245
Forensic Science International, 44 (1990) 245-255 Elsevier Scientific Publishers Ireland Ltd.
IMMUNOASSAY ANTIBODY
KIMIKO
AOKI,
Department (Received (Revision (Accepted
FOR METHAMPHETAMINE
YASUKO
of Biochemical
HIROSE
and YUKIO
Toxicology, Pharmaceutical
WITH
A NEW
KUROIWA Sciences, Showa University,
Tokyo (Japan)
February 28th, 1989) received July 4th, 1989) July 27th, 1989)
Summary p- and o-Aminomethamphetamine were synthesized as haptens to be coupled with carrier protein at the benzene ring of methamphetamine. Immunogens were prepared by the glutaraldehyde method or the MBS (N-(m-maleimidobenzoyloxy)succinimide) type cross-linking reagent method. In particular, immunization withp-aminomethamphetamine-bovine serum albumin (BSA) conjugate prepared by the glutaraldehyde method gave an anti-methamphetamine antiserum having a low cross-reactivity with methylephedrine. With the antiserum, three kinds of immunoassays for methamphetamine were established. An enzyme immunoassay (EIA) and an enzyme-linked immunosorbent assay (ELISA) were developed with alkaline phosphatase (ALP) as a label enzyme. The amount of antibody bound ALP conjugate was determined by its activity in dephosphorylating p-nitrophenyl phosphate in EIA and nicotinamide adenine dinucleotide phosphate (NADP+) in ELISA. The range of methamphetamine measurable by ELISA was 0.025-0.5 ng/well and its sensitivity was superior to that of EIA (0.3300 ng/tube). A latex agglutination inhibition reaction test (LAIRT) was also developed for the mass screening method of urine samples. The sensitivity of this method for methamphetamine was 0.1 pg/ml urine. Key words: Enzyme-linked
Methamphetamine: immunosorbent
New anti-methamphetamine antibody: assay; Latex agglutination inhibition reaction
Enzyme test
immunoassay;
Introduction Immunoassay is one of the methods of choice for the detection of drugs in biological fluids because of the simplicity of its pretreatment. We have described competitive enzyme immunoassay [l] and a latex agglutination inhibition reaction test [2] for methamphetamine in urine. However, the antibody used in these immunoassays showed a high cross-reactivity with methylephedrine, a component of proprietary medicines for the common cold. Beckett and Wilkinson reported 31.8% of methylephedrine was excreted unchanged and 8.1 yO as ephedrine in 24 h urine after oral administration in the form of its hydrochloride to a male volunteer [3]. Therefore, these immunoassays carry the risk of giving false positive results in the presence of methylephedrine. 0379-0738/90/$03.50
0 Elsevier Scientific Publishers Ireland Printed and Published in Ireland
Ltd.
246
In this paper we report the syntheses of p- and o-aminomethamphetamine as haptens and the production of anti-methamphetamine antibodies by immunization with the hapten-BSA conjugates. Enzyme immunoassays (EIAs), enzyme-linked immunosorbent assay (ELISA) and latex agglutination inhibition reaction test (LAIRT) were established with the new antibodies. Materials and Methods Materials Alkaline phosphatase (ALP) from calf intestine for enzyme immunoassay, diaphorase from microorganism and nicotinamide adenine dinucleotide phosphate (NADP+) were purchased from Boehringer Mannheim, F.R.G. Bovine serum albumin (BSA), rabbit serum albumin (RSA), alcohol dehydrogenase from bakers yeast (ADH) and p-iodonitrotetrazolium violet (INT-violet) were from Sigma Chemical Company, U.S.A. 4-(Maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (MCCS) was from Zieben Chemicals Co., Ltd., Japan. Sephadex G-200 and BrCN activated Sepharose 4B were from Pharmacia Fine Chemicals, Sweden. Protein A-cellulofine was from Seikagaku Kogyo Co., Ltd., Japan. Immunobeads (goat anti-rabbit immunoglobulin covalently bound to microspheres) were from Japan Bio-Rad Laboratories. Polystyrene microtiter plates were from S.B. Medical Co., Ltd., Japan. Carboxylate modified latex particles (diameter 0.22 pm) were kindly donated in 10% suspension from Japan Synthetic Rubber Co., Ltd. All other reagents employed were of analytical grade. Buffers and substrate solutions The composition of the buffers used in these experiments is as follows: Buffer A - 0.05 M phosphate buffer, (pH 8.0); Buffer B - buffer A containing 0.1 Y0 NaN,; Buffer C - buffer B containing 0.1%) gelatin and 0.9% NaCl; Buffer D - 0.1 M glycine buffer (glycine-KOH, pH 10.5) containing 1 mM MgCl, and 0.1 mM ZnCl,; Coating buffer - 0.01 M phosphate buffer (pH 7.4) containing 0.9% NaCl and 0.1 Y0 NaN,; Substrate A buffer D containing 6 mM p-nitrophenyl phosphate; Substrate B 0.05 M diethanolamine buffer (pH 9.5) containing 0.2 mM NADP+; Enzyme amplifier - ADH (0.17 mg) and diaphorase (0.17 mg) in 1 ml of 0.05 M phosphate buffer (pH 7.0) containing 1 mM INT-violet and 3% EtOH. Preparation of hapten The reaction products were characterized by ‘H-nuclear magnetic resonance. The following abbreviations are used; s, singlet; d, doublet; t, triplet; m, multiplet. p-Aminomethamphetamine. p-Aminomethamphetamine was prepared from paminophenylacetic acid via p-aminophenylacetone according to the method of Cavallini et al. [4]. The total yield of the product was 3.9%.
247
p-Aminophenylacetone; ‘H-NMR (CDCl,)G: 2.10(3H,s,-CH,), 354(2H,s,-CH,), 3.63(2H,broad,-NH*), 6.79(4H,q,-Ph) p-Aminomethamphetamine . HCl; ‘H-NMR(D,O)G: 1.27(3H,d,-CHCH,), 2.74(3H,s,-NHC&), 2.93(1H,q,-CH,Ph), 3.17(1H,q,-CH,Ph), 358(1H,m,-CHCH,), 7.42(4H,q,-_Ph) o-Aminomethamphetamine. Crude o-nitrophenylacetone, prepared from ofluoronitrobenzene (3.5 g, 25 mmol) according to the method of Augustine et al. [5], was purified by silica gel column chromatography (benzene/AcOEt = 4 : 1- 1: 1) to give o-nitrophenylacetone (2.77 g, 15.5 mmol). o-Nitrophenylacetone (2.77 g, 15.5 mmol), methylamine . HCl (5.2 g, 77 mmol) and NaBH,CN (0.97 g, 15.5 mmol) in absolute MeOH (45 ml) was treated according to the method of Borch et al. [6]. Crude products were purified by silica gel column chromatography (chloroform/EtOH = 9 : 1) and distillation (4 mmHg, 200°C) to give o-nitromethamphetamine (1.99 g). o-Nitromethamphetamine (1 g, 5.2 mmol) in ethanol (30 ml) was hydrogenated in the presence of Raney Ni (0.2 ml) at medium pressure and at room temperature for 20 h. The reaction mixture was filtered and 1.2 ml of cont. HCl was added to the filtrate. After the ethanol was removed in vacua, the residue was dissolved in Hz0 and washed with ether. The aqueous solution was brought to pH 10 with NaOH (solid) and extracted into ether. The ether layer washed with saturated aqueous NaCl was dried over MgS04 and evaporated. After the residue was distilled (4 mmHg, 180-185”(Z), the distillate was purified by the preparative TLC on silica gel with iso PrOH/NH,OH = 95 : 5 to give o-aminomethamphetamine (340 mg). Total yield was 16.3%. o-Nitrophenylacetone; ‘H-NMR (CDCl,) 6 : 2.32(3H,s,-CH,), 4.11(2H,s,-CL&--), 7.22-8.16(4H,m,-&) o-Nitromethamphetamine; ‘H-NMR (CDCIJ) 6 : l.O6(3H,d,-CHCH3), 2.43(3H,s,-NHCH,), 2.85(2H,m,-CH2), 3.20(1H,q,-CHCH,), 7.30-7.97(4H,m,-&) o-Aminomethamphetamine; ‘H-NMR (CDCl,) 6 : l.l1(3H,d,-CHCH,), 2.42(3H,s,-NHCH,), 2.64(2H,q,-C&-), 2.90(1H,q,-CE CH,), 3.21(3H,broad,-NH-NH,), 6.63-7.11(4H,m,--ph3 Preparation of immunogen Prepared immunogens in physiological saline were frozen until use. (I) Glutaraldehyde method. p- or o-Aminomethamphetamine . HCl(l7 mg) was coupled to BSA (45 mg) by condensation with glutaraldehyde followed by reduction with NaBH, as described previously [7]. (2) MBS [N-(m-maleimidobenzoyloxy)succinimide] type cross-linking reagent method. Acetylmercaptosuccinyl-BSA prepared by the method of Klotz and Heiney [8] was estimated to contain 16 thiol groups per BSA molecule [9]. After the hydrolysis of acetylmercaptosuccinyl-BSA, the resulting mercaptosuccinyl-BSA was coupled to p- or o-aminomethamphetamine by 4-(maleimidomethyl)
248
cyclohexane-1-carboxylic acid succinimide MBS described by Fujiwara et al. [lo].
ester (MCCS)
in the same manner as
Immunization Rabbits were immunized by the procedure described previously [7]. The antisera were stored frozen and used as an anti-methamphetamine antibody for the EIA. The IgG fraction of the antiserum was prepared with protein Acellulofine, after removal of anti-BSA antibody from the antiserum with Sepharose 4B-BSA (prepared with BrCN activated Sepharose 4B and BSA in the usual way [ 111). The lyophilized IgG fraction was used for the ELISA and the LAIRT. Preparation of methamphetamine-alkaline phosphatase conjugate (ALP conjugate) (I) Carbodiimide method. The conjugate of p- or o-aminomethamphetamine (20 mg as WC1 salt) and ALP (1 mg) was prepared with l-ethyl-3-(3. HCl by the method described previously [7]. dimethylaminopropyl)carbodiimide (2) Glutaraldehyde method [ 121. One percent glutaraldehyde aqueous solution (54 ~1) was added to o-aminomethamphetamine (7.8 mg as HCl salt) and ALP (1 mg) in 1 ml of 0.1 M phosphate buffer (pH 7.0). The reaction mixture was left for 3 h at room temperature. After the dialysis, the mixture was purified with Sephadex G-200 in the same manner as in the carbodiimide method described above. Procedure for the enzyme immunoassay (EIA) The antiserum was diluted with buffer C. The ALP conjugate was diluted with buffer A containing 0.5% BSA. Each 100 ,~l of diluted antiserum, diluted ALP conjugate solution, buffer A and sample or standard solution were allowed to stand at 4°C for 1 h. The antibody bound ALP conjugate was separated by (1) the polyethylene glycol method as described previously [13] or (2) the solid-phase second antibody method [14]. (1) Polyethylene glycol method. After addition of 50 ~1 of normal rabbit serum and 1 ml of 15 y0 polyethylene glycol aqueous solution, the mixture was centrifuged at 3000 rev./min for 20 min. The supernatant was decanted and the inner wall of the tube was wiped with tissue paper. (2) Solid-phase second antibody method. After addition of 0.2 ml of immunobeads suspension (200 mg/50 ml H,O), the mixture was allowed to stand for 2 h at 4°C then 0.5 ml of buffer D was added. The mixture was centrifuged at 3000 rev./min for 5 min and the supernatant was removed by aspiration. The precipitate was washed with 1 ml of buffer D by centrifugation. The ALP activity of the precipitate (antibody bound ALP conjugate) was measured conventionally with 1.5 ml of substrate A at 37°C for 1 h. After addition of 0.25 ml of 1N NaOH, followed by centrifugation, the absorbance of the supernatant was measured at 405 nm.
249
Procedure for the enzyme-linked immunosorbent assay (ELISA) All operations were performed at room temperature unless otherwise stated. Polystyrene microtiter wells were coated with 0.5 ,ug of IgG in 100 ~1 of coating buffer. The wells were washed twice with buffer B then filled with buffer B containing 0.1 o/0BSA. After standing for 1 h, the wells were washed with buffer A. (For storage, the wells filled with 0.1 o/0BSA solution were covered with a seal and kept at 4°C.) The ALP conjugate was diluted with 5000 ~01s. of buffer A containing 0.576 BSA. Each well was incubated with 50 ~1 of the diluted ALP conjugate solution and 50 ~1 of the sample of the standard solution for 30 min, and then washed twice with 0.1 Y0 gelatin aqueous solution. The well bound ALP activity was measured with NADP+ as a substrate according to the enzyme-amplified method of Self [ 151 and Stanley et al. [16]. Substrate B (100 ~1) was added to the well and allowed to stand for 10 min. Then 100~1 of enzyme amplifier was added, and the mixture was allowed to stand for further 5 min. After addition of 50 ~1 of 0.2 M H,S04, the absorbance was measured at 492 run with Titertek Uniskan (Flow Laboratories). Procedure for latex agglutination inhibition reaction test (LAIR T) p-Aminomethamphetamine was coupled with RSA by the glutaraldehyde method as described in the “preparation of immunogen” section for use as an antigen. Carboxylate modified latex particles were coated with the antibody (IgG fraction) or the antigen to obtain a latex-antibody or a latex-antigen reagent. The LAIRT was established with these reagents as described previously [2]. After the urine had been diluted twice and centrifuged, the supematant was used as urine sample. The latex-antibody reagent (25 ~1) was added to the 50 ~1 of the urine sample on the black glass slide and mixed for 2 min. The latex-antigen reagent (25 ~1) was added to the mixture and the agglutination reaction was visually observed. Results and Discussion Immunogens were prepared by two kinds of bifunctional cross-linking reagents; glutaraldehyde and MCCS (MBS type reagent). The amino group of the hapten and the e-amino group of lysine in BSA reacted with the aldehyde groups of glutaraldehyde. By using a MBS type reagent, the amino group of hapten and the thiol group of mercaptosuccinyl-BSA reacted with the succinimidyl group and the maleimido group of the reagent, respectively. Immunogens which could produce anti-methamphetamine antisera in rabbits were the p-aminomethamphetamine-BSA conjugate prepared by the glutaraldehyde method (I), the conjugate prepared by the MBS type reagent method (II) and the o-aminomethamphetamine-BSA conjugate prepared by the glutaraldehyde method (III). Immunogens (I, II and III) were estimated to contain 14.6, 13.3 and
250
6.3 moles of hapten per BSA molecule, respectively, by the EIAs described below. The antiserum produced with each immunogen (I, II and III) is termed antiserum I, II and III. Enzyme immunoassays (EIA-I, -11 and -111) were established with the combination of these antisera (I, II and III) and hapten-ALP conjugate. These antisera showed the reactivity only with the homologous hapten-conjugate. Especially in the case of antiserum III, the ALP conjugate needed to have a homologous hapten and a further homologous bridge. Then, thep-aminomethamphetamine-ALP conjugate was prepared by the carbodiimide method for antiserum I and II &-ALP conjugate). The o-aminomethamphetamine-ALP conjugate was prepared by the glutaraldehyde method for antiserum III (o-ALP conjugate). The antibody bound ALP conjugate was separated from the free conjugate by the polyethylene glycol method for the p-ALP conjugate and the solid-phase second antibody method for the o-ALP conjugate. A free o-ALP conjugate was also precipitated by the polyethylene glycol method, which was assumed to be because of the polymerization of ALP by glutaraldehyde. To establish the EIAs, the antiserum was diluted to exhibit approximately 50% binding of the diluted ALP conjugate. This corresponded to an absorbance 0.30.4 when the activity of the antibody bound ALP conjugate was measured at 405 nm after the incubation for 1 h at 37°C with p-nitrophenyl phosphate as a substrate. In Fig. 1, the percentage of ALP conjugate bound is plotted against the logarithm of the amount of methamphetamine. Figure 1-I shows the standard curve for EIA-I with diluted antiserum (l/6400) and diluted p-ALP conjugate (l/940). With this curve, methamphetamine could be determined in the range of 0.3lOOng/tube. Figure l-11 shows the standard curve for EIA-II with diluted antiserum (l/3200) and the diluted p-ALP conjugate (l/630). Methamphetamine could be determined in the range of l-300 ng/tube. Figure I-III shows the standard curve for EIA-III with diluted antiserum III (l/1600) and the diluted oALP conjugate (l/500). Methamphetamine could be determined in the range of 0.3-300 ng/tube. Table I shows the cross-reactivities of methamphetamine analogs in the EIA described previously [l], EIA-I, EIA-II and EIA-III. All new antisera exhibited reduced affinity for side chain analogs. Methylephedrine cross-reactivity decreased to 0.03% with antiserum I, 5.8% with antiserum II and 0.79% with antiserum III. Antiserum I and II showed a high affinity for p-hydroxymethamphetamine, a major urinary metabolite of methamphetamine, because of the similarity of the structure to that of p-aminomethamphetamine used as hapten. Antisertmr III showed a high affinity for methoxyphenamine for a similar reason. It is said to be the advantage of the MBS type reagent that self-condensation and complicated polymerization do not occur with the reagent. In our case, antiserum II was characterized by high cross-reactivities with p-hydroxymethamphetamine and methoxyphenamine, which have the same side chain structure as methamphetamine.
251
1
I
I
I
0.3
1
3
I
I
I
10 30
Methamphetamine
I
100300
(ng/tube)
Fig. 1. Standard curves for methamphetamine by the EIA-I, EIA-II and EIA-III. O-O, EIA-I with antiserum I andp-ALP conjugate; 0-0, EIA-II with antiserum II and p-ALP conjugate; H, EIAIII with antiserum III and o-ALP conjugate.
The naturally occurring amine, /I-phenylethylamine, has a maximal urinary level in many reports of 453 pg/24h [ 171. Therefore, even at this level it shows negligible inhibition in the EIAs. Antiserum I was thought to be the most suitable one for our purposes from a comparison of these antisera, because it showed low crossreactivities not only to methylephedrine but also to other analogs except for phydroxymethamphetamine and p-hydroxyephedrine. Moreover, antiserum I had higher antibody titer than the antiserum II and III. Therefore, the IgG fraction of antiserum I and the p-ALP conjugate were used in the ELISA and the LAIRT. A polystyrene microtiter well was coated with 100 ~1 of IgG solution (5 pg/ml) at 4°C overnight by simple physical adsorption. The amount of IgG on the well was presumed to be approximately 0.2 pg from the results reported by Ansari et al. [ 181. The coated well could bind about 50% of the diluted p-ALP conjugate (l/5000). Under the storage of the coated wells filled with buffer B containing 0.1 y0 BSA and covered with seal at 4°C the wells could be used as a solid-phase antibody for at least 3 months. Stanley et al. [16] applied the new calorimetric ALP detection method using enzyme amplification to EIA in order to increase both the speed and the sensitivity by measuring the formazan dye formed from INT-violet at 492nm. This ALP detection method was applied to the ELISA for methamphetamine. It took only about 1 h to finish the assay, that is 30 min for the antibody-antigen reaction, 10 min
252 TABLE
1
PERCENT
CROSS-REACTIONS
IN THE EIA”, EIA-I,
EIA-II
AND EIA-III
% Cross-reactivity Compound
Structure
0
T”3 F”3 CH2-CH -NH
Methylephedrine
06”
OH CH3 $H3 -iH -NCH3
Amphetamine
~cH~-Z-NH~
Methamphetamine
Methoxyphenamine
Ephedrine
G-
0 OH-methamphetamine
OH-ephedrine
EIA-I
EIA-II
EIA-III
100
100
100
100
0.03
5.8
0.79
4.6
0.2
3.5
1.07
0CH3 $H3 $H3 C”Z-CH -NH
1.2
7.2
Ii FH3 $I-$ H -CH -NH
4.7
0.8
0.9
0.7
-
0.01
1.8
0.05
CH,-CH2-NH2 CH3 $H3 - NH
0.2
OH CH3 $H3 - iH -NH
Forensic Science International, 44 (1990) 245-255 Elsevier Scientific Publishers Ireland Ltd.
IMMUNOASSAY ANTIBODY
KIMIKO
AOKI,
Department (Received (Revision (Accepted
FOR METHAMPHETAMINE
YASUKO
of Biochemical
HIROSE
and YUKIO
Toxicology, Pharmaceutical
WITH
A NEW
KUROIWA Sciences, Showa University,
Tokyo (Japan)
February 28th, 1989) received July 4th, 1989) July 27th, 1989)
Summary p- and o-Aminomethamphetamine were synthesized as haptens to be coupled with carrier protein at the benzene ring of methamphetamine. Immunogens were prepared by the glutaraldehyde method or the MBS (N-(m-maleimidobenzoyloxy)succinimide) type cross-linking reagent method. In particular, immunization withp-aminomethamphetamine-bovine serum albumin (BSA) conjugate prepared by the glutaraldehyde method gave an anti-methamphetamine antiserum having a low cross-reactivity with methylephedrine. With the antiserum, three kinds of immunoassays for methamphetamine were established. An enzyme immunoassay (EIA) and an enzyme-linked immunosorbent assay (ELISA) were developed with alkaline phosphatase (ALP) as a label enzyme. The amount of antibody bound ALP conjugate was determined by its activity in dephosphorylating p-nitrophenyl phosphate in EIA and nicotinamide adenine dinucleotide phosphate (NADP+) in ELISA. The range of methamphetamine measurable by ELISA was 0.025-0.5 ng/well and its sensitivity was superior to that of EIA (0.3300 ng/tube). A latex agglutination inhibition reaction test (LAIRT) was also developed for the mass screening method of urine samples. The sensitivity of this method for methamphetamine was 0.1 pg/ml urine. Key words: Enzyme-linked
Methamphetamine: immunosorbent
New anti-methamphetamine antibody: assay; Latex agglutination inhibition reaction
Enzyme test
immunoassay;
Introduction Immunoassay is one of the methods of choice for the detection of drugs in biological fluids because of the simplicity of its pretreatment. We have described competitive enzyme immunoassay [l] and a latex agglutination inhibition reaction test [2] for methamphetamine in urine. However, the antibody used in these immunoassays showed a high cross-reactivity with methylephedrine, a component of proprietary medicines for the common cold. Beckett and Wilkinson reported 31.8% of methylephedrine was excreted unchanged and 8.1 yO as ephedrine in 24 h urine after oral administration in the form of its hydrochloride to a male volunteer [3]. Therefore, these immunoassays carry the risk of giving false positive results in the presence of methylephedrine. 0379-0738/90/$03.50
0 Elsevier Scientific Publishers Ireland Printed and Published in Ireland
Ltd.
246
In this paper we report the syntheses of p- and o-aminomethamphetamine as haptens and the production of anti-methamphetamine antibodies by immunization with the hapten-BSA conjugates. Enzyme immunoassays (EIAs), enzyme-linked immunosorbent assay (ELISA) and latex agglutination inhibition reaction test (LAIRT) were established with the new antibodies. Materials and Methods Materials Alkaline phosphatase (ALP) from calf intestine for enzyme immunoassay, diaphorase from microorganism and nicotinamide adenine dinucleotide phosphate (NADP+) were purchased from Boehringer Mannheim, F.R.G. Bovine serum albumin (BSA), rabbit serum albumin (RSA), alcohol dehydrogenase from bakers yeast (ADH) and p-iodonitrotetrazolium violet (INT-violet) were from Sigma Chemical Company, U.S.A. 4-(Maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (MCCS) was from Zieben Chemicals Co., Ltd., Japan. Sephadex G-200 and BrCN activated Sepharose 4B were from Pharmacia Fine Chemicals, Sweden. Protein A-cellulofine was from Seikagaku Kogyo Co., Ltd., Japan. Immunobeads (goat anti-rabbit immunoglobulin covalently bound to microspheres) were from Japan Bio-Rad Laboratories. Polystyrene microtiter plates were from S.B. Medical Co., Ltd., Japan. Carboxylate modified latex particles (diameter 0.22 pm) were kindly donated in 10% suspension from Japan Synthetic Rubber Co., Ltd. All other reagents employed were of analytical grade. Buffers and substrate solutions The composition of the buffers used in these experiments is as follows: Buffer A - 0.05 M phosphate buffer, (pH 8.0); Buffer B - buffer A containing 0.1 Y0 NaN,; Buffer C - buffer B containing 0.1%) gelatin and 0.9% NaCl; Buffer D - 0.1 M glycine buffer (glycine-KOH, pH 10.5) containing 1 mM MgCl, and 0.1 mM ZnCl,; Coating buffer - 0.01 M phosphate buffer (pH 7.4) containing 0.9% NaCl and 0.1 Y0 NaN,; Substrate A buffer D containing 6 mM p-nitrophenyl phosphate; Substrate B 0.05 M diethanolamine buffer (pH 9.5) containing 0.2 mM NADP+; Enzyme amplifier - ADH (0.17 mg) and diaphorase (0.17 mg) in 1 ml of 0.05 M phosphate buffer (pH 7.0) containing 1 mM INT-violet and 3% EtOH. Preparation of hapten The reaction products were characterized by ‘H-nuclear magnetic resonance. The following abbreviations are used; s, singlet; d, doublet; t, triplet; m, multiplet. p-Aminomethamphetamine. p-Aminomethamphetamine was prepared from paminophenylacetic acid via p-aminophenylacetone according to the method of Cavallini et al. [4]. The total yield of the product was 3.9%.
247
p-Aminophenylacetone; ‘H-NMR (CDCl,)G: 2.10(3H,s,-CH,), 354(2H,s,-CH,), 3.63(2H,broad,-NH*), 6.79(4H,q,-Ph) p-Aminomethamphetamine . HCl; ‘H-NMR(D,O)G: 1.27(3H,d,-CHCH,), 2.74(3H,s,-NHC&), 2.93(1H,q,-CH,Ph), 3.17(1H,q,-CH,Ph), 358(1H,m,-CHCH,), 7.42(4H,q,-_Ph) o-Aminomethamphetamine. Crude o-nitrophenylacetone, prepared from ofluoronitrobenzene (3.5 g, 25 mmol) according to the method of Augustine et al. [5], was purified by silica gel column chromatography (benzene/AcOEt = 4 : 1- 1: 1) to give o-nitrophenylacetone (2.77 g, 15.5 mmol). o-Nitrophenylacetone (2.77 g, 15.5 mmol), methylamine . HCl (5.2 g, 77 mmol) and NaBH,CN (0.97 g, 15.5 mmol) in absolute MeOH (45 ml) was treated according to the method of Borch et al. [6]. Crude products were purified by silica gel column chromatography (chloroform/EtOH = 9 : 1) and distillation (4 mmHg, 200°C) to give o-nitromethamphetamine (1.99 g). o-Nitromethamphetamine (1 g, 5.2 mmol) in ethanol (30 ml) was hydrogenated in the presence of Raney Ni (0.2 ml) at medium pressure and at room temperature for 20 h. The reaction mixture was filtered and 1.2 ml of cont. HCl was added to the filtrate. After the ethanol was removed in vacua, the residue was dissolved in Hz0 and washed with ether. The aqueous solution was brought to pH 10 with NaOH (solid) and extracted into ether. The ether layer washed with saturated aqueous NaCl was dried over MgS04 and evaporated. After the residue was distilled (4 mmHg, 180-185”(Z), the distillate was purified by the preparative TLC on silica gel with iso PrOH/NH,OH = 95 : 5 to give o-aminomethamphetamine (340 mg). Total yield was 16.3%. o-Nitrophenylacetone; ‘H-NMR (CDCl,) 6 : 2.32(3H,s,-CH,), 4.11(2H,s,-CL&--), 7.22-8.16(4H,m,-&) o-Nitromethamphetamine; ‘H-NMR (CDCIJ) 6 : l.O6(3H,d,-CHCH3), 2.43(3H,s,-NHCH,), 2.85(2H,m,-CH2), 3.20(1H,q,-CHCH,), 7.30-7.97(4H,m,-&) o-Aminomethamphetamine; ‘H-NMR (CDCl,) 6 : l.l1(3H,d,-CHCH,), 2.42(3H,s,-NHCH,), 2.64(2H,q,-C&-), 2.90(1H,q,-CE CH,), 3.21(3H,broad,-NH-NH,), 6.63-7.11(4H,m,--ph3 Preparation of immunogen Prepared immunogens in physiological saline were frozen until use. (I) Glutaraldehyde method. p- or o-Aminomethamphetamine . HCl(l7 mg) was coupled to BSA (45 mg) by condensation with glutaraldehyde followed by reduction with NaBH, as described previously [7]. (2) MBS [N-(m-maleimidobenzoyloxy)succinimide] type cross-linking reagent method. Acetylmercaptosuccinyl-BSA prepared by the method of Klotz and Heiney [8] was estimated to contain 16 thiol groups per BSA molecule [9]. After the hydrolysis of acetylmercaptosuccinyl-BSA, the resulting mercaptosuccinyl-BSA was coupled to p- or o-aminomethamphetamine by 4-(maleimidomethyl)
248
cyclohexane-1-carboxylic acid succinimide MBS described by Fujiwara et al. [lo].
ester (MCCS)
in the same manner as
Immunization Rabbits were immunized by the procedure described previously [7]. The antisera were stored frozen and used as an anti-methamphetamine antibody for the EIA. The IgG fraction of the antiserum was prepared with protein Acellulofine, after removal of anti-BSA antibody from the antiserum with Sepharose 4B-BSA (prepared with BrCN activated Sepharose 4B and BSA in the usual way [ 111). The lyophilized IgG fraction was used for the ELISA and the LAIRT. Preparation of methamphetamine-alkaline phosphatase conjugate (ALP conjugate) (I) Carbodiimide method. The conjugate of p- or o-aminomethamphetamine (20 mg as WC1 salt) and ALP (1 mg) was prepared with l-ethyl-3-(3. HCl by the method described previously [7]. dimethylaminopropyl)carbodiimide (2) Glutaraldehyde method [ 121. One percent glutaraldehyde aqueous solution (54 ~1) was added to o-aminomethamphetamine (7.8 mg as HCl salt) and ALP (1 mg) in 1 ml of 0.1 M phosphate buffer (pH 7.0). The reaction mixture was left for 3 h at room temperature. After the dialysis, the mixture was purified with Sephadex G-200 in the same manner as in the carbodiimide method described above. Procedure for the enzyme immunoassay (EIA) The antiserum was diluted with buffer C. The ALP conjugate was diluted with buffer A containing 0.5% BSA. Each 100 ,~l of diluted antiserum, diluted ALP conjugate solution, buffer A and sample or standard solution were allowed to stand at 4°C for 1 h. The antibody bound ALP conjugate was separated by (1) the polyethylene glycol method as described previously [13] or (2) the solid-phase second antibody method [14]. (1) Polyethylene glycol method. After addition of 50 ~1 of normal rabbit serum and 1 ml of 15 y0 polyethylene glycol aqueous solution, the mixture was centrifuged at 3000 rev./min for 20 min. The supernatant was decanted and the inner wall of the tube was wiped with tissue paper. (2) Solid-phase second antibody method. After addition of 0.2 ml of immunobeads suspension (200 mg/50 ml H,O), the mixture was allowed to stand for 2 h at 4°C then 0.5 ml of buffer D was added. The mixture was centrifuged at 3000 rev./min for 5 min and the supernatant was removed by aspiration. The precipitate was washed with 1 ml of buffer D by centrifugation. The ALP activity of the precipitate (antibody bound ALP conjugate) was measured conventionally with 1.5 ml of substrate A at 37°C for 1 h. After addition of 0.25 ml of 1N NaOH, followed by centrifugation, the absorbance of the supernatant was measured at 405 nm.
249
Procedure for the enzyme-linked immunosorbent assay (ELISA) All operations were performed at room temperature unless otherwise stated. Polystyrene microtiter wells were coated with 0.5 ,ug of IgG in 100 ~1 of coating buffer. The wells were washed twice with buffer B then filled with buffer B containing 0.1 o/0BSA. After standing for 1 h, the wells were washed with buffer A. (For storage, the wells filled with 0.1 o/0BSA solution were covered with a seal and kept at 4°C.) The ALP conjugate was diluted with 5000 ~01s. of buffer A containing 0.576 BSA. Each well was incubated with 50 ~1 of the diluted ALP conjugate solution and 50 ~1 of the sample of the standard solution for 30 min, and then washed twice with 0.1 Y0 gelatin aqueous solution. The well bound ALP activity was measured with NADP+ as a substrate according to the enzyme-amplified method of Self [ 151 and Stanley et al. [16]. Substrate B (100 ~1) was added to the well and allowed to stand for 10 min. Then 100~1 of enzyme amplifier was added, and the mixture was allowed to stand for further 5 min. After addition of 50 ~1 of 0.2 M H,S04, the absorbance was measured at 492 run with Titertek Uniskan (Flow Laboratories). Procedure for latex agglutination inhibition reaction test (LAIR T) p-Aminomethamphetamine was coupled with RSA by the glutaraldehyde method as described in the “preparation of immunogen” section for use as an antigen. Carboxylate modified latex particles were coated with the antibody (IgG fraction) or the antigen to obtain a latex-antibody or a latex-antigen reagent. The LAIRT was established with these reagents as described previously [2]. After the urine had been diluted twice and centrifuged, the supematant was used as urine sample. The latex-antibody reagent (25 ~1) was added to the 50 ~1 of the urine sample on the black glass slide and mixed for 2 min. The latex-antigen reagent (25 ~1) was added to the mixture and the agglutination reaction was visually observed. Results and Discussion Immunogens were prepared by two kinds of bifunctional cross-linking reagents; glutaraldehyde and MCCS (MBS type reagent). The amino group of the hapten and the e-amino group of lysine in BSA reacted with the aldehyde groups of glutaraldehyde. By using a MBS type reagent, the amino group of hapten and the thiol group of mercaptosuccinyl-BSA reacted with the succinimidyl group and the maleimido group of the reagent, respectively. Immunogens which could produce anti-methamphetamine antisera in rabbits were the p-aminomethamphetamine-BSA conjugate prepared by the glutaraldehyde method (I), the conjugate prepared by the MBS type reagent method (II) and the o-aminomethamphetamine-BSA conjugate prepared by the glutaraldehyde method (III). Immunogens (I, II and III) were estimated to contain 14.6, 13.3 and
250
6.3 moles of hapten per BSA molecule, respectively, by the EIAs described below. The antiserum produced with each immunogen (I, II and III) is termed antiserum I, II and III. Enzyme immunoassays (EIA-I, -11 and -111) were established with the combination of these antisera (I, II and III) and hapten-ALP conjugate. These antisera showed the reactivity only with the homologous hapten-conjugate. Especially in the case of antiserum III, the ALP conjugate needed to have a homologous hapten and a further homologous bridge. Then, thep-aminomethamphetamine-ALP conjugate was prepared by the carbodiimide method for antiserum I and II &-ALP conjugate). The o-aminomethamphetamine-ALP conjugate was prepared by the glutaraldehyde method for antiserum III (o-ALP conjugate). The antibody bound ALP conjugate was separated from the free conjugate by the polyethylene glycol method for the p-ALP conjugate and the solid-phase second antibody method for the o-ALP conjugate. A free o-ALP conjugate was also precipitated by the polyethylene glycol method, which was assumed to be because of the polymerization of ALP by glutaraldehyde. To establish the EIAs, the antiserum was diluted to exhibit approximately 50% binding of the diluted ALP conjugate. This corresponded to an absorbance 0.30.4 when the activity of the antibody bound ALP conjugate was measured at 405 nm after the incubation for 1 h at 37°C with p-nitrophenyl phosphate as a substrate. In Fig. 1, the percentage of ALP conjugate bound is plotted against the logarithm of the amount of methamphetamine. Figure 1-I shows the standard curve for EIA-I with diluted antiserum (l/6400) and diluted p-ALP conjugate (l/940). With this curve, methamphetamine could be determined in the range of 0.3lOOng/tube. Figure l-11 shows the standard curve for EIA-II with diluted antiserum (l/3200) and the diluted p-ALP conjugate (l/630). Methamphetamine could be determined in the range of l-300 ng/tube. Figure I-III shows the standard curve for EIA-III with diluted antiserum III (l/1600) and the diluted oALP conjugate (l/500). Methamphetamine could be determined in the range of 0.3-300 ng/tube. Table I shows the cross-reactivities of methamphetamine analogs in the EIA described previously [l], EIA-I, EIA-II and EIA-III. All new antisera exhibited reduced affinity for side chain analogs. Methylephedrine cross-reactivity decreased to 0.03% with antiserum I, 5.8% with antiserum II and 0.79% with antiserum III. Antiserum I and II showed a high affinity for p-hydroxymethamphetamine, a major urinary metabolite of methamphetamine, because of the similarity of the structure to that of p-aminomethamphetamine used as hapten. Antisertmr III showed a high affinity for methoxyphenamine for a similar reason. It is said to be the advantage of the MBS type reagent that self-condensation and complicated polymerization do not occur with the reagent. In our case, antiserum II was characterized by high cross-reactivities with p-hydroxymethamphetamine and methoxyphenamine, which have the same side chain structure as methamphetamine.
251
1
I
I
I
0.3
1
3
I
I
I
10 30
Methamphetamine
I
100300
(ng/tube)
Fig. 1. Standard curves for methamphetamine by the EIA-I, EIA-II and EIA-III. O-O, EIA-I with antiserum I andp-ALP conjugate; 0-0, EIA-II with antiserum II and p-ALP conjugate; H, EIAIII with antiserum III and o-ALP conjugate.
The naturally occurring amine, /I-phenylethylamine, has a maximal urinary level in many reports of 453 pg/24h [ 171. Therefore, even at this level it shows negligible inhibition in the EIAs. Antiserum I was thought to be the most suitable one for our purposes from a comparison of these antisera, because it showed low crossreactivities not only to methylephedrine but also to other analogs except for phydroxymethamphetamine and p-hydroxyephedrine. Moreover, antiserum I had higher antibody titer than the antiserum II and III. Therefore, the IgG fraction of antiserum I and the p-ALP conjugate were used in the ELISA and the LAIRT. A polystyrene microtiter well was coated with 100 ~1 of IgG solution (5 pg/ml) at 4°C overnight by simple physical adsorption. The amount of IgG on the well was presumed to be approximately 0.2 pg from the results reported by Ansari et al. [ 181. The coated well could bind about 50% of the diluted p-ALP conjugate (l/5000). Under the storage of the coated wells filled with buffer B containing 0.1 y0 BSA and covered with seal at 4°C the wells could be used as a solid-phase antibody for at least 3 months. Stanley et al. [16] applied the new calorimetric ALP detection method using enzyme amplification to EIA in order to increase both the speed and the sensitivity by measuring the formazan dye formed from INT-violet at 492nm. This ALP detection method was applied to the ELISA for methamphetamine. It took only about 1 h to finish the assay, that is 30 min for the antibody-antigen reaction, 10 min
252 TABLE
1
PERCENT
CROSS-REACTIONS
IN THE EIA”, EIA-I,
EIA-II
AND EIA-III
% Cross-reactivity Compound
Structure
0
T”3 F”3 CH2-CH -NH
Methylephedrine
06”
OH CH3 $H3 -iH -NCH3
Amphetamine
~cH~-Z-NH~
Methamphetamine
Methoxyphenamine
Ephedrine
G-
0 OH-methamphetamine
OH-ephedrine
EIA-I
EIA-II
EIA-III
100
100
100
100
0.03
5.8
0.79
4.6
0.2
3.5
1.07
0CH3 $H3 $H3 C”Z-CH -NH
1.2
7.2
Ii FH3 $I-$ H -CH -NH
4.7
0.8
0.9
0.7
-
0.01
1.8
0.05
CH,-CH2-NH2 CH3 $H3 - NH
0.2
OH CH3 $H3 - iH -NH
