizing anfibody.
  • the actual sample used in step b) may be the same sample aliquot used in step a), but is more typically a duplicate sample aliquot, preferably subdivided or siphoned from the same sample aliquoted for step a).
  • the threshold can be determined empirically by testing a range of samples spiked with various levels of analyte and potential interfering substances, or by testing a panel of clinical samples confirmed by GC/MS. For illustration, see Examples 4 and 7.
  • immunoassay embodiments of this invention are used frequently as screening assays, it is worthwhile to get further independent confirmation of a result that indicates presence of a true analyte, especially where there are substantial consequences of a positive result.
  • the techniques of GC/MS, LC/MS, GC/MS/MS or LC/MS/MS are generally preferred, and certain combinations of liquid chromatography are also suitable where the results are definitive.
  • Quantitafion in a confirmatory assay of this invention and the ability to distinguish the analyte from the interfering substance can optionally be enhanced by measuring relative rate of enzyme catalyzed conversion of the substrate to a product between the reaction mixture for the direct test and the reaction mixture for the confirmatory test. This is illustrated in Example 7.
  • the rate of reaction in the two tests can be measured simultaneously or sequentially. Calculation of the differential rate facilitated in automated devices equipped for this purpose, such as the BM/Hitachi analyzer model 911. Procedures for performing a calculated test (in which results of multiple channels are used to calculate a final result using a pre-programmed equation) will be described in the technical manual for the device.
  • the products and methods of this invention can be applied for the determination or measurement of any analyte of interest, wherever a suitable primary detecting assay exists, and a neutralizing anfibody that meets the requirements of the invention can be obtained.
  • analytes include synthetic chemical structures including but not limited to pharmaceutical compounds, peptides, carbohydrates, lipids, environmental agents and contaminants.
  • Pharmaceutical compounds of various kinds can be measured, for example, to monitor the pharmacological profile " of a prescribed drug, to determine excretion rates in renal function tests, or to ascertain drug abuse.
  • drugs of abuse and their metabolites are both considered positive, and are distinguished from unrelated cross-reactive interfering substances.
  • the drugs of abuse are distinguished from their metabolites (see Example 7).
  • Particular abused substances of interest are those identified in the National Institute of Drug Abuse (NIDA) guidelines, which also provides cut-off levels for drug screening.
  • the following compounds are potentially interfering due to unexpected cross-reactivities and the possibility that they may be present in high concentrations, particularly in urine samples: acetaminophen, ASA, amitriptylene, amoxicillin, amphetamine, ascorbic acid, atrophine, benzoylecgonine, caffeine, captopril, chlordiazepoxide, chloroquine, cimetidine, codeine, dopamine, diazepam, digoxin, enalapril, erythromycin, estriol, fluoxetine, ibuprofen, levothyroxine (T4), methamphetamine, morphine, naproxin, nifedipine, phencyclidine, phenobarbital, ranitidine, salicyluric acid, secobarbital, tolmetin, verapamil, and tetrahydrocanabinol and its derivatives.
  • LSD assays include methamphetamine, fenfluramine, ambroxol, chlorpromazine, ergotryptine, dihydroergotamine, ecogonine and related compounds, ergonivine, ergotamine, lysergic acid, lysergol, methysergide maleate, psilocybin, psilocyn, seretonine, tryptophan, fentanyl, and 2-oxo-3-hydroxy LSD.
  • Potenfially interfering substances in PCP assays include dextramethorphan and EDDP.
  • Potenfially interfering substances in opiate assays include imipramine, rifampicin, ofloxin, meltrexone, and naloxone.
  • Potentially interfering substances in benzodiazepine assays include non-steroidal anti-inflammatory drugs such as oxyprozin.
  • Potentially interfering substances in methadone assays include EDDP, EMDP, and disopyramide.
  • Potenfially interfering substances in amphetamine assays include ephedrine, pseudoephedrine, phenylpropanolamine, phentermine, fenfluramine, 3-hydroxytyramine, norpseudoephedrine, mephentermine, and phendimetrazin.
  • Samples that can be tested using the products and methods of this invention are by no means limited to urine samples.
  • suitable samples include serum, plasma, and other blood fractions; spinal fluid, sinovial fluid, and saliva.
  • suitable samples also include tissue culture supernatants, tissue homogenates, plant products, and other types of laboratory concoctions.
  • Suitable environmental samples for testing include any aqueous sample with a detectable analyte for which it is desirable to distinguish from an interfering substance against which a neutralizing antibody can be obtained.
  • Example 1 Detecting antibodies and assays for amphetamine and methamphetamine
  • Amphetamine protein conjugates were prepared by using an activated amphetamine derivative of the following structure (BMP-amphetamine):
  • Methamphetamine protein conjugates were prepared by using an activated methamphetamine derivative of the following structure (BMP-methamphetamine):
  • the derivatives were prepared by synthesizing p-3-mercaptopropylamino-d- methamphetamine, and meta-3-mercaptopropyl-d-amphetamine according to the methods described in U.S. Patent No. 5,470,997 (Buechler et al.). U.S. Patent No. 5,470,997 is hereby incorporated herein by reference in its entirety.
  • Immunogens were prepared by linking the derivative to KLH.
  • the KLH was pre-thiolated according to the following procedure. One vial of KLH (20 mg, Pierce) was combined with 2 mL water, and vortexed to dissolve.
  • Conjugated enzyme donors were prepared by linking the activated derivative to the sulfhydryl group of the enzyme donor ED28.
  • ED28 1 mg, 102 nmole
  • BMP-amphetamine 0.44 mg, 612 nmole
  • BMP-methamphetamine 0.46 mg
  • DMF 200 ⁇ l
  • a PD-10 column was pre-equilibrated with 0.1 % TFA in water. The conjugate was desalted on this column, and further purified by HPLC.
  • the purified solution was stored at -70 °C until further use.
  • a diagnostic-grade homogeneous enzyme assay kit for detecting either amphetamine or methamphetamine in a biological sample is widely available under the name CEDIA ® DAU Amphetamines.
  • Reagent 1 contains enzyme acceptor (EA), and the test anfibodies specific for amphetamine and methamphetamine.
  • Reagent 2 contains enzyme donor (ED)-amphetamine and ED-methamphetamine conjugates and chlorophenol-red- ⁇ -D- galactopyranoside (CPRG) as substrate.
  • sample (3 ⁇ L of calibrator, control or unknown) and 130 ⁇ L of R1 are added to the cuvette of a clinical chemistry analyzer and incubated at 37° C for 5 minutes. During this incubation, any amphetamine or methamphetamine present in the sample will bind to their respective test antibodies.
  • the anfibodies for the CEDIA ® DAU Amphetamines Test are on deposit with the American Type Culture Collection (ATCC), under the following designations: Amphetamine - Safe Deposit # 2004; Methamphetamine - Safe Deposit # 2009.
  • Amphetamine conjugates linked through the aromatic ring show high cross-reactivity with ring substituted analogs. It has been found that linkage of the hapten through the nitrogen group elicits antibodies that discriminate between unsubstituted amphetamines and similar compounds.
  • the synthesis of ⁇ /-butylmaleimide-amphetamine was accomplished by first preparing the key intermediate, ⁇ /-butylamine-amphetamine (AMP-BA):
  • AMP-BA (206 mg) was dissolved in THF (10 ml) in a round bottom flask and cooled in an ice bath. Saturated NaHC0 3 solution (5 ml) was added. Ten minutes later methoxycarbonyl maleimide (155 mg) was added. After stirring for 110 minutes water (5 ml) was added and the mixture extracted with EtOAc (3 X 5 ml). The organic extract was washed with brine (5 ml) and dried wifff MgS0 4 . Trifluoroacetic acid (75 ⁇ l, TFA) was added and the solution concentrated to yield crude AMP-BM as a cream colored solid (292 mg, 73% yield).
  • KLH-2-IT-BM-AMP An immunogen (KLH-2-IT-BM-AMP) was prepared by first reacting KLH with 2-iminothiolane (2-IT) followed by reaction with ⁇ /-butylmaleimide-amphetamine:
  • DMF 200 ml
  • Example 3 Antibodies that neutralize both amphetamine and methamphetamine
  • Monoclonal anfibodies were obtained by immunizing and boosting mice with the KLH-BM- AMP immunogen. After sufficient time for an immune response to develop, mouse spleen lymphocytes were fused to mouse myeloma cells, and the resulting hybridoma cells were cloned by " limiting dilution. Antibody-secreting cell lines with the desired specificity were identified in a double screening process using a CEDIA® type immunoassay.
  • Antibodies were initially selected for their ability to bind amphetamine attached to the BM- AMP enzyme donor. Supernatants were screened ten days post-fusion for inhibifion of the corresponding ED-amphetamine-butyl maleimide conjugate in a cloned ⁇ -galactosidase enzyme donor immunoassay. 50 ⁇ L hybridoma supernatant was transferred to each well of a 96-well plate.
  • Reagents from a CEDIA® amphetamine kit were reconstituted according to manufacturer's directions.
  • R1 antibody reagent
  • the assay was performed using 6 calibrators. Hybridomas were selected that could neutralize both amphetamine and methamphetamine calibrator samples, without inhibiting the reaction between the enzyme donor and acceptor.
  • the criteria were as follows: a) a depression of rate of at least 10 mA/min for amphetamine at 1000 ng/ml compared with control; b) a depression of rate of at least 20 mA/min for methamphetamine at 1000 ng/ml compared with control; as measured; c) no significant rate shift (more than 10%) for the 0 ng/ml calibrator or the 60,000 ng/ml calibrator.
  • the criteria were set very loosely as the concentration of anfibody in these cell supernatants was unknown and we did not want to prematurely eliminate any of them.
  • a second screening step was performed to eliminate antibodies with a high cross-reactivity to fenfluramine.
  • Fenfluramine is a drug that commonly results in a false positive result in amphetamine testing. Its cross-reactivity in the CEDIA® amphetamine assay is 37%.
  • Hybridomas that survived at least three rounds of cloning and had response to amphetamine and methamphetamine were produced from ascites fluid for further evaluation.
  • Antibodies designated 7B3, 5D10, 8E5, 11G6, 14B2, and 12B5 were chosen that initially showed good amphetamine binding or neutralization, and little cross-reactivity with fenfluramine.
  • Antibody 2E1 was chosen even though there was a degree of cross-reactivity, because of an exceptional quenching activity for both amphetamine and methamphetamine.
  • Antibody 10F12 was chosen, even though it initially showed poor neutralization, because of excellent ability to distinguish amphetamine from fenfluramine.
  • Antibody was purified from ascites and tested at concentrations of 50, 100, 150, 200, and
  • Figure 1 (Upper Panel) shows a typical standard curve for amphetamine and methamphetamine using the CEDIA ® DAU Amphetamines assay. Enzyme activity, as measured by the rate of change of absorbance at 570 nm (in mAU/min) was determined as a function of amphetamine ( ⁇ ) or methamphetamine ( ⁇ ) concentration. In this test, a sample is considered positive if the rate is equal to or greater than that of the 1000 ng/ml amphetamine calibrator. A second protocol is available which uses a 6 ⁇ L sample volume; in this case, the decision point is at 500 ng/ml amphetamine.
  • the confirmation test for amphetamines was developed by addition of the absorbing anfibody to the R1 of the existing test, until the change in rate by up to 5000 ng/ml added amphetamine or methamphetamine is reduced to below the change in rate due to the cut-off (1000 ng/ml) calibrator in the unmodified test.
  • Antibody 12B5 300 ⁇ g/ml was added to R1 of the CEDIA ® DAU Amphetamines assay, and the effect on the amphetamine and methamphetamine standard curves obtained using the assay reagents was determined.
  • Figure 1 shows the effects of the absorbing anfibody on signal due to amphetamine ( ⁇ ) and methamphetamine ( ⁇ ).
  • Panel A shows the result from the unmodified assay;
  • Panel B shows the effect of including the neutralizing antibody in the reaction mixture.
  • the concentrafions of pseudoephedrine, phenylpropanolamine, phentermine and tyramine needed to give a positive result are 203, 582, 59.1 and 98.5 ⁇ g/ml, respecfively.
  • the high concentrations needed to give a positive rate indicate the high degree of specificity exhibited by the test anfibodies in the unmodified CEDIA ® DAU Amphetamines assay.
  • these compounds can be found in the urine at extremely high concentrafions, resulting in false positive results during drugs of abuse screening.
  • Very high concentrations of cross-reactants can also decrease the ability of the neutralizing antibody to lower the signal due to the target analyte.
  • concentrations of pseudoephedrine, phenylpropanolamine, phentermine and tyramine needed to give a positive result in the CEDIA ® DAU Amphetamine confirmatory test are 243, 708, 70.6 and 97.9 ⁇ g/ml, respectively. These concentrafions are equal to or slightly above the concentrations required for a positive result observed in the absence of neutralizing antibody.
  • the amphetamine confirmatory assay was tested with a panel of actual urine samples obtained from a drug screening laboratory.
  • the test samples were chosen as positive in the CEDIA ® DAU Amphetamines assay, based on a delta rate greater than that of the 1000 ng/ml amphetamine calibrator.
  • Samples in the test panel with rates between 1000 and 8000 ng/ml were pre-evaluated by GC/MS. A sample was classified as true positive if it met one of the following criteria: either > 500 ng/ml methamphetamine and > 200 ng/ml of amphetamine; or > 500 ng/ml of amphetamine. Samples were considered false positive if they failed to meet either of these criteria by GC/MS.
  • the result of the confirmatory test were calculated as the rate in the standard test channel (the normal CEDIA ® DAU Amphetamines assay) minus the rate in the confirmatory test channel (CEDIA ® DAU Amphetamines assay, plus absorbing anfibody added to R1).
  • the results are presented in the following table as ⁇ Rate Units, in mAU/min.
  • the confirmatory test approach can be used for other drug of abuse tests, including those where the results are reported as a qualitative result.
  • the method is particularly advantageous for high-volume screening methods, as exemplified by such homogeneous test methods as the CEDIA ® , Emit and latex microparticle tests, and heterogeneous test methods such as ELISA.
  • CEDIA ® DAU LSD kit contains the LSD-specific monoclonal antibody designated 19A7, and an N-1 carboxyalkyl LSD enzyme donor conjugate. The preparation of these reagents is described fully in International patent application PCT/US 96/19266.
  • N-1-(ethyl-carboxymethyl)-LSD was prepared as a starting material by treating LSD with a molar excess of sodium hydride followed by the addition of ethylbromoacetate.
  • the product was isolated by preparative HPLC on a 2.2 x 25 cm C4 column using 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 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 pooled fractions were lyophilized and re-lyophilized 2 times from water/acetonitrile 4:1 to get rid of the TEA- Ac and convert the product to a zwitterion.
  • the pooled fraction was analyzed by 1 H-NMR in acetonitrile-d 3 and identity confirmed by mass spectrometry (MS).
  • N-l-(carboxymethyl) LSD N-hydroxysuccinimide ester (N-1-CM-LSD-NHS) was prepared by dissolving a sample of N-1-carboxymethyl-LSD (6.6 mg) in 1.0 ml DMSO. To that solution N-hydroxysuccinimide (NHS) (14 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (23.2 mg) was added. The solufion was vortexed and incubated at ambient temperature overnight. The activated N-1-CM-LSD-NHS was used in for the next step without further purification.
  • NHS 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • KLH conjugates were formed as follows: N-1-CM-LSD-NHS (5.7 mg) in 1.0 ml DMSO was added to 15.0 mg KLHJn 4.0 ml phosphate buffer, vortexed gently for two minutes and allowed to stand at ambient temperature for 8.0 hours. A second conjugate was prepared using a maleimidoethylamine (MEA) derivative. A 2-fold excess of MEA HCI (6.1 mg ) in 1.5 ml phosphate buffer was added to the activated N-1-CM-LSD-NHS The progress of the reaction was monitored by HPLC. The N-1-MEA-CM-LSD was purified by HPLC.
  • MEA maleimidoethylamine
  • An LSD enzyme donor conjugate was prepared as follows: To a solution of N-1-CM-LSD- NHS (50.11 mg) an approximately equivalent number of moles of maleimidopentylamine hydrochloride (MPA) (28.78 mg) in DMF was added. To keep the reaction mixture at neutral pH, 300 ⁇ l 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 solufion of thiolated ED28 (5.0 mg) was prepared and desalted in 3.5 ml phosphate buffer.
  • MPA maleimidopentylamine hydrochloride
  • 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 min.
  • the protein conjugate N-1-MPA-CM-LSD:ED was HPLC purified.
  • Monoclonal anfibodies were raised using the KLH conjugates according to the methods described elsewhere in this disclosure.
  • a primary screening of the fusion products was first performed to evaluate the ability of the antibodies to bind to the enzyme donor conjugate. 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 anfibody.
  • the modulation assay also identified specific clones when screened against cross-reacting analytes.
  • a model CEDIA® assay for LSD was performed using the following reagents.
  • Antibody reagent test antibody, 57 ng/ml; 100 mM PIPES 100; 500 mM NaCI, 0.5% fetal bovine serum, 10 mM EGTA, 10 mM magnesium acetate, 20 mM sodium azide; pH 6.9.
  • Enzyme donor reagent 0.487 nM conjugate; 3 mg/ml of the substrate CPRG (chlorphenyl-red- ⁇ -D-galactopyranoside); 100 mM PIPES; 400 mM NaCI, 10 mM EGTA, 2 mg/ml fragmented BSA, 20 mM sodium azide; pH 6.9.
  • Enzyme acceptor reagent acceptor 880 U/ml; magnesium acetate 10 mM, NaCI 400 nM, PIPES 100 nM, EGTA 10 mM, sodium azide 20 mM, pH 6.9. Assays were performed using an HITACHI 911 analyzer. The absorbance rate at 570 nm was plotted against LSD concentration to construct a dose response curve. Several antibody clones were identified with sensitivity and specificity suitable for use in a CEDIA® format LSD assay.
  • the LSD-enzyme donor conjugate of the previous example is a conjugate in which LSD is linked through the N-1 posifion.
  • Neutralizing antibodies for LSD for use in a bidirectional type confirmation assay are prepared using an LSD derivative in which the LSD is linked to an immunogenic carrier by the alkyl amide group.
  • N-(3-Carboxypropyl)-N-ethyl lysergamide was activated to N-(0-succinimido-3- carbonylpropyl)-N-ethyl lysergamide as follows: Sure-SealTM DMSO (0.3 ml) was transferred into an appropriately sized glass vial. The solution was flushed with a flow of argon.
  • N-(3-Carboxypropyl)-N-ethyl lysergamide (4.1 mg), N- hydroxy succinimide (5.6 mg) and 1-Ethyl-3-(3-Dimethlyaminopropyl)carbodiimide Hydrochloride (EDCI) (8.2 mg) was added to the vial.
  • a dry magnetic stirrer bar was placed in the reaction mixture. The solufion was stirred at room temperature for 4.5 hrs. At this point, a 10 ⁇ l aliquot of the reaction mixture was taken, and diluted with 90 ⁇ l H 2 0 containing 0.1 % of trifluoroacefic acid. The sample (50 ⁇ l) was analyzed by HPLC. The HPLC results indicated a 88 % conversion of the starting acid to the NHS active ester. The reaction mixture was incubated for additional 1 hr.
  • the immunogen solufion was transferred into a dialysis tube and dialyzed in 800 ml of water and 200 ml of DMF in a cold room overnight.
  • the immunogen wSS dialyzed two more times, with 4000 ml of deionized water within two days.
  • the number of (3-carbonylpropyl)-N-ethyl lysergamide molecules per KLH was determined as follows: One milligram of KLH-[amino-N-(3-carbonyipropyl)-N-ethyl lysergamide]- was dissolved in
  • Initial screening is performed in a similar fashion to that described for the screening of the amphetamine neutralizing anfibody in Example 2, using an enzyme donor conjugated with LSD at the same posifion as the immunogen.
  • the selected anfibody can then be used in a homogeneous confirmation assay for LSD, similar to the amphetamine confirmafion assay described in Example 4.
  • Neutralizing anfibodies for LSD for use in an adsorption type confirmation assay are prepared by insolubilizing the same 19A7 anfibody used as the detecfing antibody.
  • Anfibody 19A7 was purified by Protein A affinity chromatography, and then attached to cyanogen bromide-activated Sepharose® CL-4B (Pharmacia, Piscataway, NJ) as recommended by the manufacturer.
  • the resin was prepared to contain approximately 0.8 mg of bound anfibody per ml of resin bed volume.
  • the procedure was as follows: The required amount of activated Sepharose® was weighed, taking into account that 1 g freeze-dried material swells to 3.5 ml gel volume. The gel was reconstituted and washed in wash buffer (1 mM HCI) (3 ml and then 200 ml per gram), and then washed in coupling buffer (0.1 M NaHC0 3 , 0.5 M NaCI, pH 8.3) (50 ml per gram) using a Buchner funnel. The anfibody was dissolved or exchanged into coupling buffer, and 5-10 mg were coupled per ml. The gel cake was transferred to a suitably sized container, the antibody was added, and the volume was adjusted to make a 50% slurry.
  • the suspension was mixed end-over-end for 2 h at room temperature, or overnight at 2 to 8 °C. (Stirring is avoided to prevent shearing of the beads.)
  • the proportion of antibody successfully coupled was determined by measuring A 280 before and after coupling. Blocking buffer was then added (1 M ethanolamine pH 8.0, or 0.2 M Tris buffer, pH 8.0), and the suspension was mixed end-over-end for a further 2 h at room temperature, or overnight at 2 to 8 °C.
  • the gel was washed in a Buchner funnel with coupling buffer, second wash buffer (0.1 M Acetate, 0.5 M NaCI, pH 4.0), and then coupling buffer (100 ml each per gram).
  • the affinity resin was diluted with prepared inactivated Sepharose® 4B to yield the desired binding capacity.
  • One and a half times the desired volume of Sepharose® 4B was transferred to a Buchner funnel, and washed with distilled water followed by storage buffer (400 ml and 200 ml per 50 ml gel). The washed gel was then added to the affinity resin.
  • the theoretical binding capacity (in ng analyte per ml gel) was calculated assuming a molecular weight of 150,000 g/mol for IgG, a valence of 2 analyte molecules per IgG, negligible loss of activity due to coupling, and a molecular weight for LSD of 323.4.
  • the resin is adjusted so that 10 ⁇ L resin provides sufficient anfibody to adsorb LSD from the samples within the working range of the assay, but not a large excess.
  • 10 ⁇ L of resin can adsorb about 17 ng LSD, meaning that a 100 ⁇ L sample could contain about 170 ng/mL LSD.
  • samples above about 100 ng/mL will show significant but only partial adsorption.
  • This example describes a solid-phase adsorption-type confirmation assay.
  • Figure 3 (Upper Panel) provides an illustrative example of the use of solid-phase anfibody 19A7 in a confirmation assay for LSD.
  • Different concentrations of LSD were added to drug-free urine samples, and then these samples were tested in an unmodified CEDIA ® DAU LSD assay ( ⁇ ).
  • solid phase anfibody (10 ⁇ L of a 1 :2 dilution of thepacked bed volume per 100 ⁇ L of sample) was added to each sample, incubated for 10 min at room temperature with mixing, and then allowed to settle. The samples were then retested in the CEDIA ® DAU LSD assay ( ⁇ ).
  • both the neutralizing antibody and the detecting antibody are monoclonal antibody 19A7.
  • the incubation of sample and resin can be carried out directly in the analyzer sample cup, by placing the sample and resin in the cup, covering with a seal (such as Parafilm ® ), and mixing, for example, on a rocking mixer such as is used in a hematology laboratory (such as the Thermolyne Vari-Mix platform mixer, available from Fisher Scientific, Pittsburgh, PA). Once the resin settles to the bottom of the reaction vessel, the supernatant can then be assayed.
  • the resin can be separated from the sample using a chromatography column (e.g., PolyPrep column, BioRad Laboratories, Richmond, CA), or using or a filter with a suitable pore diameter.
  • This type of adsorption confirmation assay can be used to distinguish LSD from certain LSD metabolites.
  • Figure 3 shows the results of an experiment in which varying concentrations of LSD (Left Panel), 2-oxo-3-hydroxy LSD (Center Panel) or a partially-purified preparation containing a mixture of 13-hydroxy LSD giucuronide and 14-hydroxy LSD giucuronide (Right Panel) were added to drug-free urine samples. The samples were then tested in the unmodified CEDIA ® DAU LSD assay ( ⁇ ) or after treatment with solid-phase anfibody ( ⁇ ).
  • the signal is completely eliminated by pretreatment with the solid phase antibody where the substance is LSD, and partially eliminated where the substance is one of the tested LSD metabolites.
  • an absorbing anfibody selected to not cross-react with metabolites could be attached to the solid phase. In this case, only parent drug would be absorbed, and samples which contained only metabolites would be read as false positive in the confirmation assay. This would be desired, for example, in the case where confirmafion of the parent drug was a legal requirement.
  • Figure 4 shows the effect of solid-phase antibody on assay response to drug cross- reactants. Different concentrations of methamphetamine (Left Panel), fenfluramine (Center Panel) or ambroxol (Right Panel) were added to drug-free urine samples. The samples were then tested in the unmodified CEDIA ® DAU LSD assay ( ⁇ ) or after treatment with solid-phase anfibody ( ⁇ ).
  • the false positive results in the initial assay can be identified by the lack of absorption of cross-reactants using the solid-phase antibody.
  • relative rates are calculated to help distinguish between true positive and false positive samples.
  • the first parameter is the net rate from the unmodified assay, and the second parameter is the difference between rates for the preadsorbed and the postadsorbed sample:
  • Figure 5 (Upper Panel) shows the effect of solid-phase antibody on assay response to LSD and metabolites. Procedures are the same as for Figure 4, but results have been calculated as
  • Figure 5 shows the effect of solid-phase antibody on assay response to drug cross-reactants. Procedures are the same as for Figure 3 (Lower Panel), but results have been calculated as ⁇ Rates as described above: ( ⁇ ) ⁇ Rate 1 (direct test); ( ⁇ ) ⁇ Rate 2 (confirmation test). Use of relative rates gives a more direct indication of the presence of LSD: • In the case of LSD, both ⁇ Rate 1 and ⁇ Rate 2 are positive, and so the sample is a true positive.
  • the secondary screening test was applied to a panel of true and false positive samples obtained from drug testing laboratories. All samples had tested positive in the CEDIA ® DAU LSD assay (apparent LSD concentrafion > 0.5 ng/ml). ⁇ Rate 2 values (assay signal in the absence trf solid-phase antibody minus signal in the presence of antibody) for all true positive samples were > 25 mAU/min, while the ⁇ Rate 2 values for all false posifive samples were ⁇ 15 mAU/min. Taken together, these results demonstrate the ability of the secondary screening test to discriminate between true posifive results (due to either parent LSD or to metabolites) from false positive results caused by drug cross-reactants.
  • the LSD secondary screening test is conducted using insolubilized neutralizing antibody that has been aliquoted for individual sample determination (50 ⁇ L).
  • the antibody is aliquoted in screw-cap microfuge tubes, and stored at 2-8°C before use.
  • the secondary screen can be conducted without recalibration, providing it is conducted within 48 hours of the direct test and the same reagents are used. Otherwise, the direct test is rerun alongside the confirmatory test.
  • the secondary screening test net rate is the difference between the ⁇ rate of the untreated sample, and the ⁇ rate of the treated sample, calculated as indicated above. A net rate of > 25 is interpreted as a true posifive for LSD.

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Abstract

L'invention concerne un système de dosage permettant une détection améliorée d'analytes, et l'aptitude à les distinguer de substances croisées. Des échantillons ayant une réaction positive dans une épreuve de dosage direct sont traités au moyen d'un anticorps neutralisant qui inhibe la réactivité de l'analyte vrai, mais non la substance parasite. Dans des dosages de confirmation de type anticorps bidirectionnel, l'anticorps neutralisant lie la substance à analyser à partir d'un orientation différente sans inhiber d'autres réactifs dans le mélange de dosage. Dans des dosages de confirmation de type adsorption, l'anticorps neutralisant est fourni en quantité suffisante pour adsorber l'analyte mais pas toute la substance parasite. Dosé une nouvelle fois en immuno-essai, l'échantillon neutralisé produit un résultat négatif s'il contient au départ l'analyte vrai. Les échantillons qui ont réagi positivement à la fois au dosage direct et au dosage de confirmation sont signalés comme contenant une substance parasite. Le dosage de confirmation permet de distinguer facilement l'analyte vrai et de réduire le pourcentage des faux positifs, même quand la substance parasite est inconnue mais présente dans des concentrations élevées.
EP98915179A 1998-03-27 1998-03-27 Dosages confirmatifs de drogues constituees de molecules de taille reduite Withdrawn EP1066523A2 (fr)

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JP4795688B2 (ja) * 2002-10-09 2011-10-19 ウオーターズ・テクノロジーズ・コーポレイシヨン サンプル中の化合物を同定する方法および装置
SG187787A1 (en) * 2010-08-10 2013-03-28 Amgen Inc Dual function in vitro target binding assay for the detection of neutralizing antibodies against target antibodies
CN102662057A (zh) * 2012-06-07 2012-09-12 广州易航生物科技有限公司 CEDIA ImmunoChip毒品检测试剂盒
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EP4397371A2 (fr) 2020-05-19 2024-07-10 Cybin IRL Limited Dérivés de tryptamine deutérés et procédés d'utilisation

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DE1066523T1 (de) 2001-09-20
WO1998026644A9 (fr) 1999-04-08
WO1998026644A2 (fr) 1998-06-25
WO1998026644A3 (fr) 1999-02-18

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