Synthesis of New d-Propoxyphene Derivatives and the Development

May 28, 1997 - ... a propoxyphene immunogen, [S-(R,S)]-4-[methyl[2-methyl-3-(1-oxopropoxy)-3,4-diphenylbutyl]amino]-1-oxobutyl-Bovine Thyroglobulin (3...
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Bioconjugate Chem. 1997, 8, 385−390

385

Synthesis of New d-Propoxyphene Derivatives and the Development of a Microparticle-Based Immunoassay for the Detection of Propoxyphene and Norpropoxyphene Robert S. Wu,* A. J. McNally, Ian A. Pilcher, and Salvatore J. Salamone Research and Development, Roche Diagnostic Systems, Inc., 1080 U.S. Highway 202, Somerville, New Jersey 08876-3771. Received May 6, 1996X

The synthesis of [S-(R,S)]-4-[[methyl[2-methyl-3-(1-oxopropoxy)-3,4-diphenylbutyl]amino]-1-oxobutoxy]2,5-pyrrolidinedione (propoxyphene active ester, 2) is described. This was used as an intermediate to prepare a propoxyphene immunogen, [S-(R,S)]-4-[methyl[2-methyl-3-(1-oxopropoxy)-3,4-diphenylbutyl]amino]-1-oxobutyl-Bovine Thyroglobulin (3). This immunogen was then used to generate antibodies which demonstrate good cross-reactivity to d-propoxyphene, d-norpropoxyphene, and other propoxyphene metabolites. In addition, these antibodies were shown to have very low cross-reactivity to methadone, a structurally related compound. The introduction of an aminomethyl benzoate spacer into the propoxyphene active ester (2), followed by the activation of the carboxylic acid, provided for a more stable active ester (5). This stable active ester, together with the antibodies generated from the propoxyphene immunogen, has led to the development of an immunoassay based on the Kinetic Interaction of Microparticles in Solution (KIMS).

INTRODUCTION

d-Propoxyphene (PPX), (2S:3R)-4-dimethylamino-1,2diphenyl-3-methyl-2-propionoxybutane, (known as Darvon) is a mild narcotic analgesic. The drug is widely prescribed, and overdose has been associated with a large number of fatalities (1). PPX by itself or in conjunction with other drugs, including alcohol, can be toxic. In addition to exerting respiratory depressant effects common to all µ agonist narcotics (2), PPX and norpropoxyphene (NPPX), its major metabolite, also act as local anesthetics for the treatment of mild to moderate pain disorders. Essentially all PPX abuse is due to oral ingestion, and peak plasma concentration occurs within 1-2 h after a single oral dose. The drug is metabolized primarily via double N-demethylation to NPPX and dinorpropoxyphene (DNPPX), both appearing as metabolites in urine. Urinary excretion (3) in a 20 h period following a 130 mg single oral dose of PPX, expressed as percent dose, was 1.1% PPX, 13.2% NPPX, and 0.7% DNPPX. The half-lives (4) of the metabolites are much longer than those of the parent compound, with a mean of 27 h. Common physical methods for analysis of PPX and its metabolites are GC/MS (5, 6) and LC (7, 8). A more convenient method of analysis is carried out by the use of commercial screening immunoassays, with several immunoassays being available. The assays include technologies that are enzyme-based (9, 10), fluorescence polarization (11), and radioimmunoassay (RIA). In this paper, we describe the synthesis of a new PPX hapten and the corresponding derivative for use as a label in a newly developed microparticle-based immunoassay (12). Like other OnLine immunoassays (13), this new assay utilizes the Kinetic Interaction of Microparticles in Solution (KIMS), as measured by changes in light transmission (14, 15). Because of the reported greater occurrence of NPPX in urine (3, 4), it is necessary to have an assay that detects both PPX and NPPX. Thus, the first two steps involved in this strategy for the development of this * Author to whom correspondence should be addressed. Telephone: 908-253-7931. X Abstract published in Advance ACS Abstracts, May 1, 1997.

S1043-1802(97)00028-1 CCC: $14.00

immunoassay are the preparation of antibodies and labels that allow for the recognition of both compounds. A PPX hapten immunogen (3) was synthesized as shown in Scheme 1 and used to develop antibodies capable of recognizing PPX and NPPX. In addition, a PPX Nhydroxysuccinimide ester bearing an aromatic spacer (5) was prepared as the label (Scheme 2). For the development of a microparticle-based assay, 5 was covalently coupled to a carrier protein, and this conjugate was then covalently linked to a microparticle. Thus, this newly developed microparticle-label conjugate allows for the competitive displacement of both PPX and NPPX in the immunoassay. EXPERIMENTAL PROCEDURES

General. All solvents were obtained from Fisher Scientific unless stated otherwise. All flash-grade silica gel and silica gel preparative TLC plates were obtained from E.M. Science. 1-Ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-N-hydroxybenzotriazole hydrate (NHB‚H2O), d-propoxyphene hydrochloride, d-norpropoxyphene hydrochloride, methadone hydrochloride, and goat IgG were obtained from Sigma. Dicyclohexyl carbodiimide (DCC), 4-bromo-ethylbutyrate, cesium carbonate, sodium cyanoborohydride (NaCNBH3), N-hydroxysuccinimide, succinic semialdehyde (15% solution in water), and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMC) were obtained from Aldrich. Trinitrobenzenesulfonic acid (TNBS) was obtained from Pierce. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded at 200 MHz or at 400 MHz on either a Varian XL-200 or a Varian XL-400 spectrometer, respectively. Coupling constants are given in hertz (Hz). The abbreviations used are: s, singlet; d, doublet; t, triplet; m, multiplet. All aqueous buffers and proteins were obtained from Sigma, and carboxyl modified microparticles were obtained from Rhone-Poulenc. The Coomassie protein assay reagent was obtained from Bio-Rad. PPX immunoassay can be performed on many different instruments: Roche COBAS MIRA, Roche COBAS FARA, Hitachi 717/747, Olympus © 1997 American Chemical Society

386 Bioconjugate Chem., Vol. 8, No. 3, 1997

Wu et al.

Scheme 1. Synthesis of the PPX Hapten 1

Scheme 2. Synthesis of the PPX Label 5

AU 800, and Olympus AU 5200 series. Described in this publication, COBAS MIRA from Roche Diagnostic Systems was used for the immunoassay. All samples were analyzed using point-to-point curve-fitting model with PPX concentrations of 0, 150, 300, and 600 ng/mL as the calibrators. Characterization of the Conjugate. The protein concentration is determined by the Coomassie protein assay and is also known as the Bradford method (16). Bovine serum albumin (BSA) was used as the reference. For immunogen, the degree of drug substitution on the protein conjugate was determined by the ability of remaining uncoupled lysine residues to react with TNBS (17, 18). Unmodified BTG at the same concentration as the conjugate was treated in the same manner with TNBS to provide a blank. This procedure produces a yellow complex with an absorbance maximum at 325 nm and was used to calculate the drug substitution expressed as percent modification. Typically, immunogens with a lysine modification of greater than 50% are used for animal immunization. The degree of drug substitution of the IgG conjugate was not determined. The resulting drug-protein conjugate was coupled to the microparticle as outlined in Scheme 3. Animal Immunization. Three sheep were placed on an immunization program according to an adaptation of a reported procedure (19). Briefly, immunogen 3 was emulsified with Complete Freund’s adjuvant, and each sheep received multiple site injections across the back using 1 mg of the immunogen. At the second week, the

Scheme 3. The Preparation microparticle Conjugate

of

PPX-protein-

animals received booster immunizations containing 1 mg of the immunogen emulsified in Incomplete Freund’s adjuvant. This injection was repeated twice, followed by a monthly injection of 0.5 mg of the immunogen/ Incomplete Freund’s adjuvant mixture for a period of 6 months. Each animal was then bled and serum was separated from the clot by centrifugation to provide the antisera. Preparation of [S-(R,S)]-4-[Methyl[2-methyl-3-(1oxopropoxy)-3,4-diphenylbutyl]amino]butanoic Acid (1). To a 15% solution of succinic semialdehyde (4.24 mL, 0.69 g, 6.8 mmol) was added tetrahydrofuran (THF) (15

Synthesis and Detection of Propoxyphenes

mL), NPPX (1.89 g, 5.23 mmol) and NaCNBH3 (328 mg, 5.23 mmol) at 0-4 °C. The mixture was stirred for 2 h while allowing the reaction to reach room temperature (RT). Thin layer chromatography (TLC) revealed a complete disappearance of the starting material (Silica Gel, 85:15 CH2Cl2/MeOH). To this was added 1 N HCl (15 mL), and the reaction mixture was stirred for an additional 2 h. The reaction flask was then placed under reduced pressure to remove THF, and the remaining aqueous phase was adjusted to pH 6-7 with 1.0 N NaOH. The aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined organic layer was washed with saturated NaHCO3 (2 × 10 mL) and H2O (2 × 10 mL), dried over anhydrous Na2SO4, and evaporated under reduced pressure to give 1.358 g (63%) of white solids with a good purity under TLC examination. Rf ) 0.37 (85:15 CH2Cl2/MeOH). IR (KBr): υ 1733 (ester), 1726 (COOH) cm-1. MSFAB: m/z 412 (M + 1). 1H-NMR (400 MHz, CDCl3): δ 7.40-7.01(m, 10H), 3.90 (d, J ) 14, 1H), 3.76 (d, J ) 14, 1H), 2.89-2.86(2 br s, 1H), 2.79 (m, 1H), 2.65 (m, 1H), 2.51 (m, 2H), 2.33 (s, 3H), 2.25 (q, J ) 7, 2H), 2.00 (t, J ) 10, 1H), 1.72 (br m, 2H), 1.16 (d, J ) 7, 3H), 1.06 (t, J ) 7, 3H). [R]D ) +73.5° (c ) 0.695%, CHCl3); dpropoxyphene [R]D ) +54.5° (c ) 1%, CHCl3). Preparation of [S-(R,S)][4-[Methyl[2-methyl-3-(1oxopropoxy)-3,4-diphenylbutyl]amino]-1-oxobutoxy]2,5-pyrrolidinedione (2). To a solution of 1 (101 mg, 0.225 mmol) in dry CH2Cl2 (5 mL of freshly distilled over CaH2) was added EDC (86 mg, 0.45 mmol) and Nhydroxysuccinimide (51.7 mg, 0.45 mmol). The reaction mixture was stirred for 18 h under argon, then washed with 0.2 N HCl (2 × 3 mL), saturated NaHCO3 (2 × 5 mL), and H2O (2 × 5 mL), and finally dried over anhydrous Na2SO4. The solvent was evaporated to dryness to afford 125 mg of white foam (92%). This active ester was found to degrade to the starting material upon a prolonged standing. An attempt to obtain an analytical grade sample by column chromatography using silica gel gave back the starting material 1. The active ester (2) was generally prepared fresh and was used immediately in the following reactions. IR (KBr): υ 3434 (broad OH), 1814 (NHS ester), 1738 (ester) cm-1. LR (+) MS-FAB: M, 508; observed, 509 (M + H). 1H-NMR (400 MHz, CDCl3): δ 7.42-7.00 (m, 10H), 3.87 and 3.76 (2d, J ) 14, 2H), 3.20-1.85 (m, 11H), 2.84 (s, 4H), 2.75-2.56 (2s, 3H, distereomeric salts), 2.69 (s, 3H), 1.22 (q, J ) 7, 3H), 1.06 (t, J ) 7, 3H). Preparation of [S-(R,S)]-1-[4-[4-[Methyl[2-methyl3-(1-oxopropoxy)-3,4-diphenylbutyl]amino]oxobutoxy]amino methyl]benzoic acid (4). A solution of 2.0 g (0.0132 mol) of 4-(aminomethyl)benzoic acid in THF (135 mL) and H2O (65 mL) was adjusted with 1 N NaOH (5 mL) to give a pH of about 9 or 10. To this was then added a solution of freshly prepared 2 (6.7 g, 0.0132 mol in 135 mL of THF). The reaction was driven to completion by the addition of 1 N NaOH over a period of 1 h (25 mL of 1 N NaOH total). The reaction was then neutralized to pH 6.5 with 6 N HCl, diluted with CH2Cl2 (500 mL) and washed with saturated brine solution (250 mL). The aqueous layer was then extracted with CH2Cl2 (250 mL). The organic layers were combined and washed with 50 mM K2HPO4, pH 8 (250 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to yield 6.7 g (93.5%) of a white amorphous solid. NMR showed this to be approximately 85-90% pure. This compound may be used without further purification. The above solid was chromatographed on silica gel (350 g) using 9:1 methylene CH2Cl2/MeOH to remove front running impurities, followed by 75:25 CH2Cl2/MeOH to

Bioconjugate Chem., Vol. 8, No. 3, 1997 387

elute the product. A yield of 5.02 g (70%) of a white amorphous solid was obtained. IR (KBr): υ 1733 (ester), 1653 (amide), 705 (C6H5) cm-1. LR (+) LSIMS: 545 (M + 1). 1H-NMR (400 MHz, MeOH-d4): δ 7.91 and 7.24 (d, J ) 8.3Hz, 4H), 7.377.01 (m, 10H), 4.36 (s, 2H), 3.84 (s, 2H), 2.72 (m,1H), 2.53 (br d, 1H), 2.30 (m, 1H), 2.26 (q, J ) 7.5 Hz, 2H), 2.18 (m, 3H), 2.07 (s, 3H), 1.67 (m, 2H), and 1.05-1.00 (m, 6H). [R]D ) +25.1° (c ) 1%, EtOH). Preparation of [S-(R,S)]-1-[[[4-[[4-[Methyl[2-methyl-3-(1-oxopropoxy)-3,4-diphenylbutyl]amino]-1-oxobutoxy]aminomethyl]phenyl]carbonyl]oxy]-2,5pyrrolidinedione (5). A round-bottom flask was charged with 170 mg (0.31 mmol) of 4, 119 mg (0.62 mmol) of EDC, 71.3 mg (0.62 mmol) of N-hydroxysuccinimide, and 6 mL of freshly distilled CH2Cl2. The reaction mixture was stirred overnight under an argon atmosphere. This was then washed with saturated NaHCO3 (2 × 5 mL) and water (2 × 5 mL) and dried over anhydrous NaSO4. Solvent was removed under reduced pressure to afford 169 mg (85%) of white foam. TLC Rf ) 0.33 (85:15 CH2Cl2/MeOH). Eighty five milligrams of this material was purified over a silica gel column (1.5 cm i.d. × 18 cm length) and eluted with a mixture of solvents consisting of 85:15 CH2Cl2/absolute EtOH to yield 72 mg (85% recovery) of white foam. IR (KBr): υ 1773 and 1742 (ester), 1668 (amide) cm-1. 1 H-NMR (400 MHz, CDCl3): δ 8.07 and 7.38 (d, J ) 8 Hz, 4H), 7.34-6.94 (m, 10H), 7.08 (br t, 1H, NH), 4.46 (d, J ) 4.8 Hz), 3.79 (dd, J ) ∼15 Hz, 2H), 2.91 (br s, 4H), 2.80 (br m, 1H), 2.42 (br d, 1H), 2.27-2.18 (m, 6H), 2.01 (s, 3H), 1.74-1.61 (m, 3H), 1.02 (t, J ) 7.8 Hz, 3H), and 0.97 (d, J ) 6.9 Hz, 3H). MS (+) FAB: 642 (M + 1). [R]D ) +29.1° (c ) 1%, CHCl3). Preparation of PPX Immunogen, [S-(R,S)]-4[Methyl[2-methyl-3-(1-oxopropoxy)-3,4-diphenylbutyl]amino]-1-oxobutyl-BTG (3). A solution of Bovine Thyroglobulin (BTG) (650 mg, 9.7 × 10-4 mmol, 18 mL of 36.2 mg/mL in 50 mM potassium phosphate (KPi) (pH ) 7.5) was cooled in an ice bath (this was to keep the protein solution cool while adding DMSO), and DMSO (54 mL) was added dropwise. The resulting homogeneous solution was set aside while letting the temperature equilibrate to RT. A freshly prepared NHS ester 2 (99.8 mg) was dissolved in dry dimethyl sulfoxide (DMSO) (1 mL). The hapten solution was immediately added dropwise to the previously prepared BTG solution. The reaction mixture remained homogeneous. This was stirred for 18 h at RT and poured into a dialysis bag of a 50 kDa cut-off. The bag was dialyzed against 50 mM KPi, pH 7.5, until an exchange of 1000000-fold was achieved. The resulting conjugate was then filtered through a 0.22 µm sterile filter. The protein concentration (Coomassie protein assay kit) was determined to be 5.6 mg/mL, and the lysine modification (17, 18) was 93% with a total protein recovery of 88%. Preparation of PPX-IgG Conjugate (6). Three milligrams of the NHS ester (5) was dissolved in 3 mL of dry DMSO (over molecular sieves) to make a 1 mg/ mL hapten solution. This was set aside. A solution of goat IgG (200 mg, 1.25 µmol) in 5 mL of 50 mM K2HPO4 (pH 7.5) was cooled with an ice bath. To this was added slowly 4.2 mL of DMSO. The temperature was allowed to equilibrate to 25 °C, and 0.8 mL of the previously prepared hapten solution (1.25 µmol) was added dropwise with stirring. Stirring was continued for 18 h at RT. The resulting conjugate was placed in a dialysis bag of 10 kDa cut-off. The dialysis was carried out according to the protocols described above. At the end of dialysis, the conjugate was filtered through a 0.22 µm sterile filter to

388 Bioconjugate Chem., Vol. 8, No. 3, 1997

yield 13 mL of the propoxyphene conjugate. Protein concentration of the conjugate was determined to be 6.7 mg/mL by the Coomassie protein assay. The degree of drug substitution was not determined. This conjugate was serially diluted with nonconjugated IgG in 50 mM bicarbonate buffer (pH 7.5) to provide molar ratios of 1:2, 1:4, 1:8, and 1:16 of conjugated IgG to nonconjugated IgG. The resulting mixture at each dilution molar ratio was coupled to the microparticle described in the following procedure. Coupling of PPX-IgG Conjugate to the Microparticle. Ten milliliters of carboxyl modified microparticle (10% solids of 0.21 µm) was first washed, by centrifugation at 10000g, with a 0.1% Tween-20 solution. To each milliliter of the particle, 20 mL of 0.1% Tweenwater was added, centrifuged (10000g), decanted, and resuspended. This process was repeated five times and the microparticle concentration was then adjusted to 3% w/v with a 0.1% Tween-20 solution. Two milliliters of 1-N-hydroxybenzotriazole hydrate (NHB, 25 mg/mL, 0.37 mmol), previously dissolved in DMSO, was added slowly to the 30 mL of microparticle suspension, under rapid stirring conditions. The suspension was then stirred for 10 min at 25 °C. To this suspension was added 2.9 mL of a freshly prepared CMC carbodiimide solution (50 mg/ mL, 0.34 mmol), and the mixture was stirred slowly for 3 h at 25 °C. The material was then washed by the method of centrifugation described above. The washed, activated microparticle (50 mL) was immediately mixed with 162.5 mg of IgG-propoxyphene conjugate diluted in 50 mM sodium bicarbonate, pH 8.6, which contains 1 mol of drug (initial molar ratio) per mol of IgG. This mixture was allowed to stir for 15 h at 25 °C. The conjugate-coupled microparticle was then mixed with 37.5 mL of 50 mM sodium bicarbonate, pH 8.6, containing 13.5 mg/mL (3.0 mmol) total IgG in order to block the unreacted sites. After blocking, the material was again washed by the method of centrifugation described using a wash solution of 10 mM potassium phosphate, pH 7.5, containing 0.09% sodium azide and 0.1% Tween-20. The washed microparticle was then resuspended in this buffer at 1.0% w/v solids. Development of the PPX Immunoassay. Four key components were needed for this assay. (1) The antibody reagent was made by placing the titered antibody in a solution of 50 mM HEPES, pH 6.5, containing 0.1% BSA, 0.5% sodium chloride, 0.09% NaN3, and 0.1% sheep IgG as the protein stabilizer. (2) A reaction buffer containing 50 mM PIPES, pH 7.0, with 2-3% polyethylene glycol (PEG, MW 15000-20000), 2% sodium chloride, and 0.09% sodium azide. (3) A microparticle reagent, diluted from a 1% stock solution to 0.2% solids in a buffer containing 10 mM potassium phosphate, pH 7.5, 0.09% sodium azide, and 0.1% Tween-20. (4) PPX calibrators at concentrations between 0 and 600 ng/mL in normal human urine containing 0.05% sodium azide. A dose response curve was generated by adjusting the antibody concentration to give maximum displacement in the appropriate concentration range. RESULTS AND DISCUSSION

Synthesis of the PPX Immunogen. It is generally accepted that the antibodies formed on injection of an antigen to an animal recognize preferentially the part of the molecule that is furthest from the attachment site of the hapten to carrier protein (20). Since we wished to have an antibody that can recognize both PPX and NPPX, it was advantageous to design one hapten derivative displaying a structure common to both drugs. PPX

Wu et al.

Figure 1. Design of the propoxyphene hapten used to prepare the immunogen.

differs from NPPX by having an additional methyl group on the amino group. In the design of the immunogen (see Figure 1), a drug linker arm was attached at the nitrogen center such that the exposed antigenic region would have the structure common to both the PPX and NPPX molecules. In addition, the linker arm must have a group to which the protein carrier can be covalently coupled. To accomplish this, NPPX appeared to be the ideal starting material, as the secondary amine group could be derivatized to allow the introduction of a tether. The introduction of a butanoic tether to prepare the PPX hapten 1 was accomplished by a reductive amination procedure as outlined in Scheme 1. This method was more direct than a two-step procedure which has been generally carried out by N-alkylation (21) of NPPX with ethyl 4-bromobutyrate, followed by saponification of the ethyl ester to the acid 1. The reductive-amination approach was more viable in that it affords 1 in a high yield. Activation of the acid 1 to the NHS ester 2 is straightforward, and upon addition of BTG, the propoxyphene immunogen 3 was prepared. Synthesis of the PPX Label Used in the Immunoassay. The aim was to synthesize an activated PPX derivative which could be linked to a water soluble protein carrier, and then this conjugate could be covalently coupled to a microparticle. This coupled microparticle could then act as a label. The coupled microparticle and selected PPX/NPPX antibodies were then used to generate an assay based on the KIMS technology. In the absence of free drug, the antibody binds the drugmicroparticle conjugate causing the formation of particle aggregates. The formation of these aggregates causes increased light scattering and is monitored by the changes in light transmission (14). When a urine sample containing the drug in question is present, this drug competes with the microparticle-bound drug derivative for antibody binding. The antibody bound to the free drug in the sample is no longer available to promote particle aggregation, and subsequent particle crosslinking is inhibited. In principle, compound 2 could be utilized as a label since this molecule contained a 4-carbon spacer that was sufficiently flexible for antibody recognition. However, the NHS ester 2 (Scheme 1) is not sufficiently stable as it hydrolyzes back to the acid 1 upon storage. This observation is also consistent with the

Bioconjugate Chem., Vol. 8, No. 3, 1997 389

Synthesis and Detection of Propoxyphenes

finding in this laboratory where an aliphatic NHS ester bearing a tertiary amine in the molecule has a very short life (21). The hydrolysis is most likely caused by the presence of residual water in the sample. Hydrolysis proceeded through a base-catalyzed process induced by the basic tertiary nitrogen group on the molecule. This problem is especially troublesome because the compound is labile and thus causes inconvenience in a routine preparation of the protein conjugate. On the other hand, an NHS ester directly attached to an aryl group appears to be resistant to hydrolysis despite the presence of a tertiary amine in the same molecule. Therefore, we made the corresponding benzoate NHS ester 5 by Scheme 2. This active ester has been found to be more stable and was suitable for our studies. Synthesis of PPX-Microparticle Conjugate. In producing the activated microparticle, drug conjugates with several different molar ratios of the drug to protein were evaluated to determine the optimal ratio that produced the best dose response curve. When working with microparticle technology, it is important that proper agglutination occurs in the absence of free antigen. Proper amounts of drug conjugate must be coupled to each microparticle so that an equilivance point can be reached allowing the crosslinking of microparticles by antibody. Excess antigen load or excess antibody in the system will prevent the formation of the large aggregates produced by crosslinking. Each substituted microparticle was then titrated against the antibody to determine the performance of the drug conjugate. The dilution ratio of the conjugate to IgG that gave the most sensitive dose response, and the lowest nonspecific binding (agglutination rate in the absence of antibody) was selected. This was determined to be a dilution ratio of 1:4. Generation of an Immunoassay for the Detection of PPX and its Assay Precision. Using the PPX conjugate coupled microparticle (0.21 µm) and the antibodies generated from the described immunogen, an immunoassay was developed for COBAS MIRA analyzer. This analyzer is an automated instrument that has the capacity of adding immunoassay reagents and samples into cuvettes to give tests results at the rate of 50 tests per h. The MIRA instrument first pipettes 10 µL of urine along with 85 µL of antibody with one syringe and 100 µL of sample diluent with a second syringe into a cuvette. The instrument then mixes the pipetted reagents, and the sample syringe pipettes 24 µL of microparticle reagent to the cuvette along with 25 µL of water. It then again mixes the reagents and monitors the reaction kinetics at 500 nm in 25 s intervals for 12 read points (5 min). An end point analysis with a point-to-point curve fit model is used to plot the difference in absorbance between each calibrator, and a dose response curve is generated. Figure 2 illustrates a typical standard curve that can be obtained using PPX calibrators as standards. In addition, Table 1A shows that when the precision of the assay was assessed, the % coefficient variation (CV) was less than 7% for several points in a single run. When the interassay precision was assessed, the % CV was less than 8% for all points, taken from five runs over five days. Table 1B shows that, in all cases tested, the positive threshold controls (120% cut-off value; 360 ng/mL) yielded positive determination, while conversely, the negative threshold controls (80% the cutoff, 240 ng/mL) were uniformly negative. Cross-Reactivity of Antibodies to PPX and NPPXLike Compounds. Three sheep were placed on an immunization program, as described in the methods section using immunogen 3 shown in Scheme 1. At the

Figure 2. Table 1 (A) intra-assay precision (n ) 20) mean ng/mLa 150 240 300 360

ng/mLb

SD

140 8.2 265 5.6 301 17.9 405 25.9 inter-assay precision (5 runs of n ) 20) 144 11.6 262 7.8 307 17.3 406 25.9 (B) qualitative precision

150 240 300 360

CV% 5.8 2.1 5.9 6.4 8.1 3.0 5.6 6.3

controlc

number tested

positive results

negative results

predicted frequency

0.8× 1.2×

200 200

0 200

200 0

100% negative reading 100% positive reading

a Concentration of propoxyphene in urine standard, theoretical spike value. b Concentration of propoxyphene measured experimentally by the assay. c 0.8× ) urine control containing propoxyphene at 80% of the cutoff concentration, and 1.2× ) urine control containing propoxyphene at 120% of the cutoff concentration.

Table 2

compound d-norpropoxyphene d-p-hydroxypropoxyphene methadone

approximate ng/mL approximate equivalent to 300 percent cross ng/mL propoxyphene reactivity 390 1 408 1 034 500

77 21 0.03

end of six months, the test sera were assayed for titer and cross reactivity to all of the structurally related crossreactants shown in Table 2. The titer, which was defined as 50% of saturation, was determined using ELISA and KIMS. Two sheep were found to respond well in binding

390 Bioconjugate Chem., Vol. 8, No. 3, 1997

Wu et al. LITERATURE CITED

Table 3 sample

theoretical recovery (ng/mL)

actual recovery (ng/mL)

spiked (ng/mL)

1 2 3 4

240 240 360 360

227 224 345 372

240 240 360 360

1 2 3 4

norpropoxyphene 270a 270a 385a 385a

242 251 350 378

350 350 500 500

propoxyphene

a

Calculated from spiked sample x% cross-reactivity.

Table 4 sample

propoxyphene GC/MS (ng/mL)

norpropoxyphene GC/MS (ng/mL)

OnLine (ng/mL)

13 15 22 23 28 32

71 76 244 202 34 224

3640 229 14 300 9360 11 900 23 600

>600 >600 >600 >600 >600 >600

to PPX with the antisera titer in the range 1:512 to 1:1024. A pool of the antisera of equal volume was made and used to develop the immunoassay. The ability of the polyclonal antibodies to bind PPX was demonstrated (Figure 2), and the cross-reactivity to NPPX, p-hydroxyPPX(4-dimethylamino-1-(4-hydroxyphenyl)-2-phenyl-3methyl-2-propionoxybutane), and methadone was evaluated as shown in Table 2. The cross-reactivity is expressed in two ways, the approximate percent cross-reactivity and the approximate nanogram per milliliter equivalence to give a positive result of 300 ng/mL. To further confirm that these antibodies detect both PPX and NPPX, urine samples were spiked with given amounts of PPX and NPPX, and values were determined by testing these samples in the immunoassay. These spike and recovery studies are reported in Table 3. This table shows the theoretical and actual value obtained based on 100% cross-reactivity to PPX and 77% cross-reactivity to NPPX. Sixty-four positive clinical samples with known PPX and NPPX values determined by GC/MS were also tested. All of these clinical samples were positive in the immunoassay. Fifty-eight of these clinical samples had GC/ MS PPX values of greater than 300 ng/mL. Six clinical samples showed levels of PPX less than 300 ng/mL and had high levels of NPPX by GC/MS analysis (Table 4). The positive response of these six samples by the immunoassay demonstrated the value of increased sensitivity for NPPX. In summary, we have synthesized a new PPX immunogen that elicited cross-reactive polyclonal antibodies directed to PPX and NPPX. This was accomplished by designing an immunogen having a linker arm tethered to the tertiary amino group of the PPX molecule. This allowed for the principal binding of the antibodies to be directed toward the structural elements common to both PPX and NPPX. In addition, we have synthesized a new label that demonstrates good stability, providing ease of use in the preparation of PPX-protein conjugates and for routine use in the OnLine assay. This microparticlebased assay provided increased sensitivity for detecting both PPX and NPPX in human urine samples compared to assays employing antibodies specific to only PPX.

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